0.10) were noted in total protein.\",\n \"Serum albumin was increased (P<0.08) on d 14 with the globulin and plasma treatment compared to the control, while that of the pepsin digested globulin group was intermediate.\",\n \"Enzyme activity, intestinal morphology, and fecal score are presented in Table 5.No differences (P>0.10) were noted in villous height and crypt depth.\",\n \"Duodenal lactase and maltase activity was increased (P<0.07) due to consumption of pepsin digested globulin compared to the control diet, while the other dietary treatments were intermediate.\",\n \"The fecal score was reduced (P<0.07; respresenting a firmer stool) due to the addition of pepsin digested globulin compared to the control while the fecal score of and plasma while globulin was intermediate.\",\n \"Tables TABLE 1 Composition of experimental diets (as fed, %).a Pepsin Digested Ingredients Control Plasma Globulin Globulin Corn 42.932 43.012 42.962 42.957 47% SBM 23.000 23.000 23.000 23.000 Dried Whey 17.000 17.000 17.000 17.000 Menhaden 8.500 3.400 3.400 Fishmeal Plasma 6.000 Globulin 3.600 Pepsin Digested 3.600 Globulin Soy Oil 4.300 5.100 4.800 4.800 Lactose 2.118 2.118 2.118 2.118 18.5% Dical 0.400 1.700 1.150 1.150 Limestone 0.070 0.435 0.290 0.290 Zinc Oxide 0.400 0.400 0.400 0.400 Mecadox 0.250 0.250 0.250 0.250 Salt 0.250 0.250 0.250 0.250 Premix 0.400 0.400 0.400 0.400 L-Lysine HCL 0.250 0.195 0.290 0.290 L-Threonine 0.090 DL-Methionine 0.040 0.140 0.090 0.095 aDiets were formulated to contain 1.60% lysine, 0.48% methionine, 14% lactose, 0.8% calcium, and 0.7% phosphorus and fed from d 0 to 14 post-weaning.\",\n \"TABLE 2 Effect of spray-dried plasma and plasma fractions on average daily gain and feed intake (kg/d).1 Pepsin Digested Treatment Control Plasma Globulin Globulin SEM ADG, kg/d D 0-6 0.037 0.094 0.080 0.073 0.029 D 0-14 0.169a 0.242b 0.234b 0.222ab 0.025 ADFI, kg/d D 0-6 0.104 0.134 0.132 0.128 0.018 D 0-14 0.213a 0.276b 0.278b 0.254ab 0.021 1Values are least squares means with 16 pigs/treatment.\",\n \"abMeans within a row without common superscript letters are different (P < 0.10).\",\n \"TABLE 3 Effect of spray-dried plasma and plasma fractions on organ weights (g/kg body weight)1 Pepsin Organ Weights, Digested g/kg BW Control Plasma Globulin Globulin SEM Intestine 44.21 50.65 50.34 44.71 3.43 Liver 32.34 31.20 30.23 32.27 1.42 Spleen 1.74 1.83 1.81 2.06 0.16 Thymus 1.45 1.39 1.32 1.36 0.20 Heart 4.93 4.89 4.94 4.73 0.22 Lung 11.26 11.28 12.14 11.95 1.03 Stomach 6.96 7.06 6.61 6.84 0.32 Kidney 4.76 5.75 5.66 5.45 0.47 Pancreas 1.93a 2.20ab 2.42b 2.34b 0.11 1Values are least squares means of 6 pigs/treatment.\",\n \"abMeans within a row without common superscript letters are different (P < 0.05).\",\n \"TABLE 4 Effect of spray-dried plasma and plasma fractions on blood parameters.1,2 Pepsin Digested Control Plasma Globulin Globulin SEM IgG, mg/mL D0 4.84a 5.70b 4.83a 5.05ab 0.34 D7 4.98 4.71 4.66 4.96 0.17 D14 4.88b 4.43ab 4.30a 4.54ab 0.24 Total Protein, g/dL D0 4.55 4.59 4.54 4.65 0.07 D7 4.39 4.37 4.35 4.47 0.08 D14 4.22 4.30 4.29 4.20 0.07 Albumin, g/dL D0 3.03 3.02 3.11 3.09 0.06 D7 2.98 3.03 3.02 3.01 0.06 D14 2.61a 2.78b 2.80b 2.71ab 0.07 1Values are least squares means of 16 pigs/treatment.\",\n \"2Day 0 used as a covariate for analysis on D7 and D14.abMeans within a row without common superscript letters are different (P < 0.08).\",\n \"TABLE 5 Effect of spray-dried plasma and plasma fractions on enzyme activities, intestinal morphology, and fecal score.1 Pepsin Digested Control Plasma Globulin Globulin SEM Maltase, umol/mg 7.97a 11.08ab 10.93ab 13.30b 1.93 prot/hr Lactase, umol/mg 1.14a 1.57ab 1.55ab 2.15b 0.31 prot/hr Villous Height, 378.7 370.7 374.0 387.7 34.4 micron Crypt Depth, micron 206.3 191.0 195.0 192.7 9.3 Fecal Score 5.12b 5.06b 4.19ab 2.88a 0.65 1Values are least squares means of 6 pigs/treatment abMeans within a row without common superscript letters are different (P < 0.07) EXAMPLE 3 Quantity and Impact of Dietary Inclusion of Variable Plasma Fractions In the second experiment the objective was to quantify the impact of dietary inclusion of different plasma fractions and the effect of separating the heavy and light chains of the IgG on average daily gain, average daily feed intake, organ weights, and blood parameters of weanling pigs.\",\n \"Materials and Methods Animals and Diets.\",\n \"Ninety-six individually penned pigs averaging 5.89 kg body weight and 21 d of age were allotted to four dietary treatments in a randomized complete block design.\",\n \"The animals were blocked by time between 3 unsanitized nursery rooms.\",\n \"Pigs were given ad libitum access to water and feed.\",\n \"Dietary treatments (Table 6) consisted of: 1) control; 2) 10% spray-dried plasma; 3) 6% spray-dried globulin; and 4) 6% globulin-rich material treated to reduce the disulfide bonds of the IgG molecule (H+L).\",\n \"Diets were corn-soybean meal-dried whey based replacing soybean meal with plasma on an equal lysine basis.\",\n \"The plasma fractions were added relative to plasma on an equal plasma fraction basis.\",\n \"Diets contained 1.60% lysine and were formulated to an ideal amino acid profile (Chung and Baker, 1992).\",\n \"Diets were meal form and fed from d 0-14 post-weaning.\",\n \"Collection of Data.\",\n \"Individual pig weights were collected on d 0, 2, 4, 6, 8, 10, 12, and 14 post-weaning.\",\n \"Feed intake and diarrhea score were collected daily from d 0 to 14 post-weaning.\",\n \"Blood was collected on d 0, 7, and 14 post-weaning.\",\n \"The blood was centrifuged and serum samples were frozen for subsequent analysis.\",\n \"Upon completion of the study (d 14), nine pigs/treatment were sacrificed to obtain organ weights (intestine, heart, liver, spleen, thymus, lung, kidney, stomach, and pancreas).\",\n \"Serum Analysis.\",\n \"Total protein, albumin, and urea nitrogen were analyzed according to ROCHE Diagnostic kits for a COBAS MIRA system.\",\n \"Serum IgG was analyzed according to Etzel et al.\",\n \"(1997).\",\n \"Statistical Analysis.\",\n \"Data were analyzed as a randomized complete block design using the GLM procedures of SAS (SAS/STAT Version 6.11 SAS Institute, Cary N.C.).\",\n \"Pigs were individually housed and the pen was the experimental unit.\",\n \"Model sum of squares consisted of block and treatment, using initial weight as a covariate.\",\n \"Least squares means for treatments are reported.\",\n \"Results From d 0-6 (Table 7), plasma increased (P<0.10) ADFI compared to control and H+L, while the globulin was intermediate.\",\n \"From d 7-14, plasma increased (P<0.10) ADFI compared to control and H+L treatments.\",\n \"Average daily feed intake of globulin fed pigs was increased compared to the control.\",\n \"From d 0-14, plasma and globulin increased (P<0.10) ADFI compared to the control and H+L dietary treatments.\",\n \"Average daily gain is presented in Table 8.Average daily gain was similar to ADFI for d 0-6.From d 7-14 and 0-14, plasma and globulin increased (P<0.10) ADG compared to the control, while H+L was intermediate.\",\n \"Blood parameters are presented in Table 9.Serum IgG and urea nitrogen (d 14) were lower (P<0.05) by the dietary inclusion of plasma and globulin compared to the control.\",\n \"The effect of H+L was intermediate.\",\n \"Dietary treatment had no effect on serum protein.\",\n \"Serum albumin (d 7) was decreased (P<0.05) due to inclusion of plasma compared to the other dietary treatments.\",\n \"No differences were noted in fecal score.\",\n \"Intestinal length and organ weights are presented in Table 10.No differences were noted in organ weights or intestinal length due to dietary treatment.\",\n \"Tables TABLE 6 Composition of experimental diets (as fed.\",\n \"%)1 Ingredients Control Plasma Globulin H + L Corn 37.937 44.96 40.006 40.034 47% Soybean Meal 18 18 18 18 Dried Whey 14 14 14 14 Lactose 6.253 6.253 6.253 6.253 Plasma 10 Globulin 6 H + L 6 Soy Protein 17.31 9.07 9.07 Concentrate Soy Oil 3.219 3.047 3.187 3.186 18.5% Dical 1.79 1.493 2.133 2.146 Limestone 0.562 0.354 0.46 0.42 Premix 0.55 0.55 0.55 0.55 Salt 0.15 0.15 0.15 0.15 DL-Methionine 0.083 0.152 0.092 0.096 L-Lysine HCL 0.146 0.041 0.099 0.095 1Diets were formulated to contain 1.60% lysine, 0.48% methionine, 16% lactose, 0.9% calcium, and 0.8% phosphorus and fed from d 0 to 14 post-weaning.\",\n \"TABLE 7 Effect of spray-dried plasma and plasma fractions on average daily feed intake (g/d).1 ADFI, g/d Control Plasma Globulin H + L SEM D 0-6 102.82a 152.43b 128.53ab 114.50a 13.44 D 7-14 280.74a 413.57c 379.21bc 319.06ab 29.07 D 0-14 193.94a 284.83b 258.55b 216.83ab 16.69 1Values are least squares means of 24 pigs/treatment.\",\n \"abcMeans within a row without common superscript letters are different (P < 0.10).\",\n \"TABLE 8 Effect of spray-dried plasma and plasma fractions on average daily gain (g/d).1 ADG, g/d Control Plasma Globulin H + L SEM D 0-6 −41.05a 27.23b −1.23ab −21.86a 20.26 D 7-14 199.38a 282.46b 302.22b 255.12ab 26.40 D 0-14 96.34a 173.07b 172.17b 136.42ab 20.56 1Values are least squares means of 24 pigs/treatment.\",\n \"abcMeans within a row without common superscript letters are different (P < 0.10).\",\n \"TABLE 9 Effects of spray-dried plasma fractions on blood parameters.1,2 Control Plasma Globulin H + L SEM IgG, g/dL D 0 0.674 0.664 0.584 0.661 0.037 D 7 0.668 0.643 0.624 0.673 0.021 D 14 0.631b 0.555a 0.545a 0.596ab 0.022 Urea N. mg/dL D 0 8.53 9.78 9.94 9.87 0.68 D 7 17.55b 14.65a 16.48ab 17.56b 1.01 D 14 17.57c 10.48a 14.73b 15.56bc 0.87 Total Protein, g/dL D 0 4.58 4.46 4.56 4.56 0.076 D 7 4.69 4.60 4.53 4.74 0.106 D 14 4.55 4.49 4.59 4.49 0.080 Albumin, g/dL D 0 2.69 2.64 2.75 2.69 0.069 D 7 2.92b 2.79a 2.92b 2.94b 0.045 D 14 2.83 2.76 2.86 2.80 0.060 1Values are least squares means of 24 pigs/treatment.\",\n \"2Day 0 used as a covariate for analysis on D 7 and D 14.abcMeans within a row without common superscript letters are different (P < 0.05).\",\n \"TABLE 10 Effect of spray-dried plasma and plasma fractions on intestinal length (inches) and organ weights (g/kg body weight)1 Control Plasma Globulin H + L SEM Int.\",\n \"length, inch 358.67 368.33 359.33 358.56 13.05 Organ weight, g/kg BW Intestine 41.48 41.79 42.82 41.04 2.16 Liver 29.61 32.61 32.29 31.09 1.10 Spleen 2.05 2.32 2.44 2.17 0.22 Thymus 1.15 1.45 1.15 1.15 0.14 Heart 6.12 6.14 5.77 5.80 0.22 Lung 12.24 12.33 13.65 11.63 0.74 Stomach 9.26 9.14 10.08 10.08 0.58 Kidney 6.18 6.57 6.10 6.30 0.21 Pancreas 2.70 2.61 2.54 2.70 0.11 1Values are least squares means of 9 pigs/treatment.\",\n \"Discussion Consistent with published research (Coffey and Cromwell, 1995) these data indicate that when included in the diet plasma and globulin increase performance (ADG, ADFI) compared to the control.\",\n \"The pepsin digested globulin and H+L fraction resulted in an intermediate improvement in performance.\",\n \"Enzyme activity (lactase and maltase) were increased and fecal score was improved with the addition of all plasma fractions (plasma, globulin, pepsin digested globulin, H&L) compared to the control.\",\n \"Serum IgG concentration and BUN were lower after consumption of plasma or globulin treatments compared to the control, pepsin digested globulin or H&L.\",\n \"The ability of oral plasma or globulin administration to elicit a systemic response as demonstrated by lower serum IgG compared to the control was unexpected.\",\n \"The noted differences between plasma and globulin fractions compared to the pepsin digested globulin or H+L is that the tertiary structure of the Fc region is intact in the plasma and globulin fractions only.\",\n \"The pepsin digested globulin has the Fc region digested, while in the H+L fraction, the Fc region remains intact but without tertiary confirmation.\",\n \"The Fab region is still intact in the pepsin digested globulin.\",\n \"The variable region is still able to bind antigen in the H+L preparation (APC, unpublished data).\",\n \"Thus, the results indicate the antibody-antigen interaction (Fab region) is important for local effects (reduced fecal score, increased lactase and maltase activity), while the intact Fab and Fc region of plasma and globulin fractions is important to modulate the systemic serum IgG response.\",\n \"EXAMPLE 4 Effect of Oral Doses of Plasma Protein on Active Immune Responses to Primary and Secondary Rotavirus and PRRS Vaccinations in Baby Pigs Overview To examine the influence of supplemental plasma protein on active immune responses following primary and secondary rotavirus and PRRS vaccinations.\",\n \"Methods Ten sows induced to farrow at a common time were utilized.\",\n \"Treatments were assigned randomly within each litter.\",\n \"Treatment delivery occurred twice weekly (3 or 4 day intervals) via a stomach tube applicator.\",\n \"A series of 7 applications occurred prior to the final vaccination and weaning.\",\n \"Treatments consisted of: control (10 mL saline) and plasma IgG (0.5 g delivered in a final volume of 8 mL).\",\n \"All pigs received a primary vaccination (orally=rotavirus; injection ═PRRS) 10 days prior to weaning.\",\n \"A secondary vaccination was given at the time of weaning via intramuscular injection.\",\n \"Blood samples were collected prior to the primary vaccination (10 d prior to weaning), prior to the secondary vaccination (at weaning), and on 3 day intervals until 12 days post-weaning.\",\n \"Results Pigs dosed with plasma protein experienced significant (P<0.05) decreases in specific antibody titers following booster vaccination.\",\n \"This response was seen for both rotavirus (FIG.\",\n \"1) and PRRS (FIG.\",\n \"2) antibody titers.\",\n \"Discussion These data provide an excellent indication of the effect of oral plasma protein in the young pig.\",\n \"Immune activation acts as a large energy and nutrient sink.\",\n \"When the immune system is activated energy and nutrients are funneled into the production of immune products (immunoglobulin, cytokines, acute phase proteins, etc.)\",\n \"and away from growth.\",\n \"Oral plasma may modulate the immune system, thereby allowing energy and nutrients to be redirected to other productive functions such as growth.\",\n \"EXAMPLE 5 Evaluation of Plasma Delivered Via Water in Turkeys Under Disease Challenge Overview To evaluate blood or fractions thereof such as serum, plasma or portions purified therefrom preferably containing immunoglobulin, when administered to animals, in particular poultry, and specifically to turkeys via their water, effects death loss in a positive manner when the turkeys are disease challenged.\",\n \"The invention demonstrates improvement in performance of turkeys specifically during the starting period if they have consumed plasma proteins in the water.\",\n \"Overall, delivery of plasma proteins via the water increases feed efficiency and percent remaining (survival) after respiratory challenge and aids in starting turkeys.\",\n \"Materials and Methods Eighty male one day old Nicholas turkey poults were randomly assigned to water treatments.\",\n \"Initial body weight was 59 g. Treatments were applied in a factorial design consisting of 1) disease challenge or no disease challenge and 2) plasma treated water or regular water.\",\n \"The turkey poults were housed as 6 or 7 turkeys per pen utilizing a total of 12 floor pens.\",\n \"The challenge turkeys were separated from the non-challenge turkeys to alleviate cross contamination.\",\n \"Body weight, feed intake and water intakes were measured daily.\",\n \"Turkeys were offered commercially available diets.\",\n \"Fresh water treatments were offered daily.\",\n \"The plasma concentrations in the treated water was altered regularly consisting of 1.3%, 0.65%, 0.325%, and 1.3% for d 0-7,7-14, 14-21, and 21-49, respectively.\",\n \"The turkeys were challenged on d 35 with pastuerella to induce a respiratory challenge.\",\n \"Clinical signs and death loss were recorded daily from d 0-49.On d 49, the study was terminated and all turkeys were necropcied.\",\n \"Data were analyzed as a factorial design using the GLM procedures of SAS (SAS/STAT Version 8, SAS Institute, Cary, N.C.).\",\n \"Model sum of squares consisted of challenge and water treatment.\",\n \"Least squares means are reported.\",\n \"Death loss after challenge was analyzed using survival analysis of SAS.\",\n \"Results Performance data before challenge is presented in Table 11.Since the turkeys were not challenged prior to d 35, only main effects are reported.\",\n \"Inclusion of plasma via the water increased (P<0.001) average daily gain (ADG) from d 0-7, while no further improvements were noted in gain to d 35.No differences (P>0.05) were noted in average daily feed intake (ADFI) from d 0-35.Water disappearance was increased (P<0.05) from d 0-7,0-14, and 0-21 from consumption of plasma via the water compared to the controls fed untreated water.\",\n \"Feed efficiency (G/F) was increased (P<0.05) from d 0-7,7-14, 0-14, and 0-28 from due to consumption of plasma treated water compared to untreated water.\",\n \"No differences (P>0.05) were noted in G/F and water disappearance during the remainder of the study till d 35.Performance data after challenge is presented in Table 12.No differences (P>0.05) were noted in ADG, ADFI, and water disappearance from consumption of plasma treated water compared to treated water for challenge or unchallenged groups.\",\n \"Feed efficiency was improved (P<0.05) in challenge turkeys from d 35-42 and d 35-49 due to consumption of plasma treated water compared to untreated water; while, the no differences (P>0.05) were noted in unchallenged turkeys due to consumption of plasma treated water.\",\n \"Body weight of untreated and plasma treated groups after challenge are demonstrated in FIGS.\",\n \"3A and 3B.\",\n \"Seven turkeys consuming untreated water after challenge were removed or died from the challenge as depicted in FIG.\",\n \"3A.\",\n \"One turkey consuming treated water after challenge lost weight and died due to the challenge as shown in FIG.\",\n \"3B.\",\n \"FIG.\",\n \"4 demonstrates percent remaining after challenge, while FIG.\",\n \"5 demonstrates percent remaining before challenge.\",\n \"No differences (P>0.05) in percent remaining were noted after the challenge period in unchallenged turkeys, while challenged turkeys consuming plasma treated water had increased (P<0.05) percent remaining compared to challenge turkeys consuming untreated water (FIG.\",\n \"4).\",\n \"No differences (P>0.05) were noted in percent remaining prior to challenge (d 0-35) due to consumption of treated water (FIG.\",\n \"5).\",\n \"Discussion The current study demonstrates improvement in performance of turkeys during the starting period due to consumption of plasma proteins in the water.\",\n \"Furthermore, after a respiratory challenge, consumption of plasma proteins via the water improved survival and decreased removals.\",\n \"Overall, delivery of plasma proteins via the water increases feed efficiency and percent remaining (survival) after respiratory challenge and aids in starting turkeys.\",\n \"Tables TABLE 11 Main Effect of water treatment on performance in turkeys.\",\n \"Water Plasma SEM P ADG D 0-7 14.38 16.62 0.42 0.0003 D 7-14 31.64 32.06 0.69 0.6587 D 14-21 50.01 51.18 1.3 0.5152 D 21-28 77.53 78.56 2.18 0.7372 D 28-35 98.85 101.85 3.39 0.5281 D 0-14 23.13 24.34 0.48 0.0728 D 0-21 32.09 33.29 0.7 0.2212 D 0-28 43.51 44.52 1.04 0.4854 D 0-35 54.57 55.99 1.42 0.4772 ADFI D 0-7 19.13 18.93 0.47 0.7757 D 7-14 39.32 37.62 1.18 0.3361 D 14-21 59.69 61.54 1.38 0.3736 D 21-28 99.82 97.44 2.14 0.455 D 28-35 162.65 161.66 4.77 0.8871 D 0-14 29.22 28.27 0.75 0.4002 D 0-21 39.38 39.36 0.9 0.9889 D 0-28 54.49 53.88 1.1 0.7081 D 0-35 76.12 75.44 1.78 0.7922 Water Disappearance D 0-7 68.58 79.8 3.21 0.0387 D 7-14 122.25 131.68 3.29 0.077 D 14-21 171.3 186.18 4.94 0.066 D 21-28 236.65 251.42 8.97 0.2779 D 28-35 313.1 339.22 11.59 0.1497 D 0-14 95.41 105.74 3 0.0407 D 0-21 120.71 132.56 3.26 0.0332 D 0-28 149.7 162.27 4.42 0.0791 D 0-35 182.38 197.66 5.43 0.0819 Gain/Feed D 0-7 0.74 0.88 0.03 0.0111 D 7-14 0.79 0.85 0.01 0.0019 D 14-21 0.84 0.83 0.02 0.9194 D 21-28 0.76 0.8 0.01 0.0897 D 28-35 0.6 0.63 0.02 0.2613 D 0-14 0.77 0.86 0.02 0.0032 D 0-21 0.8 0.85 0.02 0.0544 D 0-28 0.78 0.82 0.01 0.0272 D 0-35 0.71 0.74 0.01 0.0618 TABLE 12 Effect of water treatment and challenge on performance of turkeys.\",\n \"Unchallenge Challenge Water Plasma SEM P Water Plasma SEM P ADG D 35-42 117.92 114.77 5.89 0.6991 124.06 135.11 6.09 0.1913 D 42-49 123.04 124.58 5.36 0.8342 131.46 138.14 6.19 0.4177 D 35-49 120.45 119.69 5.28 0.9167 129.16 136.65 6.1 0.3574 ADFI D 35-42 194.51 181.67 7.54 0.2628 199.56 208.75 7.54 0.4134 D 42-49 242.85 225.3 14.99 0.4318 239.62 249.28 14.99 0.661 D 35-49 218.69 203.48 9.72 0.3011 219.59 229.02 9.72 0.5124 Water Disappearance D 35-42 472.24 400.46 29.62 0.1096 459.28 500.85 29.62 0.3187 D 42-49 507.57 516.09 29.22 0.8418 475.92 524.5 29.21 0.2735 D 35-49 489.91 450.74 31.48 0.3724 469.52 512.68 31.48 0.3291 Gain/Feed D 35-42 0.6 0.58 0.02 0.5063 0.54 0.65 0.02 0.0149 D 42-49 0.5 0.56 0.05 0.3527 0.48 0.54 0.05 0.3255 D 35-49 0.54 0.57 0.02 0.4125 0.51 0.59 0.02 0.0319 EXAMPLE 5 The Effects of Orally-administered Plasma on Immunological Functions The immunological response to plasma protein administration has not been studied.\",\n \"However, some of the individual components from colostrum or milk have been found to have immuno-modulatory effects.\",\n \"IgA and sIgA have anti-inflammatory functions in neonates.\",\n \"Eibl found that the oral administration of human immunoglobulin reduces circulating TNF-α production by isolated macrophages and also reduces immunoglobulin concentrations in young children affected by necrotizing enterocolitis.\",\n \"Schriffrin found that colostrum was effective in the modulation of experimental colitis.\",\n \"In an uncontrolled study, Schriffrin and his colleagues found that the dietary supplementation of a TGF-β2-rich casein fraction was useful in the modulation of inflammation in Crohn's disease in human subjects1.The mode of action has not been elucidated but TGF-β2 has been found to inhibit interferon-γ induced MHC Class II receptor expression in neonates.\",\n \"MHC class II receptor expression is also known to be upregulated in newly weaned animals.\",\n \"Other peptides found in milk, colostrum, and plasma could also have anti-inflammatory effects.\",\n \"Parenteral administration of TGF-β1 has also been shown to improve survival of mice challenged with salmonella.\",\n \"In addition, the oral administration of immunoglobulin from plasma proteins has been shown to improve weight gain and feed intake in young animals.\",\n \"TNF-α is a central cytokine in inflammatory processes and has negative effects on appetite and protein utilization1,1.And, it is well-known that the production of TNF-α is stimulated with exposure of phagocytes to endotoxin.\",\n \"Plasma proteins contain immunoglobulin, endotoxin-binding proteins, mannan-binding lectins, and TGF-β.\",\n \"The mixture of proteins, cytokines and other factors could play a role in reducing the exposure of the immune system to lumen-derived bacteria and endotoxin and therefore alter the activation of the immune system.\",\n \"The objective of this experiment was to study the immunomodulatory effects of plasma protein administration in animals beyond the postweaning period through measurement of: (a) respiratory burst in peripheral blood monocytes, (b) respiratory burst in peritoneal macrophages, (c) phagocytosis in peritoneal macrophages, and (d) TNF-α production of peritoneal macrophages in the presence and absence of lipopolysaccharide.\",\n \"2.0 Procedures for Experiment I and II 2.1 Animals Experiment I 60 Balb/c White female mice were received from Charles River Laboratories.\",\n \"Upon receipt, the animals were housed four per cage.\",\n \"At start of dosing the body weight range was 15-19 g. Three cages were assigned to a test diet, for a total of 12 animals per diet.\",\n \"The dosing had to be staggered on three successive days to accommodate the processing required at necropsy.\",\n \"So that on day 1 after arrival dosing was initiated on the animals in cage 1 from each treatment/control group, on day 2 the dosing was initiated in all the second cages, and on day 3 the third cages from all groups were dosed.\",\n \"Necropsy was similarly staggered so that the animals were dosed for a total of 7 days.\",\n \"All cages were labeled with the animal numbers and designated diet.\",\n \"The animal room was maintained between 66 and 82° F. The lighting was on a 12 hours on-12 hours off cycle.\",\n \"Experiment II Balb/c White female mice (73) were received from Charles River Laboratories, on Jun.\",\n \"18, 2001, and 72 animals were used in the study.\",\n \"These animals were born on May 7, 2001.Upon receipt, the animals were housed three per cage.\",\n \"At start of dosing the body weight range was 15-19 g. Three cages were assigned to a test diet, for a total of 9 animals per diet.\",\n \"The dosing had to be staggered on three successive days to accommodate the processing required at necropsy.\",\n \"So that on Day 1 after arrival dosing was initiated on the animals in cage 1 from each treatment/control group, on Day 2 the dosing was initiated in all the second cages, and on Day 3 the third cages from all groups were dosed.\",\n \"Necropsy was similarly staggered so that the animals were dosed for a total of 7 days.\",\n \"All mice were dosed by oral gavage with 100 ug LPS 2 days after the start of treatment with an individual diet and 5 days prior to the end of the study.\",\n \"All cages were labeled with the animal numbers and designated diet.\",\n \"The animal room was maintained between 66 and 82° F. The lighting was on a 12 hours on-12 hours off cycle.\",\n \"2.2 Peritoneal Lavage, Bleeding and Blood Sample Processing Cells were harvested from each animal by peritoneal lavage.\",\n \"After termination, the abdominal muscles were drawn away from the abdominal organs and 9 ml of sterile PBS was injected into the peritoneal cavity.\",\n \"The abdomen was massaged and 6-8 ml of lavage fluid was recovered.\",\n \"The four mice housed together were pooled to form one sample.\",\n \"The samples were kept on ice prior to processing.\",\n \"The cells were centrifuged and the pellet was re-suspended in 1 ml of Dulbeccco's Modified Eagle's Medium (DMEM) with Fetal Bovine Serum and Penicillin/Streptomycin.\",\n \"The cell numbers were determined using a Coulter Counter Z1.After collecting the lavage cells, the abdominal cavity was opened and blood was collected from the renal artery and transferred to a 3 ml vacutainer tube containing EDTA.\",\n \"Once again four mice were pooled to form one sample.\",\n \"The blood samples were diluted in PBS for a total volume of 8 ml.\",\n \"This mixture was then layered on top of 3 ml of Histopaque®-1077.The samples were centrifuged and the opaque interface containing the mononuclear cells was removed with a pasteur pipette.\",\n \"After a total of three washes in PBS the pellet was re-suspended in 0.5 ml PBS.\",\n \"The cell numbers were determined using a Coulter Counter Z1.2.3 Respiratory Burst After the cell counts were determined, both the monocyte and peritoneal samples were adjusted to a concentration of 1×106 cells per ml.\",\n \"All samples were assayed in triplicate.\",\n \"One hundred (100) ul of each cell suspension (1×105 cells/well) was added to a 96-well tissue culture plate.\",\n \"2,7-Dicholorofluorescein diacetate (Molecular Probes) was added to each well and the plate was incubated at 37° C. to allow uptake of the substrate by the cells.\",\n \"Following incubation, Phorbol Myristate Acetate (PMA) (Sigma) was added to triplicate wells of at a concentration of 10 ng/well in order to stimulate oxygen radical production.\",\n \"The plate was incubated at 37° C. After the 1-hour incubation, 200 ul of each 2,7-dicholorofluorescein standard (Polysciences) was added to the plate.\",\n \"The increase in fluorescent product was then measured using the Cytofluor 4000 (PerSeptive Biosystems) fluorescence microplate reader (Wavelengths: excitation—485, emission—530).\",\n \"The data was exported from the Cytofluor program into Excel.\",\n \"From Excel the plate layout was copied then pasted into a Softmax Pro file (molecular Devices), where the results were determined automatically by interpolation of the standard curve.\",\n \"2.4 Phagocytosis One hundred (100) ul of each cell suspension was added to five wells on a 96-well tissue culture plate, at a concentration of 1×106 cells per ml (1×105 cells/well).\",\n \"50 ul of medium (DMEM) was added to each well, making the final volume 150 ul.\",\n \"Five wells containing only DMEM were used as plate blanks.\",\n \"Each samples or blank was run in a set of five (5) replicates.\",\n \"The cells were incubated at 37° C. and then examined under a microscope.\",\n \"During the incubation period, the E. coli K-12 bioparticle suspension in HBSS (Molecular Probes) was prepared.\",\n \"The mixture was vortexed and sonicated.\",\n \"After the one-hour incubation period, the plates were centrifuged, and the supernate was aspirated by vacuum aspiration.\",\n \"100 ul of the E. coli/HBSS mixture was added to each well and incubated for two hours at 37° C. Following incubation, the E. coli bioparticles were aspirated by vacuum aspiration, and 100 ul of trypan blue/citrate-balanced salt solution (Molecular Probes) was added to each well.\",\n \"After approximately 1 minute, the trypan blue was removed by vacuum aspiration and the fluorescent product was measured using a Cytofluor 4000 fluorescence microplate reader (Wavelengths: excitation—485, emission—530).\",\n \"2.5 Cytokine Assay One hundred (100) uL of the 1×106 cells/mL cell suspension was added to 10 replicate wells of a 96-well tissue culture plate.\",\n \"Five of the wells contained LPS (1 ug per well), the other five wells did not have any LPS.\",\n \"The plate was incubated at 37° C. for 24 hours.\",\n \"Upon incubation, supernatants from replicate wells were pooled and stored at −20° C. until assay.\",\n \"Production of TNF- and IL-10 in the supernatant was evaluated using mouse ELISA kits from R&D Systems.\",\n \"3.0 MATERIAL The materials were as follows: Diet A—Control (Skim milk) Diet B—Porcine serum (PP) Diet C—Bovine plasma protein (BP) Diet D—Nalco-treated plasma light phase (BL) Diet E—Nalco-treated plasma heavy phase (BH) The dietary treatments for Experiment II were as follows: 1.Control (Skim milk) 2.Ig concentrate, 2.5% 3.Ig concentrate, 0.5% 4.Bovine serum, 5% 5.Bovine serum, 1% 6.Heavy phase, 0.5% 7.Activated HP, 0.5% 8.Activated, de-ashed HP, 0.1% 3.1 Storage, and Handling of Study Material The test diets were stored at 4° C. in their original ziploc bags.\",\n \"Safety glasses, gloves, and a lab coat were worn while handling.\",\n \"3.2 Application of Study Material Feeding dishes were filled twice a day and animals were allowed to feed ad lib for seven days.\",\n \"Results and Discussion According to the invention we found that plasma of either bovine or porcine species origin resulted in less TNF-α production by both stimulated and unstimulated peritoneal macrophages.\",\n \"In addition, the administration of both the heavy and the light phase of plasma treated with 5% silicon dioxide resulted in reduced TNF-α production albeit at different concentrations.\",\n \"The fractions were not evaluated at equal concentrations, however.\",\n \"The change in TNF-α that accompanied macrophage stimulation was greater when animals were fed a plasma fraction, irrespective of source or concentration.\",\n \"This observation indicates that the immunological responsiveness of the macrophage is enhanced with the addition of plasma and/or it's components to the diet of young mice.\",\n \"In the second experiment, we confirmed the suppressive effect of plasma fractions on TNF-α production by unstimulated peritoneal macrophages.\",\n \"The level of supplementation and the fraction did alter the effect however.\",\n \"The Nalco precipitate reduced TNF-α production in unstimulated cells at both 0.5 and 0.1%.\",\n \"The immunoglobulin rich fraction suppressed TNF-α production at 0.5% but not at 2.5%.\",\n \"The addition of serum suppressed TNF-α production at 5% but not at 1.0%.\",\n \"The experimental conditions in Exp.\",\n \"II differed from the previous experiment.\",\n \"The mice in this study were all challenged with endotoxin on d 1 in an attempt to prime the immune system in all animals.\",\n \"Previous reports have found that priming macrophages will reduce immunological responsiveness upon subsequent challenge.\",\n \"The results of the first experiment would seem to confirm this observation.\",\n \"Isolated macrophages from animals fed the control diet produced higher levels of TNF-α in the unstimulated state and therefore produced less TNF-α when stimulated with LPS than animals fed diets supplemented with plasma and/or fractions.\",\n \"The levels of TNF-α were markedly different in the control animals from the two experiments.\",\n \"TNF-α production was 15 fold higher in the first experiment than in the second experiment.\",\n \"Nonetheless, while immune system activation was lower in both experiments, immunological responsiveness was greater in mice fed a diet supplemented with a plasma fraction.\",\n \"Both TNF-α and IL-10 concentrations increased markedly with exposure of macrophages to LPS.\",\n \"Plasma is rich in biologically active proteins, peptides, cytokines, and other immunomodulatory substances.\",\n \"The fractions of plasma administered in these experiments differed in composition and dietary inclusion rate.\",\n \"The effect of these fractions on TNF-α production was consistent in the two experiments.\",\n \"Animals fed plasma and/or fractions thereof produced less TNF-α in an unstimulated state and therefore responded with increased TNF-α production upon stimulation with endotoxin.\",\n \"The results of these two experiments are consistent with the concept that both the immunoglobulin-rich fractions and the silicon dioxide fractions reduce the stimulation of the immune system.\",\n \"The oral administration of plasma proteins or its fractions is a novel means of reducing TNF-α production and levels.\",\n \"TABLE 13 The effects of bovine and porcine plasma protein administration on immune response measures in mice.\",\n \"TNF-α, pg/ml Respiratory Burst Treatment −LPS +LPS TNF-α change −LPS +LPS Control 1540a 1867a 322a 17.4a 23.9a Porcine plasma 70b 1156b 1085b 12.2b 13.6b Bovine plasma 28b 1135b 1107b 10.1b 11.1b Bovine plasma 136b 1260b 1101b 10.6b 13.7b (Heavy phase) Bovine plasma 34b 1135b 1124b 9.3b 11.2b (Light phase) TABLE 14 Mean Phagocytosis Results for Peritoneal Macrophages (FIG.\",\n \"5) Animal Mean Diet No.\",\n \"Result Se Control 1-12 298 47.6 PP 13-24 264 46.2 BP 25-36 311 52.1 BL 37-48 360 66.5 BH 49-60 375 63.9 TABLE 15 TNF-α production in cultured peritoneal macrophages from mice fed plasma protein components TNF-α production, pg/ml Treatment −LPS +LPS Change Control 128a 296a 169a Ig concentrate, 2.5% 107ab 308a 201ab Ig concentrate, .5% 20b 325a 306b Bovine serum, 5% 5b 371a 366b Bovine serum, 1% 130a 306a 176a Heavy phase, .5% 48ab 271a 223ab Activated HP, .5% 30ab 303a 272ab Activated, de-ashed 11b 352a 341b HP, .1% TABLE 16 IL-10 production in cultured peritoneal macrophages from mice fed plasma protein components IL-10 production, pg/ml Treatment −LPS +LPS Change Control 80a 237a 156a Ig concentrate, 2.5% 92a 366a 274a Ig concentrate, .5% 45a 374a 329ab Bovine serum, 5% 22a 369a 347b Bovine serum, 1% 116a 354a 238ab Heavy phase, .5% 64a 348a 284ab Activated HP, .5% 54ab 394a 339b Activated, de-ashed 32b 412a 381b HP, .1% Reference List Wolf H M, Eibl M M. The anti-inflammatory effect of an oral immunoglobulin (IgA-IgG) preparation and its possible relevance for the prevention of necrotizing enterocolitis.\",\n \"Acta Paediatr Suppl 1994; 396:37-40.Wolf H M, Hauber I, Gulle H, Samstag A, Fischer M B, Ahmad R U, Eibl M M. Anti-inflammatory properties of human serum IgA: induction of IL-1 receptor antagonist and Fc aR (CD89)-mediated down-regulation of tumour necrosis factor-alpha (TNF-a) and IL-6 in human monocytes.\",\n \"Clin.\",\n \"Exp.\",\n \"Immunol.\",\n \"1996; 105:537-43.Eibl M M, Wolf H M, Furnkranz H, Rosenkranz A.\",\n \"Prevention of Necrotizing Enterocolotis in low-birth-weight infants by IgA-IgG feeding.\",\n \"The New England Journal of Medicine 1988; 319 (1):1-7.Caldarini dB M, Schiffrin E J, Ogawa dF, Caccamo D V, Ledesma dP M, Celener D, Bustos-Fernandez L. Prevention of carrageenan-induced ulcerative colitis in the guinea pig by serum of bovine colostrum.\",\n \"Medicina.\",\n \"(B.\",\n \"Aires.)\",\n \"1987; 47 (3):273-7.Beattie R M, Schiffrin E J, Donnet-Hughes A, Huggett A C, Domizio P, MacDonald T T, Walker-Smith J A. Polymeric nutrition as the primary therapy in children with small bowel Crohn's disease.\",\n \"Aliment.\",\n \"Pharmacol.\",\n \"Ther.\",\n \"1994 December; 8 (6):609-15.Donnet-Hughes A, Schiffrin E J, Huggett A C. Expression of MHC antigens by intestinal epithelial cells.\",\n \"Effect of transforming growth factor-beta 2 (TGF-beta 2).\",\n \"Clin Exp.\",\n \"Immunol.\",\n \"1995 February; 99 (2):240-4.Zijlstra R T, McCracken BA, Odle J, Donovan S M, Gelberg H B, Petschow B W, Zuckermann F A, Gaskins H R. Malnutrition modifies pig small intestinal inflammatory responses to rotavirus.\",\n \"J Nutr 1999 April; 129 (4):838-43.Donnet-Hughes A, Duc N, Serrant P, Vidal K, Schiffrin E J. Bioactive molecules in milk and their role in health and disease: the role of transforming growth factor-beta.\",\n \"Immunol.\",\n \"Cell Biol.\",\n \"2000.Feb.\",\n \"; 78.(1.\",\n \"):74.-9.78 (1):74-9.Galdiero M, Marcatili A, Cipollaro dl, Nuzzo I, Bentivoglio C, Romano C C. Effect of transforming growth factor beta on experimental Salmonella typhimurium infection in mice.\",\n \"Infect.\",\n \"Immun.\",\n \"1999 March; 67 (3):1432-8.Owen K Q, Nelssen J L, Goodband R D, Tokach M D, Friesen K G, Richert B T, Smith J W, Russell L E. Effect of various fractions of spray-dried porcine plasma on performance of early weaned pigs [Abstract].\",\n \"In: J. Anim.\",\n \"Sci.\",\n \"(Suppl.)\",\n \"2000.Pierce J L, Cromwell G L, Lindemann M D, Monegue H J, Weaver E M, Russell L E. Spray-dried bovine globulin for early weaned pigs [Abstract].\",\n \"In: J. Anim.\",\n \"Sci.\",\n \"(Suppl.)\",\n \"1996.Yeh S S, Schuster M W. Geriatric cachexia: the role of cytokines.\",\n \"Am.\",\n \"J Clin Nutr 1999 August; 70 (2):183-97.Rozenfeld R A, Huang W, Hsuch W. Effects of antibiotics and germ-free environment on endotoxin (LPS)-induced injury and on intestinal group II phospholipase A2 (PLA2-II) activity [Abstract].\",\n \"In: FASEB Journal 1999; 643.5\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10470982"}}},{"rowIdx":566,"cells":{"text":{"kind":"list like","value":[["Delivery of biologically active agents","A method of delivering a biologically active agent to the cervix, the method comprising using a needleless injector.","Typically, the biologically active agent is a cervical ripening agent.","The needleless injector may be a powder injector or a liquid injector."],["1.A method of delivering a biologically active agent to the cervix, the method comprising using a needleless injector.","2.A method according to claim 1 wherein the needleless injector is a liquid injector.","3.A method according to claim 1 wherein the needless injector is a powder injector.","4.A method according to claim 1 wherein the biologically active agent is a cervical ripening agent.","5.A method according to claim 4 wherein the cervical ripening agent is any one or more of a prostaglandin, MCP-1 and IL-8.6.A method according to claim 1 wherein the biologically active agent is a vasodilator or precursor thereof, a chemokine or a cytokine which stimulates monocyte or granulocyte entry into cervical tissue.","7.A method of ripening the female cervix, the method comprising administering a cervical ripening agent to the cervix using a needleless injector.","8.A system for delivering a biologically active agent to the cervix comprising an agent which is biologically active on the cervix and a needleless injector.","9.A system according to claim 8 wherein the biologically active agent is a cervical ripening agent.","10.A system according to claim 8 wherein the needleless injector is a liquid injector.","11.A system according to claim 8 wherein the needleless injector is a powder injector.","12.A needleless injector loaded for injection with an agent which is biologically active on the cervix.","13.A needleless injector according to claim 12 wherein the biologically active agent is a cervical ripening agent.","14.A needleless injector according to claim 12 which is a liquid injector.","15.A needleless injector according to claim 12 which is a powder injector.","16.A vial for insertion into, and containing an agent for delivery by, a needleless injector wherein the agent is an agent which is biologically active on the cervix.","17.A vial according to claim 16 wherein the agent is a cervical ripening agent.","18.A method of preparing a needleless injector for use in delivering a biologically active agent to the cervix, the method comprising loading the injector with the biologically active agent.","19.A method according to claim 18 wherein the agent is loaded in a vial disposed for insertion into the needleless injector.","20.A pharmaceutical formulation comprising an agent for delivery to the cervix and a carrier suitable for use in a needleless injector.","21.A pharmaceutical formulation according to claim 20 wherein the formulation contains particles of a density between about 0.1 and about 25 g/cm3 and of a size between 0.1 and 250 μm which particles comprise the said agent.","22.A pharmaceutical formulation according to claim 20 comprising an agent which permeabilises a mucosal surface, such as dimethylsulphoxide.","23.Use of a cervical ripening agent in the manufacture of a medicament for treating a female in need of a cervical ripening agent wherein the cervical ripening agent is for delivery using a needleless injector.","24-26.","(canceled)."],["The present invention relates to the delivery of biologically active agents to the female cervix and, in particular, it relates to the delivery of cervical ripening agents.","The cervix acts as a barrier to the ingress of infection as well as a rigid barrier at the neck of the womb.","This organ has relatively little muscle with the majority of the strength of the organ derived from collagen.","In a normal, easy delivery, the cervix has to soften and stretch (eface) to allow the baby's head to pass through, if this can be achieved with little uterine contractility then subsequent contractions can deliver the baby with minimal trauma for mother and child.","Biologically active agents such as prostaglandins and chemokines are delivered to the cervix or vaginal fomix for various purposes.","In particular, agents are delivered to aid cervical ripening for a number of reasons including (a) the induction of labour, (b) to soften an unfavourable cervix during labour, (c) to assist medical termination of pregnancy, (d) to assist uterine surgery and (e) to accelerate normal parition and to reduce the accompanying risks and discomforts.","U.S. Pat.","No.","5,624,670 to Kelly et al describes the use of interleukin-8 (IL-8) for inducing cervical ripening, and U.S. Pat.","No.","5,908,829 to Kelly describes the use of monocyte chemotactic peptide-1 (MCP-1) for inducing ripening of the cervix.","Vasodilators or their precursors (such as the nitric oxide donor nitroglycerine) are also delivered to the cervix.","At present, biological agents are delivered to the cervix or vaginal fornix using gels, creams, pessaries.","A relatively large amount of drug is currently needed since diffusion through the epithelial layers of the cervix is relatively slow.","The gel can also get displaced.","The most common current method of cervical ripening is to use prostaglandin E2.This is used as a vaginal gel or tablet or as a gel placed in the cervix.","Typically, gel is placed in the vaginal fornix.","The prostaglandin has to diffuse through the epithelium of the cervix and act on the blood vessels to increase permeability.","In addition prostaglandin may act on fibroblasts to release interleukin-8 (IL-8) within the cervix.","The combination of IL-8 and PGE is very powerful in attracting neutrophils (Colditz (1990) Immunol.","Cell Biol.","68, 397-403) and neutrophils are a source of collagenase.","There are several disadvantages to the current approaches.","Firstly, in order that sufficient amounts of the biologically active agent are delivered to the site of action, relatively high concentrations of the agent need to be included in the formulation administered.","Secondly, prostaglandin gels used to ripen the cervix by administration to the vaginal fornix can stimulate the myometrium to contract excessively and, in extreme circumstances, an uterine rupture can occur (Maymon et al (1991) Am.","J. Obstet, Gynecol.","165, 368-370).","Thirdly, with prostaglandin gel the rate of diffusion into the cervix is limited and variable.","Fourthly, for certain of the biologically active agents delivered to the cervix or vaginal fornix, diffusion is relatively slow due to the increased molecular size and hydrophilic nature of the compound.","This is particularly true for peptide agents.","During its ripening, the cervix is thought to soften from the internal os.","That is likely because of the possible fetal contribution to the timing of birth.","Thus, the initial softening might involve agents (such as PGE2 and or chemokines) diffusing outward from the fetal membranes (amnion and chorion) to the point of contact with the cervix, the internal os.","Attempts to deliver chemokines has identified a problem with the diffusion of the active agent, particularly when that agent is a peptide and accordingly may not be lipophilic.","In addition, we believe that delivery to the cervix of any ripening agent should be diffuse rather than punctate and thus conventional injection using a needle and syringe is not ideal.","There remains the need to improve on delivery methods to the cervix and, as described in more detail below, the inventors believe that alternatives lo which use needleless injectors may lead to benefits over the existing methods.","Needleless injectors are well known in the art and have been used to deliver agents principally to the skin of individuals, for example in vaccinations.","As far as the inventors are aware, despite needleless injectors having been available for very many years, no-one has suggested that they would be useful in delivering agents to the cervix, and no-one has realised the potential benefits in terms of the possibility of using lower concentrations of biologically-active agents, particularly cervical-ripening agents such as prostaglandins, and benefits in terms of potentially simplifying the formulation of the active agents for delivery.","In particular, compared to other delivery methods, no gel matrix (such as a methyl cellulose or modified starch gel) is required.","Commercially available needleless injectors fall into two main categories: those which use powder formulations of the biologically active agent, and those that use the agent in a solution formulation.","These will be referred to as “powder” injectors and “liquid” injectors.","Generally, both types of injector use compressed air, or gas, or some type of mechanical configuration to “inject” the active agent into the patient.","Powderject (Oxford, UK) manufactures commercially powder injectors.","Typically, powder injectors make use of dry powder formulations and fine particles of the drug to be delivered are projected at high velocity within a brief burst of, typically, helium gas.","Transdermal application has been described and, typically, the dose is delivered over a skin area of up to 2 cm2.WO 94/24263 (Oxford Biosciences Limited) describes a needleless syringe which uses supersonic gas flow for particle delivery.","The needleless syringe has a membrane which is ruptured by gas pressure to generate a supersonic gas flow in which particles containing a therapeutic agent are injected.","WO 96/12513 (Oxford Biosciences Limited) describes a similar system except that a soft-walled capsule, such as one with a gelatine wall, is used to hold the therapeutic agent in the syringe.","WO 96/20022 (Oxford Biosciences Limited) again describes a similar device except that it has a diaphragm which snaps from inverted to everted positions to catapult particles outwardly.","WO 96/25190 (Oxford Biosciences Limited) describes a needleless powder injector with a bend.","WO 97/34652 (Powderject Research Limited) relates to a shroud which surrounds the nozzle of the syringe and reduces the noise.","WO 98/13470 (Powderject Vaccines, Inc) relates to a rotational flow element which modifies gas flow through the device.","WO 99/01168 and WO 99/01169 (Powderject Research Limited) relate to a prefilled capsule for use with a powder injector and a modified gas release mechanism, respectively.","WO 00/54827 (Powderject Research Limited) relates to a diaphragm for use with a needleless injector wherein the diaphragm provides for enhanced particle retention.","WO 00/62846 (Powderject Research Limited) relates to a needleless powder injector device that does not require particles to be between burstable membranes.","Certain other patent applications relate to the manufacture of particle compositions (WO 97/48485 and WO 00/53160; Powderject Research Limited); and cartridges for use in a needleless powder injector (WO 98/21364; Powderject Vaccines, Inc and U.S. Pat.","No.","5,780,100 to McCabe and Heinzen).","Weston Medical (Peterborough, UK) manufactures commercially liquid injectors.","WO 93/03799 (Weston) and WO 95/03844 (Weston Medical) describe a liquid injector system which is used to deliver vaccines by penetration of the epidermis for intradermal delivery.","WO 96/28202 (Weston Medical) and WO 97/37705 (Weston Medical) describe a spring-powered dispensing device which can be in the form of a needleless injector.","EP 0 520 571 A (Glaxo) describes an atomising nozzle for use with a liquid injector.","WO 96/15821 (Weston Medical) describes a glass container for use in needleless injectors, and WO 97/22375 (Weston Medical) and WO 97/36785 (Weston Medical) describe a needleless injector drug capsule and filling method.","WO 96/19252 (Weston Medical) describes a filling device for a needleless injector.","WO 00/10630 (Weston Medical) describes an actuator for a gas powered needleless injector.","WO 00/15281 (Weston Medical) describes a needleless injector cartridge.","A first aspect of the invention provides a method of delivering a biologically active agent to the cervix, the method comprising using a needleless injector.","The delivery may be directly to the cervix or may be via the vaginal fornix, which is a fold in the vagina where the cervix is located.","Thus, the end of the needleless injector may occupy the vaginal fornix and delivery may be at or through the vaginal fornix into the cervix.","Typically, injections will aim at the cervix but may be aimed from different angles.","Preferably, the site of injection is “off-centre” into the cervical lumen or into any part of the externally presenting area of the cervix.","The needleless injector may be any suitable man-made needleless injector and, conveniently, may be a liquid injector or a powder injector as described above.","Thus, suitable needleless liquid injectors include those manufactured by Weston Medical Limited, Peterborough, UK and those described in WO 93/03779, WO 95/03844, WO 96/28202, WO 97/37705 and WO 00/10630, all of which are incorporated herein by reference.","Suitable needleless powder injectors include those manufactured by Powderject Research Limited, Oxford, UK and those described in WO 94/24263, WO 96/12513, WO 96/20022, WO 96/25190, WO 97/34652, WO 98/13470, WO 99/01168, WO 99/01169, WO 00/54827 and WO 00/62846, all of which are incorporated herein by reference.","The biologically active agent may be loaded into the injector by means known in the art.","It may be useful if the needleless injector has a “bent” configuration such that delivery of the biologically active agent is to the cervix rather than to the walls of the vagina or to the entrance of the cervix.","Thus, the needleless injector may be adapted for this purpose, and the geometry of the device arranged such that there is easy passage through the vagina but that the exit from the injector (ie outlet orifice) is angled such that the biologically active agent is delivered efficiently to the desired site on the cervix.","The type of injectors described in WO 96/25190 may be particularly suitable; however, rather than the internal angle of approximately 90° shown in FIGS.","1 to 4 of WO 96/25190, it may be more appropriate if the internal angle of the head to the shaft is between 20° to 90°; preferably between 35° and 85°, for example 65°.","The arrangement of the head containing the outlet orifice shown in FIG.","16B of WO 00/54827 may also be particularly suited for use in the present invention.","Similarly, the arrangement shown in FIG.","5 of WO 00/62846, where the exit plane is not perpendicular to the longitudinal axis of the nozzle, but angled, may be particularly suited for use with the invention.","The biologically active agent may be any agent for delivery to the cervix which has a useful effect.","For example, the agent may be any agent which has a therapeutic or prophylactic effect, or which is useful in any medical procedure.","Typically, the agent is one which is useful in gynaecology or obstetrics.","Suitable agents include prostaglandins (both natural and synthetic), vasodilators or their precursors (such as the nitric oxide (NO) donor, nitroglycerine), and chemokines or any cytokine that could stimulate monocyte or granulocyte entry into the cervical tissue such as GM-CSF.","It is particularly preferred if the agent is a cervical ripening agent, such as a prostaglandin (in particular, prostaglandin E2) or interleukin-8 (IL-8) or monocyte chemotactic pepfide-1 (MCP-1) or combinations thereof.","MCP-1, IL-8 and variants thereof may be obtained by chemical synthetic methods or by recombinant DNA means as is well known in the art.","Prostaglandins, such as PGE2, are commercially available, for example from Pharmacia and Upjohn.","The use of prostaglandins in cervical ripening is well known in the art.","The use of IL-8 and MCP-1 in cervical ripening is described in U.S. Pat.","Nos.","5,624,670 and 5,908,829, respectively, incorporated herein by reference.","Variants of MCP-1 or IL-8 which are able to induce cervical ripening may also be used.","A “variant” refers to a protein wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in a protein whose basic properties, for example cervical ripening-inducing activity, thermostability, activity in a certain pH-range (pH-stability) have not significantly been changed.","“Significantly” in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein.","By “conservative substitutions” is intended combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.","Such variants may be made using the methods of protein engineering and site-directed mutagenesis as described below.","The dose of the agent administered is any suitable dose of the agent which provides the desired effect of the agent.","Typically, because of the precision with which the agent can be administered with the needleless injector, the dose may be significantly reduced compared to the dose administered by other, conventional means.","The amount may be reduced to 50% or 20% or 10% or 5% or 2% of the amount normally used, depending on the agent administered.","For example, 2 to 100 μg of IL-8 or MCP-1 dissolved in 200 μl of sterile saline may be used in a liquid injector.","Diffused delivery is achieved using needleless injectors.","The agent, once injected, distributes radially and along the length of the injection path.","Such a method of delivery is not obtainable by any other method of injection, and, although not being bound by any theory, is believed to be particularly beneficial in the context of the present invention.","Administration of lower amounts of the agent may reduce the chance of side effects.","For example, side effects of NO donors are headaches and excessive bleeding.","Injection of a low amount directly into the cervix would both accelerate the effect and reduce the side effects.","Typically, the needleless injector is loaded with the biologically active agent which is to be delivered to the cervix, the injector is inserted into the vagina such that the exit from the injector (ie outlet orifice) is in proximity to the cervix, and the injector is actuated so as to deliver the agent to the cervix.","Preferably, the exit from the injector (ie outlet orifice) is not at the rim of the cervix but is at the side of the cervix.","It will be appreciated that position or type of “head” of the needleless injector (ie that contains the outlet orifice) may be adjusted or adapted depending on the particular use.","Thus, particular positions or designs of head may be more suited to delivery when the cervix is partially dilated (eg during parturition) or may be more suited to delivery when the cervix is closed or substantially closed.","A description of the anatomy of the vagina, cervix and uterus is given in FIG.","1 and positions for possible delivery to the cervix are indicated.","The method may be used to deliver biologically active agents to any female mammal, but it is particularly preferred if the method is used to deliver agents to a human female.","However, the method may be especially useful for sheep where in vitro fertilization and embryo transfer procedures currently involve laparoscopy which brings up ethical and animal husbandry considerations, the problem being that the cervix is impenetrable.","It is believed that a diffuse injection of cervical ripening agent would give a very rapid response, allowing a wider use of in vitro fertilization.","It will be appreciated that for certain agents, in particular peptide and polypeptide agents, it may be appropriate to select the peptide or polypeptide of the mammal, or a closely related molecule for administration.","Thus, for example, it is preferred to administer human peptides or polypeptides to a human female.","Of course, the peptide or polypeptide may be produced synthetically or by recombinant DNA means but would still be considered “mammalian” or, as the case may be “human”.","Suitable IL-8 molecules and derivatives thereof are described in U.S. Pat.","No.","5,624,670, incorporated herein by reference.","Suitable MCP-1 molecules and derivatives thereof are described in U.S. Pat.","No.","5,908,829, incorporated herein by reference.","It will be appreciated that the invention includes a method of ripening the female cervix, the method comprising administering a cervical ripening agent to the cervix using a needleless injector.","The method can be used for the induction of labour at term (ie time of ordinary birth), but it may also be used for the induction of pre-term labour, and induction of labour in connection with a pathological pregnancy, or in connection with intrauterine fetal death.","It may be used to ripen dysfunctional cervices, ie when dilation stops before completion.","The method may also be used for preliminary cervical ripening prior to induction of abortion (eg in the first or second trimester abortion), and for induction of cervical ripening of a non-pregnant or pregnant female to assist surgical or diagnostic procedure such as D&C.","Cervical ripening may also be induced in the female for the purposes of treatment by in vitro fertilisation.","It is particularly preferred if the cervical ripening agent is any one or more of IL-8, MCP-1 or a prostaglandin.","The prostaglandin typically is PGE2 which is currently used in cervical ripening.","It is believed that by using the methods of the invention, which may be used to more accurately deliver the agent to a particular site on the cervix, a smaller amount of agent may be used compared to the use of gels, creams, pessaries and the like.","At least in the case of PGE2 this has the potential benefit of reducing the risk of undesirable contraction of the myometrium.","The amount of PGE2 may be reduced to 10% or 5% or 2% of the amount normally used when in a gel.","For example, 2 to 100 μg of PGE2 dissolved in 200 μl of sterile saline may be used.","The cervical ripening agent may be a combination of agents.","It is particularly preferred if the cervical ripening agent is a complex comprising a proteinaceous agent which is chemotactic for monocytes and/or neutrophils and a prostaglandin.","By “proteinaceous agent which is chemotactic for monocytes and/or neutrophils” we include any protein or polypeptide or oligopeptide or peptide which is able to encourage monocytes and/or neutrophils into a tissue where the agent is present.","Typically, the agent is a proteinaceous cervical ripening agent.","By “proteinaceous cervical ripening agent” we include any protein or polypeptide or oligopeptide or peptide which has the ability to induce ripening of the cervix when suitably administered to the female.","Thus, the proteinaceous cervical ripening agent is one which contains peptide bonds and has amino acid side chain residues.","Preferably, the proteinaceous agent which is chemotactic for monocytes and/or neutrophils is one with a pI value of 7.5 or above, preferably 8 or above and more preferably 8.5 or above.","Typically, >5% of the amino acid residues are lysines.","The prostaglandin may be any suitable prostaglandin.","It is preferred that the prostaglandin is one which is able to induce cervical ripening when suitably administered to the female.","It is preferred that the prostaglandin is PGE2 or a synthetic analogue thereof with a free acid.","Synthetic analogues include these modified at position 15 by the addition of a methyl group.","It is particularly preferred if the prostaglandin is one which has an acidic group such as a carboxylate group.","Preferably, the acidic group is one which, under low pH conditions, gives a free acid.","It is preferred that the prostaglandin is prostaglandin E2 (PGE2).","Particularly preferred proteinaceous cervical ripening agents include monocyte chemotactic peptide-1 (MCP-1) or interleukin-8 (IL-8) or variants thereof which retain the ability to induce cervical ripening.","These complexes are described in our co-pending PCT Patent Application which claims priority from UK Patent Application No 0103354.7.A second aspect of the invention provides a system for delivering a biologically active agent to the cervix comprising an agent which is biologically active on the cervix and a needleless injector.","The needleless injector may be any needleless injector suitable for delivering the agent to the cervix.","Such needleless injectors are described above and preferred injectors in the above mentioned methods of the invention are also preferred in this aspect of the invention.","The biologically active agent is an agent which is active on the cervix.","Suitably, the agents are ones which are delivered to the cervix for therapeutic or prophylactic or other medical use.","Typically, they are agents which are not ones which are administered to the skin.","Suitable such agents include PGEs, IL-8 and MCP-1 as described above.","The agents preferably include cervical ripening agents as described above.","The system may be considered to be a kit of parts comprising various components.","Thus, the kit of parts may comprise a needleless injector and, separately, the biologically active agent.","In that case, the agent may be loaded into a suitable reservoir in the needleless injector.","Alternatively, the kit of parts may comprise a needleless injector and a vial (by which we include capsule or cartridge or the like) which contains the agent wherein the vial is one which can be loaded into the injector.","Additionally, in the case of powder injectors, the kit may contain the therapeutic agent in a suitable, powdered form.","Similarly, in the case of liquid injectors, the kit may contain the therapeutic agent in a suitable solution.","It will be appreciated also that the system of this aspect of the invention may also comprise a needleless injector which is pre-loaded with the biologically active agent.","Vials (or capsules or cartridges) for use with a needleless injector are described in more detail below.","A third aspect of the invention provides a needleless injector loaded for injection with an agent which is biologically active on the cervix.","The needleless injector may be any suitable needleless injector as described above, and the agent which is biologically active on the cervix may be any agent as described above.","The needleless injector loaded with the agent according to this aspect of the invention may be formed by introducing a vial which contains the agent into the injector.","Alternatively, the agent may be introduced into an existing reservoir within the needleless injector.","A fourth aspect of the invention provides a vial for insertion into, and containing an agent for delivery by, a needleless injector wherein the agent is an agent which is biologically active on the cervix.","The term “vial” may be interchanged with “cartridge”, “capsule” and the like, unless the context indicates otherwise.","In particular, it includes the burstable membranes described in WO 94/24263 and the soft-walled capsules described in WO 96/12513.Certain needleless injectors, for example those described in WO 93/03779, WO 95/03844, WO 00/10630, WO 94/24263, WO 96/20022 and WO 96/25190 are designed to receive pre-charged vials containing the agent to be “injected”.","Thus, the vials of this aspect of the invention include such vials pre-charged with an agent which is biologically active on the cervix, such as those described above and, in particular, cervical ripening agents.","Accordingly, the vials are of the correct size and shape to fit into the appropriate needleless injector.","Also, the vials are made of appropriate material for their purpose.","Suitably, the vial contains a unit dosage of the biologically active agent.","Suitably, when used in a liquid needleless injector, the vial contains around 200 μl (for example, between 50 μl and 500 μl).","Typical unit dosages are 2 to 100 μg of the agent, such as PGE2 or MCP-1 or IL-8.Suitable vials for use with certain needleless liquid injectors, and methods of manufacture and filling the same, are described in WO 00/15281, WO 97/36785, WO 97/22375, WO 96/19252, WO 96/15821, WO 98/12121 and WO 98/13086, all of which are incorporated herein by reference.","Suitable vials for use with certain needleless powder injectors, and methods of manufacturing and filling the same, are described in WO 00/54827, WO 00/62846, U.S. Pat.","No.","5,780,100, WO 98/21364, WO 99/01169, WO 96/20022, WO 96/12513 and WO 94/24263.A fifth aspect of the invention provides a method of preparing a needleless injector for use in delivering a biologically active agent to the cervix, the method comprising loading the injector with the biologically active agent.","The biologically active agent may be loaded directly into a pre-existing eservoir within the needleless injector or it may be loaded into the injector in a pre-charged vial.","A sixth aspect of the invention provides a pharmaceutical formulation comprising an agent for delivery to the cervix and a carrier suitable for use in a needleless injector.","The pharmaceutical formulation is typically sterile and pyrogen free.","For liquid injectors the biologically active agent is typically dissolved in sterile water or sterile saline.","However, it may be advantageous to include a dispersion agent or an agent which allows for or increases transdermal or transmucosal transfer or penetration such as dimethyl sulphoxide (DMSO) and the like.","For powder injectors the powder to be delivered needs to be of a suitable quality for delivery.","Suitable powder (ie particle compositions) may be made according to the methods described in WO 97/48485 and WO 00/53160, incorporated herein by reference.","In particular, the particles (powder) for use in the present invention have an optimum particle density ranging from about 0.1 to about 25 g/cm3, preferably ranging from about 0.5 to about 3.0 g/cm3, and most preferably ranging from about 0.8 to 1.5 g/cm3.The densified particles are suitably processed to obtain particle sizes ranging from about 0.1 to 250 μm, preferably ranging from about 0.1 to 150 μm, and most preferably ranging from about 20 to about 60 μm.","Thus, the invention includes particles comprising a cervical ripening agent such as interleukin-8 or MCP-1 or PGE2 or combinations thereof with these physical parameters.","These particles may be made using the methods described in WO 97/48485, incorporated herein by reference.","The particles may be biologically inert particles onto which has been coated the biologically active agent.","Suitably, the biologically active agent may be deposited onto the particle by dissolving it in a suitable solvent and then evaporating the solvent.","A further aspect of the invention provides use of a cervical ripening agent in the manufacture of a medicament for treating a female in need of a cervical ripening agent wherein the cervical ripening agent is for delivery using a needleless injector.","Preferably, the cervical ripening agent is any one or more of IL-8, MCP-1 or prostaglandin E2.The invention will now be described in more detail by reference to the following Examples and Figures.","FIG.","1: Anatomy of the human vagina, cervix and uterus.","FIG.","2: View of dye track for three separate cervices injected with the Weston needleless injector.","Note the small ruler divisions are in mm.","All injections were performed from the face of the cervix that would normally be in the vagina.","EXAMPLE 1 Administration of MCP-1 to a Human Female Using a Needleless Syringe The patient is a human female in need of cervical ripening.","200 μl of solution of monocyte chemotactic peptide-1 (MCP-1) of 100 μg/ml in sterile saline is loaded into a helium-loaded injection device and the end of the device is placed either in the vaginal fornix against the cervical mass or against the exposed cervical lumen.","The injector is fired at one or two locations.","EXAMPLE 2 Preparation of IL-8 as a Powder for Injection Using a Needleless Injector Lyophilized recombinant human interleukin-8 powder is obtained and reprocessed using the method described in WO 97/48485.Particularly, approximately 30 mg of IL-8 was compacted under pressure using a Carver Laboratory Pellet Press (Model 3620, available from Carver, Inc., Wabash, Ind.).","The pressure of compaction was 15,000 lbs/in2, which was applied for approximately 45 seconds.","A pellet was obtained which was ground using mortar and pestle until visually broken up.","The resulting reduced pellet was then sieved using a 53 μm sieve (Endecott, London).","Particles having a size greater 53 μm were selected and appropriate dosages thereof were measured into drug cassettes for delivery from a needleless syringe.","EXAMPLE 3 Use of the Weston Needleless Injector to Evaluate Penetration of the Cervix A Weston needleless injector was loaded with 0.2 ml of dye (1 mg/ml of methylene blue {Sigma} in phosphate buffered saline).","Whole human cervix was obtained at hysterectomy from non-pregnant women of reproductive age.","The cervix was tested within 30 minutes of removal from the patient.","The needleless injector device was held against the ectocervix region.","Increased pressure on the needleless injector activated the device.","Two injections were performed for each specimen.","The cervix was dissected along the track of the injection (FIG.","2) and the length of the dye track was measured.","In four experiments the length of the track was a mean of 6.1 mm (standard error of the mean=1.9 mm).","The mean lateral spread (any signs of the blue dye) was 8.8 mm.","Liquid needleless injectors (eg Weston) penetrate skin but the liquid stream rebounds when it hits muscle.","The cervix has relatively little smooth muscle (but gets its strength from collagen) and therefore the extent to which the liquid stream would penetrate was unknown until these experiments.","As far as we can see there is relatively little tissue damage at the site of injection.","The lateral spread of the injected dye is very difficult to see in the black/white photographs but can be seen in the colour ones from which the black and white photographs are derived.","The use of the needleless injector in testing the penetration and spread of a marker dye shows that the distribution of cervical ripening product would be ideal for a rapid ripening of the cervix.","The relatively wide spread of the injected material suggests that the ripening agent would get ready access to the vasculature of the cervix.","The rapid distribution will reduce the time for any agent to start the ripening process.","Thus the use of such a device together with an effective cervical softening agent will provide the obstetrician with a useful tool to control the cervical ripening process."]],"string":"[\n [\n \"Delivery of biologically active agents\",\n \"A method of delivering a biologically active agent to the cervix, the method comprising using a needleless injector.\",\n \"Typically, the biologically active agent is a cervical ripening agent.\",\n \"The needleless injector may be a powder injector or a liquid injector.\"\n ],\n [\n \"1.A method of delivering a biologically active agent to the cervix, the method comprising using a needleless injector.\",\n \"2.A method according to claim 1 wherein the needleless injector is a liquid injector.\",\n \"3.A method according to claim 1 wherein the needless injector is a powder injector.\",\n \"4.A method according to claim 1 wherein the biologically active agent is a cervical ripening agent.\",\n \"5.A method according to claim 4 wherein the cervical ripening agent is any one or more of a prostaglandin, MCP-1 and IL-8.6.A method according to claim 1 wherein the biologically active agent is a vasodilator or precursor thereof, a chemokine or a cytokine which stimulates monocyte or granulocyte entry into cervical tissue.\",\n \"7.A method of ripening the female cervix, the method comprising administering a cervical ripening agent to the cervix using a needleless injector.\",\n \"8.A system for delivering a biologically active agent to the cervix comprising an agent which is biologically active on the cervix and a needleless injector.\",\n \"9.A system according to claim 8 wherein the biologically active agent is a cervical ripening agent.\",\n \"10.A system according to claim 8 wherein the needleless injector is a liquid injector.\",\n \"11.A system according to claim 8 wherein the needleless injector is a powder injector.\",\n \"12.A needleless injector loaded for injection with an agent which is biologically active on the cervix.\",\n \"13.A needleless injector according to claim 12 wherein the biologically active agent is a cervical ripening agent.\",\n \"14.A needleless injector according to claim 12 which is a liquid injector.\",\n \"15.A needleless injector according to claim 12 which is a powder injector.\",\n \"16.A vial for insertion into, and containing an agent for delivery by, a needleless injector wherein the agent is an agent which is biologically active on the cervix.\",\n \"17.A vial according to claim 16 wherein the agent is a cervical ripening agent.\",\n \"18.A method of preparing a needleless injector for use in delivering a biologically active agent to the cervix, the method comprising loading the injector with the biologically active agent.\",\n \"19.A method according to claim 18 wherein the agent is loaded in a vial disposed for insertion into the needleless injector.\",\n \"20.A pharmaceutical formulation comprising an agent for delivery to the cervix and a carrier suitable for use in a needleless injector.\",\n \"21.A pharmaceutical formulation according to claim 20 wherein the formulation contains particles of a density between about 0.1 and about 25 g/cm3 and of a size between 0.1 and 250 μm which particles comprise the said agent.\",\n \"22.A pharmaceutical formulation according to claim 20 comprising an agent which permeabilises a mucosal surface, such as dimethylsulphoxide.\",\n \"23.Use of a cervical ripening agent in the manufacture of a medicament for treating a female in need of a cervical ripening agent wherein the cervical ripening agent is for delivery using a needleless injector.\",\n \"24-26.\",\n \"(canceled).\"\n ],\n [\n \"The present invention relates to the delivery of biologically active agents to the female cervix and, in particular, it relates to the delivery of cervical ripening agents.\",\n \"The cervix acts as a barrier to the ingress of infection as well as a rigid barrier at the neck of the womb.\",\n \"This organ has relatively little muscle with the majority of the strength of the organ derived from collagen.\",\n \"In a normal, easy delivery, the cervix has to soften and stretch (eface) to allow the baby's head to pass through, if this can be achieved with little uterine contractility then subsequent contractions can deliver the baby with minimal trauma for mother and child.\",\n \"Biologically active agents such as prostaglandins and chemokines are delivered to the cervix or vaginal fomix for various purposes.\",\n \"In particular, agents are delivered to aid cervical ripening for a number of reasons including (a) the induction of labour, (b) to soften an unfavourable cervix during labour, (c) to assist medical termination of pregnancy, (d) to assist uterine surgery and (e) to accelerate normal parition and to reduce the accompanying risks and discomforts.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,624,670 to Kelly et al describes the use of interleukin-8 (IL-8) for inducing cervical ripening, and U.S. Pat.\",\n \"No.\",\n \"5,908,829 to Kelly describes the use of monocyte chemotactic peptide-1 (MCP-1) for inducing ripening of the cervix.\",\n \"Vasodilators or their precursors (such as the nitric oxide donor nitroglycerine) are also delivered to the cervix.\",\n \"At present, biological agents are delivered to the cervix or vaginal fornix using gels, creams, pessaries.\",\n \"A relatively large amount of drug is currently needed since diffusion through the epithelial layers of the cervix is relatively slow.\",\n \"The gel can also get displaced.\",\n \"The most common current method of cervical ripening is to use prostaglandin E2.This is used as a vaginal gel or tablet or as a gel placed in the cervix.\",\n \"Typically, gel is placed in the vaginal fornix.\",\n \"The prostaglandin has to diffuse through the epithelium of the cervix and act on the blood vessels to increase permeability.\",\n \"In addition prostaglandin may act on fibroblasts to release interleukin-8 (IL-8) within the cervix.\",\n \"The combination of IL-8 and PGE is very powerful in attracting neutrophils (Colditz (1990) Immunol.\",\n \"Cell Biol.\",\n \"68, 397-403) and neutrophils are a source of collagenase.\",\n \"There are several disadvantages to the current approaches.\",\n \"Firstly, in order that sufficient amounts of the biologically active agent are delivered to the site of action, relatively high concentrations of the agent need to be included in the formulation administered.\",\n \"Secondly, prostaglandin gels used to ripen the cervix by administration to the vaginal fornix can stimulate the myometrium to contract excessively and, in extreme circumstances, an uterine rupture can occur (Maymon et al (1991) Am.\",\n \"J. Obstet, Gynecol.\",\n \"165, 368-370).\",\n \"Thirdly, with prostaglandin gel the rate of diffusion into the cervix is limited and variable.\",\n \"Fourthly, for certain of the biologically active agents delivered to the cervix or vaginal fornix, diffusion is relatively slow due to the increased molecular size and hydrophilic nature of the compound.\",\n \"This is particularly true for peptide agents.\",\n \"During its ripening, the cervix is thought to soften from the internal os.\",\n \"That is likely because of the possible fetal contribution to the timing of birth.\",\n \"Thus, the initial softening might involve agents (such as PGE2 and or chemokines) diffusing outward from the fetal membranes (amnion and chorion) to the point of contact with the cervix, the internal os.\",\n \"Attempts to deliver chemokines has identified a problem with the diffusion of the active agent, particularly when that agent is a peptide and accordingly may not be lipophilic.\",\n \"In addition, we believe that delivery to the cervix of any ripening agent should be diffuse rather than punctate and thus conventional injection using a needle and syringe is not ideal.\",\n \"There remains the need to improve on delivery methods to the cervix and, as described in more detail below, the inventors believe that alternatives lo which use needleless injectors may lead to benefits over the existing methods.\",\n \"Needleless injectors are well known in the art and have been used to deliver agents principally to the skin of individuals, for example in vaccinations.\",\n \"As far as the inventors are aware, despite needleless injectors having been available for very many years, no-one has suggested that they would be useful in delivering agents to the cervix, and no-one has realised the potential benefits in terms of the possibility of using lower concentrations of biologically-active agents, particularly cervical-ripening agents such as prostaglandins, and benefits in terms of potentially simplifying the formulation of the active agents for delivery.\",\n \"In particular, compared to other delivery methods, no gel matrix (such as a methyl cellulose or modified starch gel) is required.\",\n \"Commercially available needleless injectors fall into two main categories: those which use powder formulations of the biologically active agent, and those that use the agent in a solution formulation.\",\n \"These will be referred to as “powder” injectors and “liquid” injectors.\",\n \"Generally, both types of injector use compressed air, or gas, or some type of mechanical configuration to “inject” the active agent into the patient.\",\n \"Powderject (Oxford, UK) manufactures commercially powder injectors.\",\n \"Typically, powder injectors make use of dry powder formulations and fine particles of the drug to be delivered are projected at high velocity within a brief burst of, typically, helium gas.\",\n \"Transdermal application has been described and, typically, the dose is delivered over a skin area of up to 2 cm2.WO 94/24263 (Oxford Biosciences Limited) describes a needleless syringe which uses supersonic gas flow for particle delivery.\",\n \"The needleless syringe has a membrane which is ruptured by gas pressure to generate a supersonic gas flow in which particles containing a therapeutic agent are injected.\",\n \"WO 96/12513 (Oxford Biosciences Limited) describes a similar system except that a soft-walled capsule, such as one with a gelatine wall, is used to hold the therapeutic agent in the syringe.\",\n \"WO 96/20022 (Oxford Biosciences Limited) again describes a similar device except that it has a diaphragm which snaps from inverted to everted positions to catapult particles outwardly.\",\n \"WO 96/25190 (Oxford Biosciences Limited) describes a needleless powder injector with a bend.\",\n \"WO 97/34652 (Powderject Research Limited) relates to a shroud which surrounds the nozzle of the syringe and reduces the noise.\",\n \"WO 98/13470 (Powderject Vaccines, Inc) relates to a rotational flow element which modifies gas flow through the device.\",\n \"WO 99/01168 and WO 99/01169 (Powderject Research Limited) relate to a prefilled capsule for use with a powder injector and a modified gas release mechanism, respectively.\",\n \"WO 00/54827 (Powderject Research Limited) relates to a diaphragm for use with a needleless injector wherein the diaphragm provides for enhanced particle retention.\",\n \"WO 00/62846 (Powderject Research Limited) relates to a needleless powder injector device that does not require particles to be between burstable membranes.\",\n \"Certain other patent applications relate to the manufacture of particle compositions (WO 97/48485 and WO 00/53160; Powderject Research Limited); and cartridges for use in a needleless powder injector (WO 98/21364; Powderject Vaccines, Inc and U.S. Pat.\",\n \"No.\",\n \"5,780,100 to McCabe and Heinzen).\",\n \"Weston Medical (Peterborough, UK) manufactures commercially liquid injectors.\",\n \"WO 93/03799 (Weston) and WO 95/03844 (Weston Medical) describe a liquid injector system which is used to deliver vaccines by penetration of the epidermis for intradermal delivery.\",\n \"WO 96/28202 (Weston Medical) and WO 97/37705 (Weston Medical) describe a spring-powered dispensing device which can be in the form of a needleless injector.\",\n \"EP 0 520 571 A (Glaxo) describes an atomising nozzle for use with a liquid injector.\",\n \"WO 96/15821 (Weston Medical) describes a glass container for use in needleless injectors, and WO 97/22375 (Weston Medical) and WO 97/36785 (Weston Medical) describe a needleless injector drug capsule and filling method.\",\n \"WO 96/19252 (Weston Medical) describes a filling device for a needleless injector.\",\n \"WO 00/10630 (Weston Medical) describes an actuator for a gas powered needleless injector.\",\n \"WO 00/15281 (Weston Medical) describes a needleless injector cartridge.\",\n \"A first aspect of the invention provides a method of delivering a biologically active agent to the cervix, the method comprising using a needleless injector.\",\n \"The delivery may be directly to the cervix or may be via the vaginal fornix, which is a fold in the vagina where the cervix is located.\",\n \"Thus, the end of the needleless injector may occupy the vaginal fornix and delivery may be at or through the vaginal fornix into the cervix.\",\n \"Typically, injections will aim at the cervix but may be aimed from different angles.\",\n \"Preferably, the site of injection is “off-centre” into the cervical lumen or into any part of the externally presenting area of the cervix.\",\n \"The needleless injector may be any suitable man-made needleless injector and, conveniently, may be a liquid injector or a powder injector as described above.\",\n \"Thus, suitable needleless liquid injectors include those manufactured by Weston Medical Limited, Peterborough, UK and those described in WO 93/03779, WO 95/03844, WO 96/28202, WO 97/37705 and WO 00/10630, all of which are incorporated herein by reference.\",\n \"Suitable needleless powder injectors include those manufactured by Powderject Research Limited, Oxford, UK and those described in WO 94/24263, WO 96/12513, WO 96/20022, WO 96/25190, WO 97/34652, WO 98/13470, WO 99/01168, WO 99/01169, WO 00/54827 and WO 00/62846, all of which are incorporated herein by reference.\",\n \"The biologically active agent may be loaded into the injector by means known in the art.\",\n \"It may be useful if the needleless injector has a “bent” configuration such that delivery of the biologically active agent is to the cervix rather than to the walls of the vagina or to the entrance of the cervix.\",\n \"Thus, the needleless injector may be adapted for this purpose, and the geometry of the device arranged such that there is easy passage through the vagina but that the exit from the injector (ie outlet orifice) is angled such that the biologically active agent is delivered efficiently to the desired site on the cervix.\",\n \"The type of injectors described in WO 96/25190 may be particularly suitable; however, rather than the internal angle of approximately 90° shown in FIGS.\",\n \"1 to 4 of WO 96/25190, it may be more appropriate if the internal angle of the head to the shaft is between 20° to 90°; preferably between 35° and 85°, for example 65°.\",\n \"The arrangement of the head containing the outlet orifice shown in FIG.\",\n \"16B of WO 00/54827 may also be particularly suited for use in the present invention.\",\n \"Similarly, the arrangement shown in FIG.\",\n \"5 of WO 00/62846, where the exit plane is not perpendicular to the longitudinal axis of the nozzle, but angled, may be particularly suited for use with the invention.\",\n \"The biologically active agent may be any agent for delivery to the cervix which has a useful effect.\",\n \"For example, the agent may be any agent which has a therapeutic or prophylactic effect, or which is useful in any medical procedure.\",\n \"Typically, the agent is one which is useful in gynaecology or obstetrics.\",\n \"Suitable agents include prostaglandins (both natural and synthetic), vasodilators or their precursors (such as the nitric oxide (NO) donor, nitroglycerine), and chemokines or any cytokine that could stimulate monocyte or granulocyte entry into the cervical tissue such as GM-CSF.\",\n \"It is particularly preferred if the agent is a cervical ripening agent, such as a prostaglandin (in particular, prostaglandin E2) or interleukin-8 (IL-8) or monocyte chemotactic pepfide-1 (MCP-1) or combinations thereof.\",\n \"MCP-1, IL-8 and variants thereof may be obtained by chemical synthetic methods or by recombinant DNA means as is well known in the art.\",\n \"Prostaglandins, such as PGE2, are commercially available, for example from Pharmacia and Upjohn.\",\n \"The use of prostaglandins in cervical ripening is well known in the art.\",\n \"The use of IL-8 and MCP-1 in cervical ripening is described in U.S. Pat.\",\n \"Nos.\",\n \"5,624,670 and 5,908,829, respectively, incorporated herein by reference.\",\n \"Variants of MCP-1 or IL-8 which are able to induce cervical ripening may also be used.\",\n \"A “variant” refers to a protein wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in a protein whose basic properties, for example cervical ripening-inducing activity, thermostability, activity in a certain pH-range (pH-stability) have not significantly been changed.\",\n \"“Significantly” in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein.\",\n \"By “conservative substitutions” is intended combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.\",\n \"Such variants may be made using the methods of protein engineering and site-directed mutagenesis as described below.\",\n \"The dose of the agent administered is any suitable dose of the agent which provides the desired effect of the agent.\",\n \"Typically, because of the precision with which the agent can be administered with the needleless injector, the dose may be significantly reduced compared to the dose administered by other, conventional means.\",\n \"The amount may be reduced to 50% or 20% or 10% or 5% or 2% of the amount normally used, depending on the agent administered.\",\n \"For example, 2 to 100 μg of IL-8 or MCP-1 dissolved in 200 μl of sterile saline may be used in a liquid injector.\",\n \"Diffused delivery is achieved using needleless injectors.\",\n \"The agent, once injected, distributes radially and along the length of the injection path.\",\n \"Such a method of delivery is not obtainable by any other method of injection, and, although not being bound by any theory, is believed to be particularly beneficial in the context of the present invention.\",\n \"Administration of lower amounts of the agent may reduce the chance of side effects.\",\n \"For example, side effects of NO donors are headaches and excessive bleeding.\",\n \"Injection of a low amount directly into the cervix would both accelerate the effect and reduce the side effects.\",\n \"Typically, the needleless injector is loaded with the biologically active agent which is to be delivered to the cervix, the injector is inserted into the vagina such that the exit from the injector (ie outlet orifice) is in proximity to the cervix, and the injector is actuated so as to deliver the agent to the cervix.\",\n \"Preferably, the exit from the injector (ie outlet orifice) is not at the rim of the cervix but is at the side of the cervix.\",\n \"It will be appreciated that position or type of “head” of the needleless injector (ie that contains the outlet orifice) may be adjusted or adapted depending on the particular use.\",\n \"Thus, particular positions or designs of head may be more suited to delivery when the cervix is partially dilated (eg during parturition) or may be more suited to delivery when the cervix is closed or substantially closed.\",\n \"A description of the anatomy of the vagina, cervix and uterus is given in FIG.\",\n \"1 and positions for possible delivery to the cervix are indicated.\",\n \"The method may be used to deliver biologically active agents to any female mammal, but it is particularly preferred if the method is used to deliver agents to a human female.\",\n \"However, the method may be especially useful for sheep where in vitro fertilization and embryo transfer procedures currently involve laparoscopy which brings up ethical and animal husbandry considerations, the problem being that the cervix is impenetrable.\",\n \"It is believed that a diffuse injection of cervical ripening agent would give a very rapid response, allowing a wider use of in vitro fertilization.\",\n \"It will be appreciated that for certain agents, in particular peptide and polypeptide agents, it may be appropriate to select the peptide or polypeptide of the mammal, or a closely related molecule for administration.\",\n \"Thus, for example, it is preferred to administer human peptides or polypeptides to a human female.\",\n \"Of course, the peptide or polypeptide may be produced synthetically or by recombinant DNA means but would still be considered “mammalian” or, as the case may be “human”.\",\n \"Suitable IL-8 molecules and derivatives thereof are described in U.S. Pat.\",\n \"No.\",\n \"5,624,670, incorporated herein by reference.\",\n \"Suitable MCP-1 molecules and derivatives thereof are described in U.S. Pat.\",\n \"No.\",\n \"5,908,829, incorporated herein by reference.\",\n \"It will be appreciated that the invention includes a method of ripening the female cervix, the method comprising administering a cervical ripening agent to the cervix using a needleless injector.\",\n \"The method can be used for the induction of labour at term (ie time of ordinary birth), but it may also be used for the induction of pre-term labour, and induction of labour in connection with a pathological pregnancy, or in connection with intrauterine fetal death.\",\n \"It may be used to ripen dysfunctional cervices, ie when dilation stops before completion.\",\n \"The method may also be used for preliminary cervical ripening prior to induction of abortion (eg in the first or second trimester abortion), and for induction of cervical ripening of a non-pregnant or pregnant female to assist surgical or diagnostic procedure such as D&C.\",\n \"Cervical ripening may also be induced in the female for the purposes of treatment by in vitro fertilisation.\",\n \"It is particularly preferred if the cervical ripening agent is any one or more of IL-8, MCP-1 or a prostaglandin.\",\n \"The prostaglandin typically is PGE2 which is currently used in cervical ripening.\",\n \"It is believed that by using the methods of the invention, which may be used to more accurately deliver the agent to a particular site on the cervix, a smaller amount of agent may be used compared to the use of gels, creams, pessaries and the like.\",\n \"At least in the case of PGE2 this has the potential benefit of reducing the risk of undesirable contraction of the myometrium.\",\n \"The amount of PGE2 may be reduced to 10% or 5% or 2% of the amount normally used when in a gel.\",\n \"For example, 2 to 100 μg of PGE2 dissolved in 200 μl of sterile saline may be used.\",\n \"The cervical ripening agent may be a combination of agents.\",\n \"It is particularly preferred if the cervical ripening agent is a complex comprising a proteinaceous agent which is chemotactic for monocytes and/or neutrophils and a prostaglandin.\",\n \"By “proteinaceous agent which is chemotactic for monocytes and/or neutrophils” we include any protein or polypeptide or oligopeptide or peptide which is able to encourage monocytes and/or neutrophils into a tissue where the agent is present.\",\n \"Typically, the agent is a proteinaceous cervical ripening agent.\",\n \"By “proteinaceous cervical ripening agent” we include any protein or polypeptide or oligopeptide or peptide which has the ability to induce ripening of the cervix when suitably administered to the female.\",\n \"Thus, the proteinaceous cervical ripening agent is one which contains peptide bonds and has amino acid side chain residues.\",\n \"Preferably, the proteinaceous agent which is chemotactic for monocytes and/or neutrophils is one with a pI value of 7.5 or above, preferably 8 or above and more preferably 8.5 or above.\",\n \"Typically, >5% of the amino acid residues are lysines.\",\n \"The prostaglandin may be any suitable prostaglandin.\",\n \"It is preferred that the prostaglandin is one which is able to induce cervical ripening when suitably administered to the female.\",\n \"It is preferred that the prostaglandin is PGE2 or a synthetic analogue thereof with a free acid.\",\n \"Synthetic analogues include these modified at position 15 by the addition of a methyl group.\",\n \"It is particularly preferred if the prostaglandin is one which has an acidic group such as a carboxylate group.\",\n \"Preferably, the acidic group is one which, under low pH conditions, gives a free acid.\",\n \"It is preferred that the prostaglandin is prostaglandin E2 (PGE2).\",\n \"Particularly preferred proteinaceous cervical ripening agents include monocyte chemotactic peptide-1 (MCP-1) or interleukin-8 (IL-8) or variants thereof which retain the ability to induce cervical ripening.\",\n \"These complexes are described in our co-pending PCT Patent Application which claims priority from UK Patent Application No 0103354.7.A second aspect of the invention provides a system for delivering a biologically active agent to the cervix comprising an agent which is biologically active on the cervix and a needleless injector.\",\n \"The needleless injector may be any needleless injector suitable for delivering the agent to the cervix.\",\n \"Such needleless injectors are described above and preferred injectors in the above mentioned methods of the invention are also preferred in this aspect of the invention.\",\n \"The biologically active agent is an agent which is active on the cervix.\",\n \"Suitably, the agents are ones which are delivered to the cervix for therapeutic or prophylactic or other medical use.\",\n \"Typically, they are agents which are not ones which are administered to the skin.\",\n \"Suitable such agents include PGEs, IL-8 and MCP-1 as described above.\",\n \"The agents preferably include cervical ripening agents as described above.\",\n \"The system may be considered to be a kit of parts comprising various components.\",\n \"Thus, the kit of parts may comprise a needleless injector and, separately, the biologically active agent.\",\n \"In that case, the agent may be loaded into a suitable reservoir in the needleless injector.\",\n \"Alternatively, the kit of parts may comprise a needleless injector and a vial (by which we include capsule or cartridge or the like) which contains the agent wherein the vial is one which can be loaded into the injector.\",\n \"Additionally, in the case of powder injectors, the kit may contain the therapeutic agent in a suitable, powdered form.\",\n \"Similarly, in the case of liquid injectors, the kit may contain the therapeutic agent in a suitable solution.\",\n \"It will be appreciated also that the system of this aspect of the invention may also comprise a needleless injector which is pre-loaded with the biologically active agent.\",\n \"Vials (or capsules or cartridges) for use with a needleless injector are described in more detail below.\",\n \"A third aspect of the invention provides a needleless injector loaded for injection with an agent which is biologically active on the cervix.\",\n \"The needleless injector may be any suitable needleless injector as described above, and the agent which is biologically active on the cervix may be any agent as described above.\",\n \"The needleless injector loaded with the agent according to this aspect of the invention may be formed by introducing a vial which contains the agent into the injector.\",\n \"Alternatively, the agent may be introduced into an existing reservoir within the needleless injector.\",\n \"A fourth aspect of the invention provides a vial for insertion into, and containing an agent for delivery by, a needleless injector wherein the agent is an agent which is biologically active on the cervix.\",\n \"The term “vial” may be interchanged with “cartridge”, “capsule” and the like, unless the context indicates otherwise.\",\n \"In particular, it includes the burstable membranes described in WO 94/24263 and the soft-walled capsules described in WO 96/12513.Certain needleless injectors, for example those described in WO 93/03779, WO 95/03844, WO 00/10630, WO 94/24263, WO 96/20022 and WO 96/25190 are designed to receive pre-charged vials containing the agent to be “injected”.\",\n \"Thus, the vials of this aspect of the invention include such vials pre-charged with an agent which is biologically active on the cervix, such as those described above and, in particular, cervical ripening agents.\",\n \"Accordingly, the vials are of the correct size and shape to fit into the appropriate needleless injector.\",\n \"Also, the vials are made of appropriate material for their purpose.\",\n \"Suitably, the vial contains a unit dosage of the biologically active agent.\",\n \"Suitably, when used in a liquid needleless injector, the vial contains around 200 μl (for example, between 50 μl and 500 μl).\",\n \"Typical unit dosages are 2 to 100 μg of the agent, such as PGE2 or MCP-1 or IL-8.Suitable vials for use with certain needleless liquid injectors, and methods of manufacture and filling the same, are described in WO 00/15281, WO 97/36785, WO 97/22375, WO 96/19252, WO 96/15821, WO 98/12121 and WO 98/13086, all of which are incorporated herein by reference.\",\n \"Suitable vials for use with certain needleless powder injectors, and methods of manufacturing and filling the same, are described in WO 00/54827, WO 00/62846, U.S. Pat.\",\n \"No.\",\n \"5,780,100, WO 98/21364, WO 99/01169, WO 96/20022, WO 96/12513 and WO 94/24263.A fifth aspect of the invention provides a method of preparing a needleless injector for use in delivering a biologically active agent to the cervix, the method comprising loading the injector with the biologically active agent.\",\n \"The biologically active agent may be loaded directly into a pre-existing eservoir within the needleless injector or it may be loaded into the injector in a pre-charged vial.\",\n \"A sixth aspect of the invention provides a pharmaceutical formulation comprising an agent for delivery to the cervix and a carrier suitable for use in a needleless injector.\",\n \"The pharmaceutical formulation is typically sterile and pyrogen free.\",\n \"For liquid injectors the biologically active agent is typically dissolved in sterile water or sterile saline.\",\n \"However, it may be advantageous to include a dispersion agent or an agent which allows for or increases transdermal or transmucosal transfer or penetration such as dimethyl sulphoxide (DMSO) and the like.\",\n \"For powder injectors the powder to be delivered needs to be of a suitable quality for delivery.\",\n \"Suitable powder (ie particle compositions) may be made according to the methods described in WO 97/48485 and WO 00/53160, incorporated herein by reference.\",\n \"In particular, the particles (powder) for use in the present invention have an optimum particle density ranging from about 0.1 to about 25 g/cm3, preferably ranging from about 0.5 to about 3.0 g/cm3, and most preferably ranging from about 0.8 to 1.5 g/cm3.The densified particles are suitably processed to obtain particle sizes ranging from about 0.1 to 250 μm, preferably ranging from about 0.1 to 150 μm, and most preferably ranging from about 20 to about 60 μm.\",\n \"Thus, the invention includes particles comprising a cervical ripening agent such as interleukin-8 or MCP-1 or PGE2 or combinations thereof with these physical parameters.\",\n \"These particles may be made using the methods described in WO 97/48485, incorporated herein by reference.\",\n \"The particles may be biologically inert particles onto which has been coated the biologically active agent.\",\n \"Suitably, the biologically active agent may be deposited onto the particle by dissolving it in a suitable solvent and then evaporating the solvent.\",\n \"A further aspect of the invention provides use of a cervical ripening agent in the manufacture of a medicament for treating a female in need of a cervical ripening agent wherein the cervical ripening agent is for delivery using a needleless injector.\",\n \"Preferably, the cervical ripening agent is any one or more of IL-8, MCP-1 or prostaglandin E2.The invention will now be described in more detail by reference to the following Examples and Figures.\",\n \"FIG.\",\n \"1: Anatomy of the human vagina, cervix and uterus.\",\n \"FIG.\",\n \"2: View of dye track for three separate cervices injected with the Weston needleless injector.\",\n \"Note the small ruler divisions are in mm.\",\n \"All injections were performed from the face of the cervix that would normally be in the vagina.\",\n \"EXAMPLE 1 Administration of MCP-1 to a Human Female Using a Needleless Syringe The patient is a human female in need of cervical ripening.\",\n \"200 μl of solution of monocyte chemotactic peptide-1 (MCP-1) of 100 μg/ml in sterile saline is loaded into a helium-loaded injection device and the end of the device is placed either in the vaginal fornix against the cervical mass or against the exposed cervical lumen.\",\n \"The injector is fired at one or two locations.\",\n \"EXAMPLE 2 Preparation of IL-8 as a Powder for Injection Using a Needleless Injector Lyophilized recombinant human interleukin-8 powder is obtained and reprocessed using the method described in WO 97/48485.Particularly, approximately 30 mg of IL-8 was compacted under pressure using a Carver Laboratory Pellet Press (Model 3620, available from Carver, Inc., Wabash, Ind.).\",\n \"The pressure of compaction was 15,000 lbs/in2, which was applied for approximately 45 seconds.\",\n \"A pellet was obtained which was ground using mortar and pestle until visually broken up.\",\n \"The resulting reduced pellet was then sieved using a 53 μm sieve (Endecott, London).\",\n \"Particles having a size greater 53 μm were selected and appropriate dosages thereof were measured into drug cassettes for delivery from a needleless syringe.\",\n \"EXAMPLE 3 Use of the Weston Needleless Injector to Evaluate Penetration of the Cervix A Weston needleless injector was loaded with 0.2 ml of dye (1 mg/ml of methylene blue {Sigma} in phosphate buffered saline).\",\n \"Whole human cervix was obtained at hysterectomy from non-pregnant women of reproductive age.\",\n \"The cervix was tested within 30 minutes of removal from the patient.\",\n \"The needleless injector device was held against the ectocervix region.\",\n \"Increased pressure on the needleless injector activated the device.\",\n \"Two injections were performed for each specimen.\",\n \"The cervix was dissected along the track of the injection (FIG.\",\n \"2) and the length of the dye track was measured.\",\n \"In four experiments the length of the track was a mean of 6.1 mm (standard error of the mean=1.9 mm).\",\n \"The mean lateral spread (any signs of the blue dye) was 8.8 mm.\",\n \"Liquid needleless injectors (eg Weston) penetrate skin but the liquid stream rebounds when it hits muscle.\",\n \"The cervix has relatively little smooth muscle (but gets its strength from collagen) and therefore the extent to which the liquid stream would penetrate was unknown until these experiments.\",\n \"As far as we can see there is relatively little tissue damage at the site of injection.\",\n \"The lateral spread of the injected dye is very difficult to see in the black/white photographs but can be seen in the colour ones from which the black and white photographs are derived.\",\n \"The use of the needleless injector in testing the penetration and spread of a marker dye shows that the distribution of cervical ripening product would be ideal for a rapid ripening of the cervix.\",\n \"The relatively wide spread of the injected material suggests that the ripening agent would get ready access to the vasculature of the cervix.\",\n \"The rapid distribution will reduce the time for any agent to start the ripening process.\",\n \"Thus the use of such a device together with an effective cervical softening agent will provide the obstetrician with a useful tool to control the cervical ripening process.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10470984"}}},{"rowIdx":567,"cells":{"text":{"kind":"list like","value":[["System and method for fabricating bragg gratings","A novel method and apparatus for fabrication of blazed and slanted fiber Bragg gratings is disclosed.","The method comprises the step of simultaneously exposing the fiber with two mutually coherent light beams so as to create an interference pattern along a longitudinal axis of the fiber, wherein each one of said beams is brought into a line focus, which coincides with the core of the fiber.","Further, the plane comprising the beams is rotated to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements."],["1.A method for photo-inducing a blazed grating in an optical fiber, comprising: simultaneously exposing the fiber with two mutually coherent light beams which intersects with a first angle in a plane comprising the beams and which interfere in a predetermined region of the fiber so as to create an interference pattern along a longitudinal axis of the fiber, wherein each one of said beams is brought into a line focus, which coincides with the core of the fiber, and wherein said plane comprising the beams, at least in the vicinity of the fiber, is rotated to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.","2.The method according to claim 1, wherein the beams are focused on the fiber by at least one cylindrical lens and the rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said lens.","3.The method according to claim 2, wherein the beam incidence position is displaced in a direction essentially perpendicular to the fiber direction.","4.The method according to claim 2, wherein the incidence positions of both beams are displaced.","5.The method according to claim 4, wherein the incidence positions are displaced essentially symmetrically.","6.The method according to claim 1, wherein the beams are focused on the fiber by at least one curved mirror and rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said mirror.","7.The method according to claim 6, wherein the beam incidence position is displaced in a direction essentially perpendicular to the fiber direction.","8.The method according to claim 6, wherein the incidence positions of both beams are displaced.","9.The method according to claim 8, wherein the incidence positions are displaced essentially symmetrically.","10.The method according to claim 1, wherein the fiber is translated through the exposure area where the beams intersect.","11.An apparatus for photo-inducing a blazed grating in an optical fiber, comprising: a source for emitting light; a beam splitter for forming two mutually coherent light beams; a fiber holder for holding the fiber during exposure; and a projection system for making the beams intersect with a first angle in the exposure area and thereby to interfere in a predetermined region of the fiber so as to create an interference pattern along the longitudinal axis of the fiber, wherein the projection system further comprises means for focusing the beams so that each one of said beams is brought into a line focus, which coincides with the core of the fiber, said means for focusing the beams further comprising means for rotating the plane comprising the two light beams relative to the fiber, at least in the vicinity of the fiber, to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.","12.The apparatus according to claim 11, wherein the means for focusing the beams comprises at least one lens for focusing the beams on the fiber, the means for rotating the beam plane comprising means for displacing the beam incidence position on said lens for at least one of the beams.","13.The apparatus according to claim 12, wherein the means for displacing the beam incidence position comprises at least one reflecting mirror, which is at least one of displaceable and rotatable, arranged in the beam path for said beam.","14.The apparatus according to claim 12, wherein the means for displacing the beam incidence position on said lens is adapted to displace the incidence positions of both beams.","15.The apparatus according to claim 12, wherein the means for displacing the beam incidence comprises means for parallel displacement of the beams.","16.The apparatus according to claim 11, wherein the means for focusing the beams comprises at least one curved mirror, and means for rotating the beam plane comprising means for displacing the beam incidence position on said mirror for at least one of the beams.","17.The apparatus according to claim 16, wherein the means for displacing the beam incidence position is adapted to displace the incidence position in a direction essentially perpendicular to the fiber direction.","18.The apparatus according to claim 16, wherein the means for displacing the incidence positions is adapted to displace the incidence position of both beams.","19.The apparatus according to claim 18, wherein the means for displacing the incidence positions displaces the incidence positions of the beams essentially symmetrically.","20.The apparatus according to claim 11, further comprising: means for moving the fiber essentially in the direction of a longitudinal axis of the fiber through the exposing area where the beams intersect.","21.The method according to claim 3, wherein the incidence positions of both beams are displaced.","22.The method according to claim 7, wherein the incidence positions of both beams are displaced.","23.The apparatus according to claim 13, wherein the means for displacing the beam incidence position on the lens is adapted to displace the incidence positions of both beams.","24.The apparatus according to claim 13, wherein the means for displacing the beam incidence includes means for parallel displacement of the beams.","25.The apparatus according to claim 14, wherein the means for displacing the beam incidence includes means for parallel displacement of the beams.","26.The apparatus according to claim 17, wherein the means for displacing the incidence positions is adapted to displace the incidence position of both beams."],["<SOH> BACKGROUND OF THE INVENTION <EOH>There is a rapidly growing demand for high-quality optical Bragg gratings with arbitrary phase and index profiles, as these gratings are key elements in many components that are used in WDM networks.","Over the past few years, several methods that improve the quality and the flexibility in the grating fabrication process have been developed.","A straightforward approach is to scan a UV beam over a long phase mask in a fixed relative position to the fiber.","Non-uniform profiles can in this case be fabricated either by post processing the illuminated region or by using a phase mask that contains the appropriate structure.","Complex grating structures can also be synthesized by moving the fiber slightly relative to the phase mask during the scan.","In 1995, a novel versatile sequential technique for venting long and complex fiber gratings was demonstrated by R. Stubbe, B. Sahlgren, S. Sandgren and A. Asseh, in “Novel technique for writing long superstructured fiber Bragg gratings”, in Photosensitivity and Quadratic Nonlinearity in Glass Waveguides (Fundamentals and Applications), Portland, PD1 (1995) and by A. Asseh, H. Stormy, B. E. Sahlgren, S. Sandgren and R. A. H. Stubbe, in “A writing technique for long fiber Bragg gratings with complex reflectivity profiles”, J. Lightw.","Techn.","15, 1419-1423 (1997).","The idea was to expose a large number of small partially overlapping subgratings—each containing a few hundred periods or less—in sequence; where advanced properties such as chirp, phase shifts and apodization were introduced by adjusting the phase offset and pitch of the subgratings.","In the setup that was used in the above-mentioned references, each subgrating was created by exposing the fiber with a short UV pulse while the fiber itself was translated at a constant speed.","The UV pulses were triggered by the position of the fiber relative the UV beams, which was measured by a standard helium-neon laser interferometer.","Bragg gratings normally have their grating elements aligned normal to the waveguide axis.","However, there is an increasing interest in producing gratings which have their elements at an angle to the waveguide axis, known as blazed Bragg gratings.","Such blazed Bragg gratings are difficult to fabricate efficiently and with appropriate precision with previously know methods and apparatuses.","For example, U.S. Pat.","No.","5,730,888 and U.S. Pat.","No.","5,042,897 both relates to methods and apparatuses to photo-induce blazed gratings in optical fibers.","The blazed gratings are formed by tilting the projection system relative to the fiber.","However, there are several problems with these known methods.","To be able to tilt the projection system relative to the fiber the equipment becomes complex and costly.","Further, it is difficult to achieve an adequate focus on the fiber in the whole exposure area.","Hereby, the known methods becomes slow and inefficient, with a low through-put.","Still further, with the known methods it is only possible to produce blazed gratings with a limited blazing angle, whereas blazed gratings with larger angles of inclination is very complicated to produce, or even not possible to produce at all."],["<SOH> SUMMARY OF THE INVENTION <EOH>It is therefore an object of the present invention to provide a method and an apparatus for photo-inducing a blazed grating in an optical fiber, which alleviates the above-mentioned problems of the prior art.","This object is achieved with an apparatus and a method as defined in the appended claims.","According to the invention there is provided a method for photo-inducing a blazed grating in an optical fiber comprising the step of simultaneously exposing the fiber with two mutually coherent light beams, which intersects with an first angle in a plane comprising the beams and interfere in a predetermined region of the fiber so as to create an interference pattern along a longitudinal axis of the fiber.","Further, each one of said beams is brought into a line focus, which coincides with the core of the fiber.","The invention presents a novel method for fabrication of advanced blazed fiber Bragg gratings.","As opposed to prior art methods, the method according to the invention does not tilt the projection system relative to the fiber, but provides a blazed interference pattern in line focus with the fiber.","Especially, the method could be used when said plane comprising the beams, at least in the vicinity of the fiber, is rotated to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.","According to the inventive method, a blazing angle could be chosen arbitrarily, without any equipment restrictions.","Further, the quality and precision of the photo-induced pattern is improved, since the line focus coincides with the fiber even at large blazing angles.","Still further, the control of the fabrication process becomes simplified, rendering the fabrication more efficient and less costly, and with a shortening of fabrication times for complex gratings.","Light is in the context of the application not limited to mean visible light, but a wide range of wavelengths from infrared (IR) to extreme UV.","Further, with optical fiber is in the meaning of this application to be understood any kind of optical waveguide made of a material which has a refractive index that can be permanently changed by exposure to light of at least one predetermined wavelength.","By photo-induction is to be understood the process of exposing the optical fiber of the above-mentioned type with light of the predetermined wavelength so as to form a permanent refractive index variation in the fiber.","According to one embodiment of the invention, the beams are focused on the fiber by means of at least one lens and the rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said lens.","The beam incidence position could preferably be displaced in a direction essentially perpendicular to the fiber direction.","Hereby, the beams could be displaced on the lens, and still be focused on the fiber.","Hence, the angle of incidence relative to the fiber could be varied without affecting the focus.","In an alternative embodiment, the beams are focused on the fiber by means of at least one curved mirror and rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said mirror.","The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction.","Hereby, the beams could be displaced on the mirror, and still be focused on the fiber.","Hence, the angle of incidence relative to the fiber could be varied without affecting the focus even in this embodiment.","The invention also relates to an apparatus for photo-inducing a blazed grating in an optical fiber comprising a source for emitting light; a beam splitter for forming two mutually coherent light beams; a fiber holder for holding the fiber during exposure; and a projection system for making the beams intersect with a first angle in the exposure area and thereby to interfere in a predetermined region of the fiber so as to create an interference pattern along the longitudinal axis of the fiber.","The projection system further comprises means for focusing the beams so that each one of said beams is brought into a line focus, which coincides with the core of the fiber.","With this apparatus, the method discussed above could be executed.","Accordingly, a novel apparatus is presented for fabrication of advanced blazed fiber Bragg gratings.","As opposed to prior art equipment, the apparatus according to the invention does not tilt the projection system relative to the fiber, but provides a blazed interference pattern in line focus with the fiber.","Especially, the means for focussing the beams could further comprise means for rotating the plane comprising the two light beams relative to the fiber, at least in the vicinity of the fiber, to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.","According to the invention, a blazing angle could be chosen arbitrarily, without any equipment restrictions.","Further, the quality and precision of the photo-induced pattern is improved, since the line focus coincides with the fiber even at large blazing angles.","Still further, the control of the fabrication process becomes simplified, rendering the fabrication more efficient and less costly, and with a shortening of fabrication times for complex gratings.","In one embodiment, the means for focussing the beams comprises at least one lens for focusing the beams on the fiber, the means for rotating the beam plane comprising means for displacing the beam incidence position on said lens for at least one of the beams.","The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction.","Hereby, the beams could be displaced on the lens, and still be focused on the fiber.","Hence, the angle of incidence relative to the fiber could be varied without affecting the focus.","The means for displacing the beam incidence position could comprise at least one displaceable or rotatable reflecting mirror arranged in the beam path for said beam, and preferably allowing parallax displacement of the beams.","In an alternative embodiment, the means for focusing the beams comprises at least one curved mirror, and means for rotating the beam plane comprising means for displacing the beam incidence position on said mirror for at least one of the beams.","The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction.","Hereby, the beams could be displaced on the mirror, and still be focused on the fiber.","Hence, the angle of incidence relative to the fiber could be varied without affecting the focus even in this embodiment."],["FIELD OF THE INVENTION The present invention relates to a method for photo-inducing a blazed grating in an optical fiber comprising the step of simultaneously exposing the fiber with two mutually coherent light beams, which intersects with an first angle in a plane comprising the beams and interfere in a predetermined region of the fiber so as to create an interference pattern along a longitudinal axis of the fiber.","The invention further relates to an apparatus for performing the method.","BACKGROUND OF THE INVENTION There is a rapidly growing demand for high-quality optical Bragg gratings with arbitrary phase and index profiles, as these gratings are key elements in many components that are used in WDM networks.","Over the past few years, several methods that improve the quality and the flexibility in the grating fabrication process have been developed.","A straightforward approach is to scan a UV beam over a long phase mask in a fixed relative position to the fiber.","Non-uniform profiles can in this case be fabricated either by post processing the illuminated region or by using a phase mask that contains the appropriate structure.","Complex grating structures can also be synthesized by moving the fiber slightly relative to the phase mask during the scan.","In 1995, a novel versatile sequential technique for venting long and complex fiber gratings was demonstrated by R. Stubbe, B. Sahlgren, S. Sandgren and A. Asseh, in “Novel technique for writing long superstructured fiber Bragg gratings”, in Photosensitivity and Quadratic Nonlinearity in Glass Waveguides (Fundamentals and Applications), Portland, PD1 (1995) and by A. Asseh, H. Stormy, B. E. Sahlgren, S. Sandgren and R. A. H. Stubbe, in “A writing technique for long fiber Bragg gratings with complex reflectivity profiles”, J. Lightw.","Techn.","15, 1419-1423 (1997).","The idea was to expose a large number of small partially overlapping subgratings—each containing a few hundred periods or less—in sequence; where advanced properties such as chirp, phase shifts and apodization were introduced by adjusting the phase offset and pitch of the subgratings.","In the setup that was used in the above-mentioned references, each subgrating was created by exposing the fiber with a short UV pulse while the fiber itself was translated at a constant speed.","The UV pulses were triggered by the position of the fiber relative the UV beams, which was measured by a standard helium-neon laser interferometer.","Bragg gratings normally have their grating elements aligned normal to the waveguide axis.","However, there is an increasing interest in producing gratings which have their elements at an angle to the waveguide axis, known as blazed Bragg gratings.","Such blazed Bragg gratings are difficult to fabricate efficiently and with appropriate precision with previously know methods and apparatuses.","For example, U.S. Pat.","No.","5,730,888 and U.S. Pat.","No.","5,042,897 both relates to methods and apparatuses to photo-induce blazed gratings in optical fibers.","The blazed gratings are formed by tilting the projection system relative to the fiber.","However, there are several problems with these known methods.","To be able to tilt the projection system relative to the fiber the equipment becomes complex and costly.","Further, it is difficult to achieve an adequate focus on the fiber in the whole exposure area.","Hereby, the known methods becomes slow and inefficient, with a low through-put.","Still further, with the known methods it is only possible to produce blazed gratings with a limited blazing angle, whereas blazed gratings with larger angles of inclination is very complicated to produce, or even not possible to produce at all.","SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a method and an apparatus for photo-inducing a blazed grating in an optical fiber, which alleviates the above-mentioned problems of the prior art.","This object is achieved with an apparatus and a method as defined in the appended claims.","According to the invention there is provided a method for photo-inducing a blazed grating in an optical fiber comprising the step of simultaneously exposing the fiber with two mutually coherent light beams, which intersects with an first angle in a plane comprising the beams and interfere in a predetermined region of the fiber so as to create an interference pattern along a longitudinal axis of the fiber.","Further, each one of said beams is brought into a line focus, which coincides with the core of the fiber.","The invention presents a novel method for fabrication of advanced blazed fiber Bragg gratings.","As opposed to prior art methods, the method according to the invention does not tilt the projection system relative to the fiber, but provides a blazed interference pattern in line focus with the fiber.","Especially, the method could be used when said plane comprising the beams, at least in the vicinity of the fiber, is rotated to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.","According to the inventive method, a blazing angle could be chosen arbitrarily, without any equipment restrictions.","Further, the quality and precision of the photo-induced pattern is improved, since the line focus coincides with the fiber even at large blazing angles.","Still further, the control of the fabrication process becomes simplified, rendering the fabrication more efficient and less costly, and with a shortening of fabrication times for complex gratings.","Light is in the context of the application not limited to mean visible light, but a wide range of wavelengths from infrared (IR) to extreme UV.","Further, with optical fiber is in the meaning of this application to be understood any kind of optical waveguide made of a material which has a refractive index that can be permanently changed by exposure to light of at least one predetermined wavelength.","By photo-induction is to be understood the process of exposing the optical fiber of the above-mentioned type with light of the predetermined wavelength so as to form a permanent refractive index variation in the fiber.","According to one embodiment of the invention, the beams are focused on the fiber by means of at least one lens and the rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said lens.","The beam incidence position could preferably be displaced in a direction essentially perpendicular to the fiber direction.","Hereby, the beams could be displaced on the lens, and still be focused on the fiber.","Hence, the angle of incidence relative to the fiber could be varied without affecting the focus.","In an alternative embodiment, the beams are focused on the fiber by means of at least one curved mirror and rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said mirror.","The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction.","Hereby, the beams could be displaced on the mirror, and still be focused on the fiber.","Hence, the angle of incidence relative to the fiber could be varied without affecting the focus even in this embodiment.","The invention also relates to an apparatus for photo-inducing a blazed grating in an optical fiber comprising a source for emitting light; a beam splitter for forming two mutually coherent light beams; a fiber holder for holding the fiber during exposure; and a projection system for making the beams intersect with a first angle in the exposure area and thereby to interfere in a predetermined region of the fiber so as to create an interference pattern along the longitudinal axis of the fiber.","The projection system further comprises means for focusing the beams so that each one of said beams is brought into a line focus, which coincides with the core of the fiber.","With this apparatus, the method discussed above could be executed.","Accordingly, a novel apparatus is presented for fabrication of advanced blazed fiber Bragg gratings.","As opposed to prior art equipment, the apparatus according to the invention does not tilt the projection system relative to the fiber, but provides a blazed interference pattern in line focus with the fiber.","Especially, the means for focussing the beams could further comprise means for rotating the plane comprising the two light beams relative to the fiber, at least in the vicinity of the fiber, to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.","According to the invention, a blazing angle could be chosen arbitrarily, without any equipment restrictions.","Further, the quality and precision of the photo-induced pattern is improved, since the line focus coincides with the fiber even at large blazing angles.","Still further, the control of the fabrication process becomes simplified, rendering the fabrication more efficient and less costly, and with a shortening of fabrication times for complex gratings.","In one embodiment, the means for focussing the beams comprises at least one lens for focusing the beams on the fiber, the means for rotating the beam plane comprising means for displacing the beam incidence position on said lens for at least one of the beams.","The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction.","Hereby, the beams could be displaced on the lens, and still be focused on the fiber.","Hence, the angle of incidence relative to the fiber could be varied without affecting the focus.","The means for displacing the beam incidence position could comprise at least one displaceable or rotatable reflecting mirror arranged in the beam path for said beam, and preferably allowing parallax displacement of the beams.","In an alternative embodiment, the means for focusing the beams comprises at least one curved mirror, and means for rotating the beam plane comprising means for displacing the beam incidence position on said mirror for at least one of the beams.","The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction.","Hereby, the beams could be displaced on the mirror, and still be focused on the fiber.","Hence, the angle of incidence relative to the fiber could be varied without affecting the focus even in this embodiment.","BRIEF DESCRIPTION OF THE DRAWINGS For exemplifying purposes, the invention will be described in closer detail in the following with reference to embodiments thereof illustrated in the attached drawings, wherein: FIG.","1 is a schematic drawing of a fiber Bragg grating fabrication system according to an embodiment according to the invention.","FIG.","2 is a schematic drawing of a projection system in the exposure area according to a first embodiment of the invention with the beams not being displaced, where FIG.","2a is seen perpendicular to the fiber and 2b is seen in the longitudinal direction of the fiber.","FIG.","3 is a schematic drawing of the projection system in FIG.","2, but where the beams are displaced, where FIG.","3a is seen perpendicular to the fiber and 3b is seen in the longitudinal direction of the fiber.","FIG.","4 is a schematic drawing of a projection system in the exposure area according to a second embodiment of the invention, where FIG.","4a is seen perpendicular to the fiber, 4b is seen in the longitudinal direction of the fiber, and 4c is a side view from a direction perpendicular to the view in 4a.","DESCRIPTION OF PREFERRED EMBODIMENTS Further scope of the applicability of the present invention will become apparent from the detailed description given hereinafter.","However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.","The setup according to an embodiment of the invention is illustrated in FIG.","1.The base for the system according to the invention could e.g.","be a system as described in “Fiber Bragg Gratings” by Raman Kashyap, p. 55-101.Especially the system described with reference to FIG.","3.21 serves as a good base.","This description is hereby incorporated by reference.","However, other types of photo-inducing systems are conceivable for the invention.","A system according to the invention comprises in general a light source, a beam splitter of any kind, a projection system and a fiber holder.","The projection system could preferably comprise a mirror arrangement, and sees to that the beams interfere with another in an exposure area so as to form a interference pattern in the fiber and hence corresponding grating elements.","The light source could be a continuous or pulsed wave laser.","When a pulsed laser is used, the laser is preferably actuated when the distance moved by the fiber equals one grating period.","However, preferably a continuous laser is used, whereby the interference pattern is caused to move in accordance with the fiber during certain time periods.","Between such time periods, the pattern is rapidly reversed to the starting point so as to continue the grating writing.","Hence, a generally un-interrupted writing process is achieved.","Such movement of the interference pattern may be achieved by alteration of the phase difference between the beams, e.g.","by displacing mirrors in the beam path.","In a preferred embodiment of the invention, the fiber 1 to be exposed is placed in a fiber holder 2 mounted on an airbearing born carriage 3, which is translated by a feedback-controlled linear drive.","The position of the translator stage relative the UV interference pattern is measured with a heterodyne interference detection system 4 utilizing a He-Ne laser as light source.","The resulting spatial resolution is approximately 0.6 nm, available over the translation length of about half a meter.","A light source 5, e.g.","a frequency-doubled argon-ion laser emits light, such as 100-mW radiation of wavelength 244 nm, into a beam splitter, such as a half transparent mirror or prism, or a phase-mask, where it is divided into two coherent beams.","Thereafter, the beams are launched into a projection system, such as a double Sagnac interferometer 6, which generates the interference pattern forming the grating.","In the projection system, one or several cylindrical lenses focus the two interfering beams into a line focus that coincides with the core of the fiber 1.Longitudinally, the focus could extend over about 100 microns, which roughly corresponds to 200 fringes for a Bragg wavelength of 1550 nm resonance wavelength.","The apparatus is controlled by a control unit 8, which e.g.","could be electronically implemented in hardware.","This control unit could in turn be controlled by a software control unit 7, such as an conventional personal computer.","Preferably, first displacing means 61 are provided, comprising e.g.","a step motor, for controlling the angle between the interfering beams in a plane comprising the fiber.","This could be used to change the period of the interference fringes to match the desired local pitch of the grating.","The angle change is preferably performed symmetrically for both beams so that the center fringe does not move its position.","In a preferred setup, the resolution for this pitch variation is about 1.4 μm in resonant wavelength.","Hereby, the chirp or distance between individual grating elements could be accurately controlled.","In order to prevent unwanted exposure outside the actual grating, the system preferably also comprises a controllable shutter 9 in the UV beam path that is only open within the grating region during the writing.","Introduction of Controlled Blazing According to the invention, the writing system further comprises means 62 for focusing the beams so that each one of said beams is brought into a line focus, which coincides with the core of the fiber.","Said means for focusing the beams further comprises means for rotating the plane comprising the two light beams relative to the fiber, at least in the vicinity of the fiber, to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.","However, the line focus is still maintained during said rotation.","The rotation could be provided by second displacing means 63 for displacing the beams, and preferably in a direction being oblique relative to the fiber direction, and most preferably in a direction essentially perpendicular to the fiber direction.","In a first embodiment, as illustrated in FIGS.","1 and 2, the rotation is provided by a lens 621, whereby the beam incidence position on said lens is displaceable for at least one of the beams.","In FIG.","2, the beams are not displaced, whereas they are displaced in FIG.","3.The displacement could be provided by displaceable or rotatable mirrors 622 arranged in the beam path.","Hereby, the beams may be parallax displaced.","However, as will be appreciated by someone skilled in the art, other means for displacing the beams are conceivable as well.","The displacement of the beams does not influence the focusing of the beams on the fiber.","However, the beams being displaced provides an angle between the beams in a plane perpendicular to the fiber direction, as is illustrated in FIG.","3b.","Hence, the grating being photo-induced will be slanted, i.e.","blazed, in a plane comprising the fiber but being perpendicular to the plane illustrated in FIG.","3a.","According to an alternative embodiment, as is illustrated in FIG.","4, the rotation is provided by a curved mirror 623, whereby the beam incidence position on said mirror is displaceable for at least one of the beams.","The displacement could be provided in the same way as is discussed above.","Accordingly, the grating being photo-induced will be slanted, i.e.","blazed, in a plane comprising the fiber but being perpendicular to the plane illustrated in FIG.","4a, as is illustrated in FIG.","4c.","The displacement is preferably performed on both beams, and most preferably in an essentially symmetrical fashion.","CONCLUSIONS The invention provides a novel technique for fabrication of high quality blazed fiber Bragg gratings.","By the inventive method, a precisely controllable blazing may be introduced, without tilting the projection system relative to the fiber or the like.","As a result, the fabrication time for all kinds of customized gratings is greatly reduced as compared to earlier methods.","The invention further allows for a very precise control of the grating formation, and especially the introduction of blazing.","The invention has now been described by way of embodiments.","However, many alternatives are possible.","For example, different types of beam splitters are feasible, other types of means for translating the fiber could be used etc.","Further, other means for displacing the beams are feasible.","Such alternatives are known from the prior art.","It should be appreciated by someone skilled in the art that such alternatives are part of the invention, such as it is defined by the appended claims."]],"string":"[\n [\n \"System and method for fabricating bragg gratings\",\n \"A novel method and apparatus for fabrication of blazed and slanted fiber Bragg gratings is disclosed.\",\n \"The method comprises the step of simultaneously exposing the fiber with two mutually coherent light beams so as to create an interference pattern along a longitudinal axis of the fiber, wherein each one of said beams is brought into a line focus, which coincides with the core of the fiber.\",\n \"Further, the plane comprising the beams is rotated to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.\"\n ],\n [\n \"1.A method for photo-inducing a blazed grating in an optical fiber, comprising: simultaneously exposing the fiber with two mutually coherent light beams which intersects with a first angle in a plane comprising the beams and which interfere in a predetermined region of the fiber so as to create an interference pattern along a longitudinal axis of the fiber, wherein each one of said beams is brought into a line focus, which coincides with the core of the fiber, and wherein said plane comprising the beams, at least in the vicinity of the fiber, is rotated to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.\",\n \"2.The method according to claim 1, wherein the beams are focused on the fiber by at least one cylindrical lens and the rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said lens.\",\n \"3.The method according to claim 2, wherein the beam incidence position is displaced in a direction essentially perpendicular to the fiber direction.\",\n \"4.The method according to claim 2, wherein the incidence positions of both beams are displaced.\",\n \"5.The method according to claim 4, wherein the incidence positions are displaced essentially symmetrically.\",\n \"6.The method according to claim 1, wherein the beams are focused on the fiber by at least one curved mirror and rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said mirror.\",\n \"7.The method according to claim 6, wherein the beam incidence position is displaced in a direction essentially perpendicular to the fiber direction.\",\n \"8.The method according to claim 6, wherein the incidence positions of both beams are displaced.\",\n \"9.The method according to claim 8, wherein the incidence positions are displaced essentially symmetrically.\",\n \"10.The method according to claim 1, wherein the fiber is translated through the exposure area where the beams intersect.\",\n \"11.An apparatus for photo-inducing a blazed grating in an optical fiber, comprising: a source for emitting light; a beam splitter for forming two mutually coherent light beams; a fiber holder for holding the fiber during exposure; and a projection system for making the beams intersect with a first angle in the exposure area and thereby to interfere in a predetermined region of the fiber so as to create an interference pattern along the longitudinal axis of the fiber, wherein the projection system further comprises means for focusing the beams so that each one of said beams is brought into a line focus, which coincides with the core of the fiber, said means for focusing the beams further comprising means for rotating the plane comprising the two light beams relative to the fiber, at least in the vicinity of the fiber, to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.\",\n \"12.The apparatus according to claim 11, wherein the means for focusing the beams comprises at least one lens for focusing the beams on the fiber, the means for rotating the beam plane comprising means for displacing the beam incidence position on said lens for at least one of the beams.\",\n \"13.The apparatus according to claim 12, wherein the means for displacing the beam incidence position comprises at least one reflecting mirror, which is at least one of displaceable and rotatable, arranged in the beam path for said beam.\",\n \"14.The apparatus according to claim 12, wherein the means for displacing the beam incidence position on said lens is adapted to displace the incidence positions of both beams.\",\n \"15.The apparatus according to claim 12, wherein the means for displacing the beam incidence comprises means for parallel displacement of the beams.\",\n \"16.The apparatus according to claim 11, wherein the means for focusing the beams comprises at least one curved mirror, and means for rotating the beam plane comprising means for displacing the beam incidence position on said mirror for at least one of the beams.\",\n \"17.The apparatus according to claim 16, wherein the means for displacing the beam incidence position is adapted to displace the incidence position in a direction essentially perpendicular to the fiber direction.\",\n \"18.The apparatus according to claim 16, wherein the means for displacing the incidence positions is adapted to displace the incidence position of both beams.\",\n \"19.The apparatus according to claim 18, wherein the means for displacing the incidence positions displaces the incidence positions of the beams essentially symmetrically.\",\n \"20.The apparatus according to claim 11, further comprising: means for moving the fiber essentially in the direction of a longitudinal axis of the fiber through the exposing area where the beams intersect.\",\n \"21.The method according to claim 3, wherein the incidence positions of both beams are displaced.\",\n \"22.The method according to claim 7, wherein the incidence positions of both beams are displaced.\",\n \"23.The apparatus according to claim 13, wherein the means for displacing the beam incidence position on the lens is adapted to displace the incidence positions of both beams.\",\n \"24.The apparatus according to claim 13, wherein the means for displacing the beam incidence includes means for parallel displacement of the beams.\",\n \"25.The apparatus according to claim 14, wherein the means for displacing the beam incidence includes means for parallel displacement of the beams.\",\n \"26.The apparatus according to claim 17, wherein the means for displacing the incidence positions is adapted to displace the incidence position of both beams.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>There is a rapidly growing demand for high-quality optical Bragg gratings with arbitrary phase and index profiles, as these gratings are key elements in many components that are used in WDM networks.\",\n \"Over the past few years, several methods that improve the quality and the flexibility in the grating fabrication process have been developed.\",\n \"A straightforward approach is to scan a UV beam over a long phase mask in a fixed relative position to the fiber.\",\n \"Non-uniform profiles can in this case be fabricated either by post processing the illuminated region or by using a phase mask that contains the appropriate structure.\",\n \"Complex grating structures can also be synthesized by moving the fiber slightly relative to the phase mask during the scan.\",\n \"In 1995, a novel versatile sequential technique for venting long and complex fiber gratings was demonstrated by R. Stubbe, B. Sahlgren, S. Sandgren and A. Asseh, in “Novel technique for writing long superstructured fiber Bragg gratings”, in Photosensitivity and Quadratic Nonlinearity in Glass Waveguides (Fundamentals and Applications), Portland, PD1 (1995) and by A. Asseh, H. Stormy, B. E. Sahlgren, S. Sandgren and R. A. H. Stubbe, in “A writing technique for long fiber Bragg gratings with complex reflectivity profiles”, J. Lightw.\",\n \"Techn.\",\n \"15, 1419-1423 (1997).\",\n \"The idea was to expose a large number of small partially overlapping subgratings—each containing a few hundred periods or less—in sequence; where advanced properties such as chirp, phase shifts and apodization were introduced by adjusting the phase offset and pitch of the subgratings.\",\n \"In the setup that was used in the above-mentioned references, each subgrating was created by exposing the fiber with a short UV pulse while the fiber itself was translated at a constant speed.\",\n \"The UV pulses were triggered by the position of the fiber relative the UV beams, which was measured by a standard helium-neon laser interferometer.\",\n \"Bragg gratings normally have their grating elements aligned normal to the waveguide axis.\",\n \"However, there is an increasing interest in producing gratings which have their elements at an angle to the waveguide axis, known as blazed Bragg gratings.\",\n \"Such blazed Bragg gratings are difficult to fabricate efficiently and with appropriate precision with previously know methods and apparatuses.\",\n \"For example, U.S. Pat.\",\n \"No.\",\n \"5,730,888 and U.S. Pat.\",\n \"No.\",\n \"5,042,897 both relates to methods and apparatuses to photo-induce blazed gratings in optical fibers.\",\n \"The blazed gratings are formed by tilting the projection system relative to the fiber.\",\n \"However, there are several problems with these known methods.\",\n \"To be able to tilt the projection system relative to the fiber the equipment becomes complex and costly.\",\n \"Further, it is difficult to achieve an adequate focus on the fiber in the whole exposure area.\",\n \"Hereby, the known methods becomes slow and inefficient, with a low through-put.\",\n \"Still further, with the known methods it is only possible to produce blazed gratings with a limited blazing angle, whereas blazed gratings with larger angles of inclination is very complicated to produce, or even not possible to produce at all.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>It is therefore an object of the present invention to provide a method and an apparatus for photo-inducing a blazed grating in an optical fiber, which alleviates the above-mentioned problems of the prior art.\",\n \"This object is achieved with an apparatus and a method as defined in the appended claims.\",\n \"According to the invention there is provided a method for photo-inducing a blazed grating in an optical fiber comprising the step of simultaneously exposing the fiber with two mutually coherent light beams, which intersects with an first angle in a plane comprising the beams and interfere in a predetermined region of the fiber so as to create an interference pattern along a longitudinal axis of the fiber.\",\n \"Further, each one of said beams is brought into a line focus, which coincides with the core of the fiber.\",\n \"The invention presents a novel method for fabrication of advanced blazed fiber Bragg gratings.\",\n \"As opposed to prior art methods, the method according to the invention does not tilt the projection system relative to the fiber, but provides a blazed interference pattern in line focus with the fiber.\",\n \"Especially, the method could be used when said plane comprising the beams, at least in the vicinity of the fiber, is rotated to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.\",\n \"According to the inventive method, a blazing angle could be chosen arbitrarily, without any equipment restrictions.\",\n \"Further, the quality and precision of the photo-induced pattern is improved, since the line focus coincides with the fiber even at large blazing angles.\",\n \"Still further, the control of the fabrication process becomes simplified, rendering the fabrication more efficient and less costly, and with a shortening of fabrication times for complex gratings.\",\n \"Light is in the context of the application not limited to mean visible light, but a wide range of wavelengths from infrared (IR) to extreme UV.\",\n \"Further, with optical fiber is in the meaning of this application to be understood any kind of optical waveguide made of a material which has a refractive index that can be permanently changed by exposure to light of at least one predetermined wavelength.\",\n \"By photo-induction is to be understood the process of exposing the optical fiber of the above-mentioned type with light of the predetermined wavelength so as to form a permanent refractive index variation in the fiber.\",\n \"According to one embodiment of the invention, the beams are focused on the fiber by means of at least one lens and the rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said lens.\",\n \"The beam incidence position could preferably be displaced in a direction essentially perpendicular to the fiber direction.\",\n \"Hereby, the beams could be displaced on the lens, and still be focused on the fiber.\",\n \"Hence, the angle of incidence relative to the fiber could be varied without affecting the focus.\",\n \"In an alternative embodiment, the beams are focused on the fiber by means of at least one curved mirror and rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said mirror.\",\n \"The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction.\",\n \"Hereby, the beams could be displaced on the mirror, and still be focused on the fiber.\",\n \"Hence, the angle of incidence relative to the fiber could be varied without affecting the focus even in this embodiment.\",\n \"The invention also relates to an apparatus for photo-inducing a blazed grating in an optical fiber comprising a source for emitting light; a beam splitter for forming two mutually coherent light beams; a fiber holder for holding the fiber during exposure; and a projection system for making the beams intersect with a first angle in the exposure area and thereby to interfere in a predetermined region of the fiber so as to create an interference pattern along the longitudinal axis of the fiber.\",\n \"The projection system further comprises means for focusing the beams so that each one of said beams is brought into a line focus, which coincides with the core of the fiber.\",\n \"With this apparatus, the method discussed above could be executed.\",\n \"Accordingly, a novel apparatus is presented for fabrication of advanced blazed fiber Bragg gratings.\",\n \"As opposed to prior art equipment, the apparatus according to the invention does not tilt the projection system relative to the fiber, but provides a blazed interference pattern in line focus with the fiber.\",\n \"Especially, the means for focussing the beams could further comprise means for rotating the plane comprising the two light beams relative to the fiber, at least in the vicinity of the fiber, to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.\",\n \"According to the invention, a blazing angle could be chosen arbitrarily, without any equipment restrictions.\",\n \"Further, the quality and precision of the photo-induced pattern is improved, since the line focus coincides with the fiber even at large blazing angles.\",\n \"Still further, the control of the fabrication process becomes simplified, rendering the fabrication more efficient and less costly, and with a shortening of fabrication times for complex gratings.\",\n \"In one embodiment, the means for focussing the beams comprises at least one lens for focusing the beams on the fiber, the means for rotating the beam plane comprising means for displacing the beam incidence position on said lens for at least one of the beams.\",\n \"The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction.\",\n \"Hereby, the beams could be displaced on the lens, and still be focused on the fiber.\",\n \"Hence, the angle of incidence relative to the fiber could be varied without affecting the focus.\",\n \"The means for displacing the beam incidence position could comprise at least one displaceable or rotatable reflecting mirror arranged in the beam path for said beam, and preferably allowing parallax displacement of the beams.\",\n \"In an alternative embodiment, the means for focusing the beams comprises at least one curved mirror, and means for rotating the beam plane comprising means for displacing the beam incidence position on said mirror for at least one of the beams.\",\n \"The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction.\",\n \"Hereby, the beams could be displaced on the mirror, and still be focused on the fiber.\",\n \"Hence, the angle of incidence relative to the fiber could be varied without affecting the focus even in this embodiment.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates to a method for photo-inducing a blazed grating in an optical fiber comprising the step of simultaneously exposing the fiber with two mutually coherent light beams, which intersects with an first angle in a plane comprising the beams and interfere in a predetermined region of the fiber so as to create an interference pattern along a longitudinal axis of the fiber.\",\n \"The invention further relates to an apparatus for performing the method.\",\n \"BACKGROUND OF THE INVENTION There is a rapidly growing demand for high-quality optical Bragg gratings with arbitrary phase and index profiles, as these gratings are key elements in many components that are used in WDM networks.\",\n \"Over the past few years, several methods that improve the quality and the flexibility in the grating fabrication process have been developed.\",\n \"A straightforward approach is to scan a UV beam over a long phase mask in a fixed relative position to the fiber.\",\n \"Non-uniform profiles can in this case be fabricated either by post processing the illuminated region or by using a phase mask that contains the appropriate structure.\",\n \"Complex grating structures can also be synthesized by moving the fiber slightly relative to the phase mask during the scan.\",\n \"In 1995, a novel versatile sequential technique for venting long and complex fiber gratings was demonstrated by R. Stubbe, B. Sahlgren, S. Sandgren and A. Asseh, in “Novel technique for writing long superstructured fiber Bragg gratings”, in Photosensitivity and Quadratic Nonlinearity in Glass Waveguides (Fundamentals and Applications), Portland, PD1 (1995) and by A. Asseh, H. Stormy, B. E. Sahlgren, S. Sandgren and R. A. H. Stubbe, in “A writing technique for long fiber Bragg gratings with complex reflectivity profiles”, J. Lightw.\",\n \"Techn.\",\n \"15, 1419-1423 (1997).\",\n \"The idea was to expose a large number of small partially overlapping subgratings—each containing a few hundred periods or less—in sequence; where advanced properties such as chirp, phase shifts and apodization were introduced by adjusting the phase offset and pitch of the subgratings.\",\n \"In the setup that was used in the above-mentioned references, each subgrating was created by exposing the fiber with a short UV pulse while the fiber itself was translated at a constant speed.\",\n \"The UV pulses were triggered by the position of the fiber relative the UV beams, which was measured by a standard helium-neon laser interferometer.\",\n \"Bragg gratings normally have their grating elements aligned normal to the waveguide axis.\",\n \"However, there is an increasing interest in producing gratings which have their elements at an angle to the waveguide axis, known as blazed Bragg gratings.\",\n \"Such blazed Bragg gratings are difficult to fabricate efficiently and with appropriate precision with previously know methods and apparatuses.\",\n \"For example, U.S. Pat.\",\n \"No.\",\n \"5,730,888 and U.S. Pat.\",\n \"No.\",\n \"5,042,897 both relates to methods and apparatuses to photo-induce blazed gratings in optical fibers.\",\n \"The blazed gratings are formed by tilting the projection system relative to the fiber.\",\n \"However, there are several problems with these known methods.\",\n \"To be able to tilt the projection system relative to the fiber the equipment becomes complex and costly.\",\n \"Further, it is difficult to achieve an adequate focus on the fiber in the whole exposure area.\",\n \"Hereby, the known methods becomes slow and inefficient, with a low through-put.\",\n \"Still further, with the known methods it is only possible to produce blazed gratings with a limited blazing angle, whereas blazed gratings with larger angles of inclination is very complicated to produce, or even not possible to produce at all.\",\n \"SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a method and an apparatus for photo-inducing a blazed grating in an optical fiber, which alleviates the above-mentioned problems of the prior art.\",\n \"This object is achieved with an apparatus and a method as defined in the appended claims.\",\n \"According to the invention there is provided a method for photo-inducing a blazed grating in an optical fiber comprising the step of simultaneously exposing the fiber with two mutually coherent light beams, which intersects with an first angle in a plane comprising the beams and interfere in a predetermined region of the fiber so as to create an interference pattern along a longitudinal axis of the fiber.\",\n \"Further, each one of said beams is brought into a line focus, which coincides with the core of the fiber.\",\n \"The invention presents a novel method for fabrication of advanced blazed fiber Bragg gratings.\",\n \"As opposed to prior art methods, the method according to the invention does not tilt the projection system relative to the fiber, but provides a blazed interference pattern in line focus with the fiber.\",\n \"Especially, the method could be used when said plane comprising the beams, at least in the vicinity of the fiber, is rotated to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.\",\n \"According to the inventive method, a blazing angle could be chosen arbitrarily, without any equipment restrictions.\",\n \"Further, the quality and precision of the photo-induced pattern is improved, since the line focus coincides with the fiber even at large blazing angles.\",\n \"Still further, the control of the fabrication process becomes simplified, rendering the fabrication more efficient and less costly, and with a shortening of fabrication times for complex gratings.\",\n \"Light is in the context of the application not limited to mean visible light, but a wide range of wavelengths from infrared (IR) to extreme UV.\",\n \"Further, with optical fiber is in the meaning of this application to be understood any kind of optical waveguide made of a material which has a refractive index that can be permanently changed by exposure to light of at least one predetermined wavelength.\",\n \"By photo-induction is to be understood the process of exposing the optical fiber of the above-mentioned type with light of the predetermined wavelength so as to form a permanent refractive index variation in the fiber.\",\n \"According to one embodiment of the invention, the beams are focused on the fiber by means of at least one lens and the rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said lens.\",\n \"The beam incidence position could preferably be displaced in a direction essentially perpendicular to the fiber direction.\",\n \"Hereby, the beams could be displaced on the lens, and still be focused on the fiber.\",\n \"Hence, the angle of incidence relative to the fiber could be varied without affecting the focus.\",\n \"In an alternative embodiment, the beams are focused on the fiber by means of at least one curved mirror and rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said mirror.\",\n \"The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction.\",\n \"Hereby, the beams could be displaced on the mirror, and still be focused on the fiber.\",\n \"Hence, the angle of incidence relative to the fiber could be varied without affecting the focus even in this embodiment.\",\n \"The invention also relates to an apparatus for photo-inducing a blazed grating in an optical fiber comprising a source for emitting light; a beam splitter for forming two mutually coherent light beams; a fiber holder for holding the fiber during exposure; and a projection system for making the beams intersect with a first angle in the exposure area and thereby to interfere in a predetermined region of the fiber so as to create an interference pattern along the longitudinal axis of the fiber.\",\n \"The projection system further comprises means for focusing the beams so that each one of said beams is brought into a line focus, which coincides with the core of the fiber.\",\n \"With this apparatus, the method discussed above could be executed.\",\n \"Accordingly, a novel apparatus is presented for fabrication of advanced blazed fiber Bragg gratings.\",\n \"As opposed to prior art equipment, the apparatus according to the invention does not tilt the projection system relative to the fiber, but provides a blazed interference pattern in line focus with the fiber.\",\n \"Especially, the means for focussing the beams could further comprise means for rotating the plane comprising the two light beams relative to the fiber, at least in the vicinity of the fiber, to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.\",\n \"According to the invention, a blazing angle could be chosen arbitrarily, without any equipment restrictions.\",\n \"Further, the quality and precision of the photo-induced pattern is improved, since the line focus coincides with the fiber even at large blazing angles.\",\n \"Still further, the control of the fabrication process becomes simplified, rendering the fabrication more efficient and less costly, and with a shortening of fabrication times for complex gratings.\",\n \"In one embodiment, the means for focussing the beams comprises at least one lens for focusing the beams on the fiber, the means for rotating the beam plane comprising means for displacing the beam incidence position on said lens for at least one of the beams.\",\n \"The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction.\",\n \"Hereby, the beams could be displaced on the lens, and still be focused on the fiber.\",\n \"Hence, the angle of incidence relative to the fiber could be varied without affecting the focus.\",\n \"The means for displacing the beam incidence position could comprise at least one displaceable or rotatable reflecting mirror arranged in the beam path for said beam, and preferably allowing parallax displacement of the beams.\",\n \"In an alternative embodiment, the means for focusing the beams comprises at least one curved mirror, and means for rotating the beam plane comprising means for displacing the beam incidence position on said mirror for at least one of the beams.\",\n \"The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction.\",\n \"Hereby, the beams could be displaced on the mirror, and still be focused on the fiber.\",\n \"Hence, the angle of incidence relative to the fiber could be varied without affecting the focus even in this embodiment.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS For exemplifying purposes, the invention will be described in closer detail in the following with reference to embodiments thereof illustrated in the attached drawings, wherein: FIG.\",\n \"1 is a schematic drawing of a fiber Bragg grating fabrication system according to an embodiment according to the invention.\",\n \"FIG.\",\n \"2 is a schematic drawing of a projection system in the exposure area according to a first embodiment of the invention with the beams not being displaced, where FIG.\",\n \"2a is seen perpendicular to the fiber and 2b is seen in the longitudinal direction of the fiber.\",\n \"FIG.\",\n \"3 is a schematic drawing of the projection system in FIG.\",\n \"2, but where the beams are displaced, where FIG.\",\n \"3a is seen perpendicular to the fiber and 3b is seen in the longitudinal direction of the fiber.\",\n \"FIG.\",\n \"4 is a schematic drawing of a projection system in the exposure area according to a second embodiment of the invention, where FIG.\",\n \"4a is seen perpendicular to the fiber, 4b is seen in the longitudinal direction of the fiber, and 4c is a side view from a direction perpendicular to the view in 4a.\",\n \"DESCRIPTION OF PREFERRED EMBODIMENTS Further scope of the applicability of the present invention will become apparent from the detailed description given hereinafter.\",\n \"However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.\",\n \"The setup according to an embodiment of the invention is illustrated in FIG.\",\n \"1.The base for the system according to the invention could e.g.\",\n \"be a system as described in “Fiber Bragg Gratings” by Raman Kashyap, p. 55-101.Especially the system described with reference to FIG.\",\n \"3.21 serves as a good base.\",\n \"This description is hereby incorporated by reference.\",\n \"However, other types of photo-inducing systems are conceivable for the invention.\",\n \"A system according to the invention comprises in general a light source, a beam splitter of any kind, a projection system and a fiber holder.\",\n \"The projection system could preferably comprise a mirror arrangement, and sees to that the beams interfere with another in an exposure area so as to form a interference pattern in the fiber and hence corresponding grating elements.\",\n \"The light source could be a continuous or pulsed wave laser.\",\n \"When a pulsed laser is used, the laser is preferably actuated when the distance moved by the fiber equals one grating period.\",\n \"However, preferably a continuous laser is used, whereby the interference pattern is caused to move in accordance with the fiber during certain time periods.\",\n \"Between such time periods, the pattern is rapidly reversed to the starting point so as to continue the grating writing.\",\n \"Hence, a generally un-interrupted writing process is achieved.\",\n \"Such movement of the interference pattern may be achieved by alteration of the phase difference between the beams, e.g.\",\n \"by displacing mirrors in the beam path.\",\n \"In a preferred embodiment of the invention, the fiber 1 to be exposed is placed in a fiber holder 2 mounted on an airbearing born carriage 3, which is translated by a feedback-controlled linear drive.\",\n \"The position of the translator stage relative the UV interference pattern is measured with a heterodyne interference detection system 4 utilizing a He-Ne laser as light source.\",\n \"The resulting spatial resolution is approximately 0.6 nm, available over the translation length of about half a meter.\",\n \"A light source 5, e.g.\",\n \"a frequency-doubled argon-ion laser emits light, such as 100-mW radiation of wavelength 244 nm, into a beam splitter, such as a half transparent mirror or prism, or a phase-mask, where it is divided into two coherent beams.\",\n \"Thereafter, the beams are launched into a projection system, such as a double Sagnac interferometer 6, which generates the interference pattern forming the grating.\",\n \"In the projection system, one or several cylindrical lenses focus the two interfering beams into a line focus that coincides with the core of the fiber 1.Longitudinally, the focus could extend over about 100 microns, which roughly corresponds to 200 fringes for a Bragg wavelength of 1550 nm resonance wavelength.\",\n \"The apparatus is controlled by a control unit 8, which e.g.\",\n \"could be electronically implemented in hardware.\",\n \"This control unit could in turn be controlled by a software control unit 7, such as an conventional personal computer.\",\n \"Preferably, first displacing means 61 are provided, comprising e.g.\",\n \"a step motor, for controlling the angle between the interfering beams in a plane comprising the fiber.\",\n \"This could be used to change the period of the interference fringes to match the desired local pitch of the grating.\",\n \"The angle change is preferably performed symmetrically for both beams so that the center fringe does not move its position.\",\n \"In a preferred setup, the resolution for this pitch variation is about 1.4 μm in resonant wavelength.\",\n \"Hereby, the chirp or distance between individual grating elements could be accurately controlled.\",\n \"In order to prevent unwanted exposure outside the actual grating, the system preferably also comprises a controllable shutter 9 in the UV beam path that is only open within the grating region during the writing.\",\n \"Introduction of Controlled Blazing According to the invention, the writing system further comprises means 62 for focusing the beams so that each one of said beams is brought into a line focus, which coincides with the core of the fiber.\",\n \"Said means for focusing the beams further comprises means for rotating the plane comprising the two light beams relative to the fiber, at least in the vicinity of the fiber, to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements.\",\n \"However, the line focus is still maintained during said rotation.\",\n \"The rotation could be provided by second displacing means 63 for displacing the beams, and preferably in a direction being oblique relative to the fiber direction, and most preferably in a direction essentially perpendicular to the fiber direction.\",\n \"In a first embodiment, as illustrated in FIGS.\",\n \"1 and 2, the rotation is provided by a lens 621, whereby the beam incidence position on said lens is displaceable for at least one of the beams.\",\n \"In FIG.\",\n \"2, the beams are not displaced, whereas they are displaced in FIG.\",\n \"3.The displacement could be provided by displaceable or rotatable mirrors 622 arranged in the beam path.\",\n \"Hereby, the beams may be parallax displaced.\",\n \"However, as will be appreciated by someone skilled in the art, other means for displacing the beams are conceivable as well.\",\n \"The displacement of the beams does not influence the focusing of the beams on the fiber.\",\n \"However, the beams being displaced provides an angle between the beams in a plane perpendicular to the fiber direction, as is illustrated in FIG.\",\n \"3b.\",\n \"Hence, the grating being photo-induced will be slanted, i.e.\",\n \"blazed, in a plane comprising the fiber but being perpendicular to the plane illustrated in FIG.\",\n \"3a.\",\n \"According to an alternative embodiment, as is illustrated in FIG.\",\n \"4, the rotation is provided by a curved mirror 623, whereby the beam incidence position on said mirror is displaceable for at least one of the beams.\",\n \"The displacement could be provided in the same way as is discussed above.\",\n \"Accordingly, the grating being photo-induced will be slanted, i.e.\",\n \"blazed, in a plane comprising the fiber but being perpendicular to the plane illustrated in FIG.\",\n \"4a, as is illustrated in FIG.\",\n \"4c.\",\n \"The displacement is preferably performed on both beams, and most preferably in an essentially symmetrical fashion.\",\n \"CONCLUSIONS The invention provides a novel technique for fabrication of high quality blazed fiber Bragg gratings.\",\n \"By the inventive method, a precisely controllable blazing may be introduced, without tilting the projection system relative to the fiber or the like.\",\n \"As a result, the fabrication time for all kinds of customized gratings is greatly reduced as compared to earlier methods.\",\n \"The invention further allows for a very precise control of the grating formation, and especially the introduction of blazing.\",\n \"The invention has now been described by way of embodiments.\",\n \"However, many alternatives are possible.\",\n \"For example, different types of beam splitters are feasible, other types of means for translating the fiber could be used etc.\",\n \"Further, other means for displacing the beams are feasible.\",\n \"Such alternatives are known from the prior art.\",\n \"It should be appreciated by someone skilled in the art that such alternatives are part of the invention, such as it is defined by the appended claims.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471027"}}},{"rowIdx":568,"cells":{"text":{"kind":"list like","value":[["Non-isothermal method for fabricating hollow composite parts","A process for making hollow composite structures or vessels which includes the steps of: A) heating a mixture of thermoplastic matrix and reinforcing fibres wrapped over a rigid or semi-rigid thermoplastic liner or bladder above the melting point of the thermoplastic composite matrix outside of a moulding tool; B) transferring the heated assembly to a mould that is maintained below the melting temperature of the thermoplastic matrix of the composite; C) closure of the mould and application of internal fluid pressure to the liner or bladder to apply pressure to the thermoplastic matrix and reinforcing fibres; D) optionally the use of a special coupling system for rapid connection of the internal pressure; E) cooling of the liner or bladder and thermoplastic matrix and reinforcing fibre assembly in contact with the cold or warm mould while consolidation of the assembly occurs; F) opening of the mould and removal of the finished assembly.","Suitable thermoplastic materials for the liner/bladder and thermoplastic composite matrix material include: polypropylene, polyamide, polyethylene, cross-linked polyethylene, polybutylene terephthalate, polyethylene terephthalate, polyoxymethylene, polyphenylene sulfide and polyetheretherketone."],["1.A non-isothermal process for fabricating a hollow composite structure, comprising the steps of: preforming at least one thermoplastic liner; over-wrapping the at least one thermoplastic liner with thermoplastic composite material to form at least one over-wrapped liner; heating the at least one over-wrapped liner in an oven to a first temperature above a melting temperature (Tm) of the thermoplastic composite material to form a heated over-wrapped liner; placing the heated over-wrapped liner into a moulding tool that is maintained below the melting temperature (Tm) of the thermoplastic composite material; applying an internal pressure to an interior of the heated over-wrapped liner while the moulding tool is closed to consolidate the composite material and a bladder against a geometry defined by the mould; maintaining the internal pressure to the interior of over-wrapped liner until a second temperature of the thermoplastic composite material is below the thermoplastic composite melting temperature (Tm); and removing the over-wrapped liner from the moulding tool.","2.The process according to claim 1, wherein a plurality of over-wrapping phase inserts are positioned on the thermoplastic liner after the step of preforming the thermoplastic liner, and the over-wrapping phase inserts are consolidated with the over-wrapped thermoplastic comnposite material before the step of heating the over-wrapped thermoplastic liner.","3.The process according to claim 2, wherein the over-wrapping phase inserts have an operating temperature such that critical dimensions of the over-wrapping phase inserts are not distorted by the first temperature during the step of heating.","4.The process according to claim 2, wherein the thermoplastic composite material is locally consolidated onto the over-wrapping phase inserts.","5.The process according to claim 1, wherein a plurality of the thermoplastic liners are included during the step of overwrapping the at least one thermoplastic liner to form a continuous process.","6.The process according to claim 1, wherein the step of applying an internal pressure includes a rapid coupling system.","7.The process according to claim 6, wherein the rapid coupling system includes a rapid gas connection system having an integral pneumatic cylinder comprising a conical member having an axial hole that is pushed into the heated over-wrapped liner against a block having an internally profiled axial hole to create a seal against the heated over-wrapped liner.","8.The process according to claim 1, wherein at least one of the thermoplastic liner and the thermoplastic composite material is selected from a group comprising polypropylene, polyamide, particularly polyamide 12, polyethylene, cross-linked polyethylene, polybutylene terephthalate, polyethylene terephthalate, polyoxymethylene, polyphenylene sulfide, and polyetheretherketone.","9.A The process according to claim 1, wherein the step of preforming includes at least one of extrusion blow moulding and rotary casting.","10.The process according to claim 1, wherein the step of preforming includes high molecular weight polymers to increase a melt strength of the thermoplastic liner.","11.The process according to claim 1, wherein the step of preforming includes discontinuous reinforcing fibres to increase a melt strength of the thermoplastic liner.","12.The process according to claim 1, wherein the step of preforming includes discontinuous reinforcing fibres to perform a load bearing function of the hollow composite structure.","13.The process according to claim 1, wherein the step of over-wrapping includes one of filament winding, braiding, tailored braiding, 3-D braiding, and over-wrapping of one of fabrics and wide tapes.","14.The process according to claim 1, wherein the step of over-wrapping includes a final layer of material to tailor surface properties.","15.The process according to claim 1, further comprising a step of heating the at least one over-wrapped liner to a third temperature above the first temperature before the step of heating the at least one over-wrapped liner to the first temperature.","16.The process according to claim 1, wherein the step of heating the at least one over-wrapped liner includes inducing a temperature gradient such that a third temperature of an outer layer of the thermoplastic composite material is above the melting temperature (Tm) of the thermoplastic composite material and the over-wrapped liner is maintained at a fourth temperature above the melting point of the thermoplastic liner.","17.The process according to claim 1, wherein the step of heating the at least one over-wrapped liner includes an oven system comprising a combined forced hot gas oven and an infra red oven.","18.The process according to claim 1, wherein the internal pressure is about 2 bar to maintain a shape of the at least one over-wrapped liner.","19.The process according to claim 1, wherein the step of heating the at least one over-wrapped liner includes maintaining the liner at a geometry using a frame that enables heat transfer into the moulding tool.","20.The process according to claim 1, wherein during the step of applying an internal pressure the heated over-wrapped liner is located between two mould halves of the moulding tool such that contact of both mould halves occurs substantially simultaneously.","21.The process according to claim 1, wherein during the step of placing the heated over-wrapped liner the over-wrapped liner is heated to a third temperature above the melting temperature (Tm) of the thermoplastic composite material such that partial impregnation of the thermoplastic liner occurs and the bulk of the thermoplastic composite material is reduced.","22.The process according to claim 1, further comprising at least one step of connecting external features to the hollow composite structure.","23.The process according to claim 1, wherein the hollow composite structure includes an integral thermoplastic liner."],["<SOH> BACKGROUND OF THE INVENTION <EOH>Hollow composite structures, or pressure vessels, here referred to as ‘vessels’, such as used to store fluids and solids, particularly under pressure, such as pressurized gas tanks, or more generally in space frame or tubular, load bearing, assemblies have traditionally been fabricated from metals such as steel or aluminium.","However, in recent years, the use of composite vessels has become more prevalent.","Such vessels are manufactured by a variety of processes, which include filament winding, resin transfer moulding and bladder-assisted moulding.","The technology of filament winding is the process of impregnating dry reinforcing fibres, such as fibreglass or carbon strands, with an activated resin prior to application to a mandrel.","Preimpregnated materials may also be used.","The mixture of reinforcing fibres and matrix is then applied to the mandrel.","In the case of thermoset based matrices, a cure cycle follows, while for thermoplastic matrices the polymer is softened (melted) before application to the mandrel where fusion bonding then occurs between layers of the wound material that forms the structure.","Either the cure cycle with thermosets or cooling of the material as it is wound with thermoplastics produces a hollow structure or shell that is then removed from the mandrel to give the component.","To produce hollow structures or vessels, the technology of resin transfer moulding uses a mandrel and an outer mould, where a mixture of activated matrix resin is injected in the cavity between the mandrel and the outer mould where reinforcing fibres of typically glass, carbon or Kevlar have been placed.","Impregnation of the reinforcing fibres then occurs, with cross-linking of thermoset polymers or polymerisation of thermoplastic monomer forming the matrix polymer and the finished shell-like or hollow component.","The mandrel could then be removed.","The technology of bladder-assisted moulding, for the case of thermoplastic matrices, uses an outer mould and inner bladder to define the shape of the part.","To melt the thermoplastic matrix and hence impregnate the reinforcing fibres, of typically glass, carbon or Kevlar, the thermoplastic is heated above its melting temperature and a pressure is then applied to the bladder to consolidate the material.","The thermoplastic material must then be cooled, while under pressure, to form the hard shell-like or hollow composite component.","Bladder materials comprising rubber-like materials, typically silicon rubber, are used to apply the compaction force, whereby the bladder stretches or elongates under the influence of the applied internal pressure.","Alternate bladders consist of in-situ extrusion blow moulded polymeric materials.","The thermoplastic matrix is typically heated by heating the outer mould, often metallic, above the matrix melting temperature before the pressure is applied for the required period, after which the mould is cooled sufficiently below the matrix melting temperature for solidification and crystallisation to occur.","Alternately, additional or separate heating may be applied by passing hot oil through the bladder or by using embedded electric heaters in the bladder, to heat the composite from the inside, thus eliminating the need to cycle the mould temperature.","Conductive reinforcing materials, such as carbon fibre based composites or composites containing a proportion of conductive fibres such as carbon or steel, may be heated in the mould through induction heating or electrical resistance heating.","In-situ extrusion blow moulding has been previously claimed to provide heat to aid the melting of the thermoplastic matrix composite, but in practice additional temperature cycling of the mould above and below the matrix melting temperature is also needed.","The technology of bladder-assisted moulding, for the case of thermoset based matrices, uses an outer mould and inner bladder to define the shape of the part.","A preimpregnated mixture of reinforcing fibres and activated matrix are placed over the bladder and the bladder with the surrounding preimpregnated material is placed into the outer mould.","The mould temperature must be high enough to induce cure of the thermosetting matrix and after cure, cooled to allow part release.","The bladder may also be heated, either through the passage of a hot fluid, or by embedded electrical heaters, to apply the heat necessary for cure of the impregnated material.","The technology also consists of bladder-assisted resin transfer moulding where reinforcement substantially without matrix is placed over the bladder and the bladder coated with reinforcement is then placed into the mould.","Activated resin is then applied that impregnates the reinforcing fibres with the application of internal pressure via the bladder, thereby aiding impregnation.","Such techniques for fabricating hollow structures or vessels, particularly if a rapid cycle time is required (which is the case for many applications of hollow composite structures or vessels as it reduces the cost of the component) have well known drawbacks which include: for thermoplastic filament winding based processes, the thermoplastic matrix based reinforcement must be heated prior to the application to a mandrel to ensure the thermal conditions necessary for fusion bonding.","Hence high processing speeds, notably the application of the thermoplastic matrix based reinforcement to the mandrel, creates a limit on the speed of the process and hence the cycle time.","High drag or friction forces induced by the placement of thermoplastic matrix based reinforcement create further quality problems whereby segregation of the thermoplastic matrix and reinforcing fibres may occur, creating an unfavourable microstructure.","Additionally, the geometry of the component is limited where there is a need to remove the mandrel after processing.","Where the mandrel remains in the component after processing, the mandrel must be compatible with the thermoplastic matrix based reinforcement and be able to sustain the high radial crush forces induced by hot fibre and matrix mixtures applied under high tension forces.","The sequential application of composite layers to the mandrel is known to create high internal stresses in the component, affecting dimensional stability and the eventual durability of the component.","The filament winding process also places restrictions on geometries that can be produced, where concave curvature requires in-situ bonding of the thermoplastic based composite material to the mandrel or preceding layer of composite; for thermoset based filament winding processes, the thermoset matrix must be cured onto the mandrel requiring a heating cycle either directly as the thermoset matrix based reinforcement is placed, or subsequent heating of the placed material and the mandrel.","The process cycle time depends on the winding speed and hence this creates a limit on the speed of the process and thereby the cycle time.","High drag or friction forces induced by the placement of thermoset matrix based reinforcement create further quality problems whereby segregation of the thermosetting matrix and reinforcing fibres may occur, creating an unfavourable microstructure.","Additionally, the geometry of the component is limited where there is a need to remove the mandrel after processing.","Where the mandrel remains in the component after processing, the mandrel must be compatible with the thermoset matrix based reinforcement and be able to sustaining the high radial crush forces induced by the fibre and matrix mixtures applied under high tension forces; for the resin transfer moulding based process, as used with thermoset based reinforced material, a preform must be assembled over the mandrel and resin injected between the mould and the mandrel.","The cycle time is limited by the need to inject the resin into this space, where pressures are limited where a permanent hollow mandrel is used such that the pressure of the incoming fluid, here the activated resin, does not distort the geometry of the mandrel, hence limiting processes to lower rate and pressure injection rather than the shorter cycle time, high pressure process of structural reaction injection moulding.","Furthermore, the cure reaction of the thermoset matrix either limits the cycle time or, where tailored to occur at a higher speed, induces high stresses in the composite.","Unless complex and expensive preforms of reinforcement are prefabricated, parts of a constant wall thickness must be produced unless a local decrease in the reinforcing fibre fraction can be tolerated.","Where the mandrel needs to be removed, expensive technologies, such as low melting temperature alloys, must be used where part geometries are not to be impaired.","for the resin transfer moulding based process, for the case of thermoplastic based reinforced material, a preform must be assembled over the mandrel and activated monomer injected between the mould and the mandrel.","The cycle time is limited by the need to inject the resin into this space, where pressures are limited where a permanent hollow mandrel is used such that the pressure of the incoming fluid, here the activated resin, does not distort the geometry of the mandrel, hence limiting processes to lower rate and pressure injection rather than the shorter cycle time, high pressure process of structural reaction injection moulding.","Furthermore, the polymerisation reaction of the monomer either limits the cycle time or, where tailored to occur at a higher speed, places limitations on bladder materials.","Thermal cycling above the monomer melting temperature for polymerisation to occur most rapidly and cooling after polymerisation to below the polymer melting temperature are also often required, requiring cycling of the mould temperature and/or mandrel temperatures for certain thermoplastic resin transfer moulding material systems.","Unless complex and expensive preforms of reinforcement are prefabricated, parts of a constant wall thickness must be produced unless a local decrease in the reinforcing fibre fraction can be tolerated.","Where the mandrel needs to be removed, expensive technologies, such as low melting temperature alloys, must be used where part geometries are not to be impaired.","for the bladder assisted moulding process, for the case of thermoset based reinforced material, the need to cure the thermoset matrix material creates problems with chemical attack with rubber-like bladder materials, typically styrene with silicon rubbers, and hence bladder life is reduced.","Bladder materials are generally removable due to the difficulty of achieving a bladder that is flexible enough to enable compaction of the impregnated reinforcing fibres prior and during cure while forming a reliable bond between the bladder and cured composite with the lack of high internal stresses in the finished component.","Heating of the impregnated reinforcement is also required that is accomplished either through cycling of the mould temperature or heating the bladder material.","Cycling the mould temperature is energy inefficient and time consuming, limiting the potential to reduce cycle times, and the fabrication of heated bladder systems is uneconomic for components made in large quantities while also being fragile and of limited life span.","Additional internal inspection is also required in the case of removable bladders to ensure that small particles of the bladder material do not remain on the inside wall of the hollow composite or vessel and that the bladder integrity has been maintained such that it is safely usable for further moulding trials.","for the bladder assisted moulding process, for the case of thermoplastic based reinforced material, the thermoplastic composite must be heated above the melt temperature of the matrix and cooled below the melt temperature before demoulding.","This occurs either by heating the outer mould temperature above the matrix melting temperature and then cooling the mould below the matrix melting temperature, whereby the thermoplastic matrix composite is also heated above and below the thermoplastic composite matrix melting temperature during which time a compaction force is applied by the bladder, or by heating the bladder material such that the thermoplastic matrix is heated sufficiently above the melting temperature of the matrix for adequate impregnation to occur, followed by cooling of the composite structure sufficiently below the matrix melting temperature for stable component release from the mould.","The currently available techniques outlined above have inherent limitations that reduce the potential of the process for applications where a minimum cycle time is required.","Heating the (often metallic) outer mould is both time consuming and requires large amounts of energy.","The use of embedded electrical heaters in a silicon rubber bladder creates a bladder system that is both fragile and difficult to extract from the cooled hollow composite structure or vessel, thereby limiting the geometry of applications.","Furthermore, at the higher processing temperatures required for certain thermoplastics, for example PA12 and PET, the life span of the often costly silicon rubber bladders is limited.","Previously disclosed inventions have claimed that in-situ extrusion blow moulding can form a bladder material that renders the thermoplastic composite matrix fluid, but where additional heating of the mould is required to ensure full melting.","Where the outer layers of the thermoplastic composite are not heated sufficiently above the melting temperature of the thermoplastic matrix, inadequate impregnation occurs, and hence an unfavourable set of mechanical properties results and surface quality is diminished.","Thermosetting materials have additional disadvantages compared with thermoplastic based materials, notably for higher rate production, whereby a limited temperature capability results, unsatisfactory finished product aesthetics, lack of extended durability, lack of appropriateness for recycling and manufacturing related issues such as downtime due to clean-up and material handling costs and sub-ambient storage.","Further, there are environmental concerns arising from worker exposure to vapour, overspray, emissions, etc., encountered during the fabrication process.","Some engineered thermoset resins improve material performance through higher temperature capacity, but unacceptable material costs are associated with them.","Thermoplastic based materials have overcome many of the above problems but the processing techniques available are limited in cycle time due to complex heating and cooling cycles, either of the mould or of expensive and fragile bladder materials and bladders based assemblies with a heating ability.","Additionally, high energy costs are associated with processes that require a cycling of the mould temperature above and below the matrix melting temperature or complex bladder based heating systems."],["<SOH> SUMMARY OF THE INVENTION <EOH>It is therefore the object of the present invention to provide an improved process for the fabrication of hollow composite parts and vessels.","This object is achieved by a process as outlined in claim 1 .","This non-isothermal process for fabricating hollow composite structures or vessels comprises the steps of: a) preforming of a thermoplastic liner; b) over-wrapping the thermoplastic liner with thermoplastic composite material; c) heating the over-wrapped liner in an oven above the melting temperature of the thermoplastic composite material; d) placing the heated over-wrapped liner into a mould that is maintained below the melting temperature of the thermoplastic composite material; e) applying an internal pressure inside the heated over-wrapped liner while the moulding tool is closed to consolidate the composite material and bladder against a geometry defined by the mould; f) maintaining the internal pressure inside the cooling over-wrapped liner until the thermoplastic composite material is sufficiently below the thermoplastic composite melting temperature; and g) removing the formed hollow composite structure or vessel from the mould.","Such an improved process enjoys advantages including, as opposed to prior art processes of fabricating hollow composite parts and vessels: reduced cycle times due to the method of assembling the fibre, matrix and bladder prior to insertion into the mould, reduced cycle times due to the method of assembling the fibre, matrix and bladder in the mould, a reduction in the number of manufacturing steps, faster changeover times, faster start-ups, potential labour saving due to less material handling, floor space reduction, adaptability to automation, simplification of material storage and handling, a safer environment for employees, lower training costs, recyclable scrap materials, reduced cycle times from the thermal conditions required for moulding, reduced energy usage from the thermal conditions required for moulding, better distributions of fibers and matrix in the composite, the ability to mould in metallic or polymeric based inserts for connection of additional items to the vessel, the ability to have an integral liner of the same or different polymer to the matrix material of the composite, the ability to tailor barrier properties at the inside of the vessel, the ability to tailor the reinforcing fibre content through the wall thickness of the hollow or vessel component, etc.","The resulting hollow composite parts or vessels can be used for many applications, as typical diameters of these vessels could be 25 cm or larger.","These applications include for example the use as containers for compressed air e.g.","for brake systems of cars and trucks, breathing apparatus, water storage, hollow automotive or aerospace space frame structures, and generally everywhere where lightweight is of advantage.","According to another preferential mode of the invention, after step a) before the over-wrapping phase inserts are positioned on the liner, and before step c) the over-wrapped composite is consolidated onto the positioned inserts.","Even more preferentially, it is possible to choose the inserts to have an operating temperature such that the critical dimensions are not distorted by the temperature of the heating phase during step c).","Additionally, the over-wrapped composite can be locally consolidated onto the positioned inserts.","According to a further preferential mode of the invention, during step b) one or more liners are placed on the same over-wrapping line to form a continuous process and/or in step e) a rapid coupling device is used for rapid connection of the internal pressure.","Additional modes of realization are described in the dependent claims.","Material forms that may be considered for this invention combine either or both discontinuous and continuous reinforcing fibres with a thermoplastic matrix material with different reinforcing fibre architectures including both random and different degrees of reinforcing fibre alignment.","Suitable arrangements of materials with a suitable mingling quality of reinforcement and matrix that may be used include tows or yarns, tapes, braids, weaves, knits, fabrics and other textile processes suitable for manipulating or combining reinforcing fibre and matrix.","Materials may be used in either or both an unconsolidated state, a partially consolidated state or a fully consolidated state where the material becomes flexible for the moulding process when the combination of matrix and reinforcing fibre material is heated using the principle of this invention to a forming temperature.","Suitable thermoplastic materials include: polypropylene, polyamide, in particular polyamide 12, polyethylene, cross-linked polyethylene, polybutylene terephthalate, polyethylene terephthalate and polyoxymethylene.","Suitable reinforcing fibres include: glass, carbon, Kevlar, ultra high molecular weight polyethylene, ultra high molecular weight polypropylene and steel.","Techniques for combining reinforcing fibres and thermoplastic matrix materials, in both continuous and discontinuous fibre lengths, which are applicable for use with the above process include: melt impregnation, film stacking, solvent impregnation, commingling, powder impregnating using both aqueous slurries and electrostatic methods of coating, powder impregnated fibres with an unmelted sheath around the powder impregnated bundle for textile processing, standard fabrics impregnated by powder to form a semifinished product, the utilization of existing textile processing techniques applied to the warp knitting of split polymer films with weft insertion of reinforcement fibres, weft-inserted multi-axial warp knitting of reinforcing fibres and thermoplastic matrix ribbons, co-weaving yarns of the two fibres into a form possessing good drape characteristics, using a polymer fibre woven around a bundle of reinforcement fibres in the plied matrix technique, reinforcing and matrix yarns inter-dispersed on line during fabric formation to produce drapable stitch bonded fabrics, heating of fibres impregnated with monomers that polymerize in-situ to form the composite, wire coating processes adapted to coating reinforcing fibres or bundles of fibres with matrix, pultrusion of fibres and matrix materials together and any other such process that achieves the required combination of fibre and matrix in a form between fully consolidated and unconsolidated forms.","Commingled materials, which are a preferred material for this moulding process, can be formed either directly as described in U.S. Pat.","No.","5,011,523 and U.S. Pat.","No.","5,316,561 where the reinforcing fibers are drawn from the molten state through a die where the reinforcing glass fibers are combined with a drawing head where the thermoplastic matrix is extruded and drawn mechanically onto the glass fibres or indirectly by separating fibre and matrix fibre yarns (U.S. Pat.","No.","5,241,731) into open ribbons where the matrix fibres are electrically charged and then drawn into a flat open bundle via a ribboning bar to the commingling bar where the Carbon fibre yarn is opened with an air curtain into an open flat ribbon that is then combined with the PEEK ribbon at the commingling bar or in the example shown herein of the material used to illustrate this invention where stretch broken Carbon fibres (80 mm average length) are blended with polyamide 12 (PA12) staple fibres using a textile spinning technique (U.S. Pat.","No.","4,825,635) with the addition of a wrapping filament (U.S. Pat.","No.","5,910,361)."],["FIELD OF THE INVENTION This invention relates to the field of fabricating hollow composite parts, especially space frame structures or vessels, and more particularly to improved methods for fabricating space frame structures or vessels and to composite vessels made in accordance with the improved methods.","BACKGROUND OF THE INVENTION Hollow composite structures, or pressure vessels, here referred to as ‘vessels’, such as used to store fluids and solids, particularly under pressure, such as pressurized gas tanks, or more generally in space frame or tubular, load bearing, assemblies have traditionally been fabricated from metals such as steel or aluminium.","However, in recent years, the use of composite vessels has become more prevalent.","Such vessels are manufactured by a variety of processes, which include filament winding, resin transfer moulding and bladder-assisted moulding.","The technology of filament winding is the process of impregnating dry reinforcing fibres, such as fibreglass or carbon strands, with an activated resin prior to application to a mandrel.","Preimpregnated materials may also be used.","The mixture of reinforcing fibres and matrix is then applied to the mandrel.","In the case of thermoset based matrices, a cure cycle follows, while for thermoplastic matrices the polymer is softened (melted) before application to the mandrel where fusion bonding then occurs between layers of the wound material that forms the structure.","Either the cure cycle with thermosets or cooling of the material as it is wound with thermoplastics produces a hollow structure or shell that is then removed from the mandrel to give the component.","To produce hollow structures or vessels, the technology of resin transfer moulding uses a mandrel and an outer mould, where a mixture of activated matrix resin is injected in the cavity between the mandrel and the outer mould where reinforcing fibres of typically glass, carbon or Kevlar have been placed.","Impregnation of the reinforcing fibres then occurs, with cross-linking of thermoset polymers or polymerisation of thermoplastic monomer forming the matrix polymer and the finished shell-like or hollow component.","The mandrel could then be removed.","The technology of bladder-assisted moulding, for the case of thermoplastic matrices, uses an outer mould and inner bladder to define the shape of the part.","To melt the thermoplastic matrix and hence impregnate the reinforcing fibres, of typically glass, carbon or Kevlar, the thermoplastic is heated above its melting temperature and a pressure is then applied to the bladder to consolidate the material.","The thermoplastic material must then be cooled, while under pressure, to form the hard shell-like or hollow composite component.","Bladder materials comprising rubber-like materials, typically silicon rubber, are used to apply the compaction force, whereby the bladder stretches or elongates under the influence of the applied internal pressure.","Alternate bladders consist of in-situ extrusion blow moulded polymeric materials.","The thermoplastic matrix is typically heated by heating the outer mould, often metallic, above the matrix melting temperature before the pressure is applied for the required period, after which the mould is cooled sufficiently below the matrix melting temperature for solidification and crystallisation to occur.","Alternately, additional or separate heating may be applied by passing hot oil through the bladder or by using embedded electric heaters in the bladder, to heat the composite from the inside, thus eliminating the need to cycle the mould temperature.","Conductive reinforcing materials, such as carbon fibre based composites or composites containing a proportion of conductive fibres such as carbon or steel, may be heated in the mould through induction heating or electrical resistance heating.","In-situ extrusion blow moulding has been previously claimed to provide heat to aid the melting of the thermoplastic matrix composite, but in practice additional temperature cycling of the mould above and below the matrix melting temperature is also needed.","The technology of bladder-assisted moulding, for the case of thermoset based matrices, uses an outer mould and inner bladder to define the shape of the part.","A preimpregnated mixture of reinforcing fibres and activated matrix are placed over the bladder and the bladder with the surrounding preimpregnated material is placed into the outer mould.","The mould temperature must be high enough to induce cure of the thermosetting matrix and after cure, cooled to allow part release.","The bladder may also be heated, either through the passage of a hot fluid, or by embedded electrical heaters, to apply the heat necessary for cure of the impregnated material.","The technology also consists of bladder-assisted resin transfer moulding where reinforcement substantially without matrix is placed over the bladder and the bladder coated with reinforcement is then placed into the mould.","Activated resin is then applied that impregnates the reinforcing fibres with the application of internal pressure via the bladder, thereby aiding impregnation.","Such techniques for fabricating hollow structures or vessels, particularly if a rapid cycle time is required (which is the case for many applications of hollow composite structures or vessels as it reduces the cost of the component) have well known drawbacks which include: for thermoplastic filament winding based processes, the thermoplastic matrix based reinforcement must be heated prior to the application to a mandrel to ensure the thermal conditions necessary for fusion bonding.","Hence high processing speeds, notably the application of the thermoplastic matrix based reinforcement to the mandrel, creates a limit on the speed of the process and hence the cycle time.","High drag or friction forces induced by the placement of thermoplastic matrix based reinforcement create further quality problems whereby segregation of the thermoplastic matrix and reinforcing fibres may occur, creating an unfavourable microstructure.","Additionally, the geometry of the component is limited where there is a need to remove the mandrel after processing.","Where the mandrel remains in the component after processing, the mandrel must be compatible with the thermoplastic matrix based reinforcement and be able to sustain the high radial crush forces induced by hot fibre and matrix mixtures applied under high tension forces.","The sequential application of composite layers to the mandrel is known to create high internal stresses in the component, affecting dimensional stability and the eventual durability of the component.","The filament winding process also places restrictions on geometries that can be produced, where concave curvature requires in-situ bonding of the thermoplastic based composite material to the mandrel or preceding layer of composite; for thermoset based filament winding processes, the thermoset matrix must be cured onto the mandrel requiring a heating cycle either directly as the thermoset matrix based reinforcement is placed, or subsequent heating of the placed material and the mandrel.","The process cycle time depends on the winding speed and hence this creates a limit on the speed of the process and thereby the cycle time.","High drag or friction forces induced by the placement of thermoset matrix based reinforcement create further quality problems whereby segregation of the thermosetting matrix and reinforcing fibres may occur, creating an unfavourable microstructure.","Additionally, the geometry of the component is limited where there is a need to remove the mandrel after processing.","Where the mandrel remains in the component after processing, the mandrel must be compatible with the thermoset matrix based reinforcement and be able to sustaining the high radial crush forces induced by the fibre and matrix mixtures applied under high tension forces; for the resin transfer moulding based process, as used with thermoset based reinforced material, a preform must be assembled over the mandrel and resin injected between the mould and the mandrel.","The cycle time is limited by the need to inject the resin into this space, where pressures are limited where a permanent hollow mandrel is used such that the pressure of the incoming fluid, here the activated resin, does not distort the geometry of the mandrel, hence limiting processes to lower rate and pressure injection rather than the shorter cycle time, high pressure process of structural reaction injection moulding.","Furthermore, the cure reaction of the thermoset matrix either limits the cycle time or, where tailored to occur at a higher speed, induces high stresses in the composite.","Unless complex and expensive preforms of reinforcement are prefabricated, parts of a constant wall thickness must be produced unless a local decrease in the reinforcing fibre fraction can be tolerated.","Where the mandrel needs to be removed, expensive technologies, such as low melting temperature alloys, must be used where part geometries are not to be impaired.","for the resin transfer moulding based process, for the case of thermoplastic based reinforced material, a preform must be assembled over the mandrel and activated monomer injected between the mould and the mandrel.","The cycle time is limited by the need to inject the resin into this space, where pressures are limited where a permanent hollow mandrel is used such that the pressure of the incoming fluid, here the activated resin, does not distort the geometry of the mandrel, hence limiting processes to lower rate and pressure injection rather than the shorter cycle time, high pressure process of structural reaction injection moulding.","Furthermore, the polymerisation reaction of the monomer either limits the cycle time or, where tailored to occur at a higher speed, places limitations on bladder materials.","Thermal cycling above the monomer melting temperature for polymerisation to occur most rapidly and cooling after polymerisation to below the polymer melting temperature are also often required, requiring cycling of the mould temperature and/or mandrel temperatures for certain thermoplastic resin transfer moulding material systems.","Unless complex and expensive preforms of reinforcement are prefabricated, parts of a constant wall thickness must be produced unless a local decrease in the reinforcing fibre fraction can be tolerated.","Where the mandrel needs to be removed, expensive technologies, such as low melting temperature alloys, must be used where part geometries are not to be impaired.","for the bladder assisted moulding process, for the case of thermoset based reinforced material, the need to cure the thermoset matrix material creates problems with chemical attack with rubber-like bladder materials, typically styrene with silicon rubbers, and hence bladder life is reduced.","Bladder materials are generally removable due to the difficulty of achieving a bladder that is flexible enough to enable compaction of the impregnated reinforcing fibres prior and during cure while forming a reliable bond between the bladder and cured composite with the lack of high internal stresses in the finished component.","Heating of the impregnated reinforcement is also required that is accomplished either through cycling of the mould temperature or heating the bladder material.","Cycling the mould temperature is energy inefficient and time consuming, limiting the potential to reduce cycle times, and the fabrication of heated bladder systems is uneconomic for components made in large quantities while also being fragile and of limited life span.","Additional internal inspection is also required in the case of removable bladders to ensure that small particles of the bladder material do not remain on the inside wall of the hollow composite or vessel and that the bladder integrity has been maintained such that it is safely usable for further moulding trials.","for the bladder assisted moulding process, for the case of thermoplastic based reinforced material, the thermoplastic composite must be heated above the melt temperature of the matrix and cooled below the melt temperature before demoulding.","This occurs either by heating the outer mould temperature above the matrix melting temperature and then cooling the mould below the matrix melting temperature, whereby the thermoplastic matrix composite is also heated above and below the thermoplastic composite matrix melting temperature during which time a compaction force is applied by the bladder, or by heating the bladder material such that the thermoplastic matrix is heated sufficiently above the melting temperature of the matrix for adequate impregnation to occur, followed by cooling of the composite structure sufficiently below the matrix melting temperature for stable component release from the mould.","The currently available techniques outlined above have inherent limitations that reduce the potential of the process for applications where a minimum cycle time is required.","Heating the (often metallic) outer mould is both time consuming and requires large amounts of energy.","The use of embedded electrical heaters in a silicon rubber bladder creates a bladder system that is both fragile and difficult to extract from the cooled hollow composite structure or vessel, thereby limiting the geometry of applications.","Furthermore, at the higher processing temperatures required for certain thermoplastics, for example PA12 and PET, the life span of the often costly silicon rubber bladders is limited.","Previously disclosed inventions have claimed that in-situ extrusion blow moulding can form a bladder material that renders the thermoplastic composite matrix fluid, but where additional heating of the mould is required to ensure full melting.","Where the outer layers of the thermoplastic composite are not heated sufficiently above the melting temperature of the thermoplastic matrix, inadequate impregnation occurs, and hence an unfavourable set of mechanical properties results and surface quality is diminished.","Thermosetting materials have additional disadvantages compared with thermoplastic based materials, notably for higher rate production, whereby a limited temperature capability results, unsatisfactory finished product aesthetics, lack of extended durability, lack of appropriateness for recycling and manufacturing related issues such as downtime due to clean-up and material handling costs and sub-ambient storage.","Further, there are environmental concerns arising from worker exposure to vapour, overspray, emissions, etc., encountered during the fabrication process.","Some engineered thermoset resins improve material performance through higher temperature capacity, but unacceptable material costs are associated with them.","Thermoplastic based materials have overcome many of the above problems but the processing techniques available are limited in cycle time due to complex heating and cooling cycles, either of the mould or of expensive and fragile bladder materials and bladders based assemblies with a heating ability.","Additionally, high energy costs are associated with processes that require a cycling of the mould temperature above and below the matrix melting temperature or complex bladder based heating systems.","SUMMARY OF THE INVENTION It is therefore the object of the present invention to provide an improved process for the fabrication of hollow composite parts and vessels.","This object is achieved by a process as outlined in claim 1.This non-isothermal process for fabricating hollow composite structures or vessels comprises the steps of: a) preforming of a thermoplastic liner; b) over-wrapping the thermoplastic liner with thermoplastic composite material; c) heating the over-wrapped liner in an oven above the melting temperature of the thermoplastic composite material; d) placing the heated over-wrapped liner into a mould that is maintained below the melting temperature of the thermoplastic composite material; e) applying an internal pressure inside the heated over-wrapped liner while the moulding tool is closed to consolidate the composite material and bladder against a geometry defined by the mould; f) maintaining the internal pressure inside the cooling over-wrapped liner until the thermoplastic composite material is sufficiently below the thermoplastic composite melting temperature; and g) removing the formed hollow composite structure or vessel from the mould.","Such an improved process enjoys advantages including, as opposed to prior art processes of fabricating hollow composite parts and vessels: reduced cycle times due to the method of assembling the fibre, matrix and bladder prior to insertion into the mould, reduced cycle times due to the method of assembling the fibre, matrix and bladder in the mould, a reduction in the number of manufacturing steps, faster changeover times, faster start-ups, potential labour saving due to less material handling, floor space reduction, adaptability to automation, simplification of material storage and handling, a safer environment for employees, lower training costs, recyclable scrap materials, reduced cycle times from the thermal conditions required for moulding, reduced energy usage from the thermal conditions required for moulding, better distributions of fibers and matrix in the composite, the ability to mould in metallic or polymeric based inserts for connection of additional items to the vessel, the ability to have an integral liner of the same or different polymer to the matrix material of the composite, the ability to tailor barrier properties at the inside of the vessel, the ability to tailor the reinforcing fibre content through the wall thickness of the hollow or vessel component, etc.","The resulting hollow composite parts or vessels can be used for many applications, as typical diameters of these vessels could be 25 cm or larger.","These applications include for example the use as containers for compressed air e.g.","for brake systems of cars and trucks, breathing apparatus, water storage, hollow automotive or aerospace space frame structures, and generally everywhere where lightweight is of advantage.","According to another preferential mode of the invention, after step a) before the over-wrapping phase inserts are positioned on the liner, and before step c) the over-wrapped composite is consolidated onto the positioned inserts.","Even more preferentially, it is possible to choose the inserts to have an operating temperature such that the critical dimensions are not distorted by the temperature of the heating phase during step c).","Additionally, the over-wrapped composite can be locally consolidated onto the positioned inserts.","According to a further preferential mode of the invention, during step b) one or more liners are placed on the same over-wrapping line to form a continuous process and/or in step e) a rapid coupling device is used for rapid connection of the internal pressure.","Additional modes of realization are described in the dependent claims.","Material forms that may be considered for this invention combine either or both discontinuous and continuous reinforcing fibres with a thermoplastic matrix material with different reinforcing fibre architectures including both random and different degrees of reinforcing fibre alignment.","Suitable arrangements of materials with a suitable mingling quality of reinforcement and matrix that may be used include tows or yarns, tapes, braids, weaves, knits, fabrics and other textile processes suitable for manipulating or combining reinforcing fibre and matrix.","Materials may be used in either or both an unconsolidated state, a partially consolidated state or a fully consolidated state where the material becomes flexible for the moulding process when the combination of matrix and reinforcing fibre material is heated using the principle of this invention to a forming temperature.","Suitable thermoplastic materials include: polypropylene, polyamide, in particular polyamide 12, polyethylene, cross-linked polyethylene, polybutylene terephthalate, polyethylene terephthalate and polyoxymethylene.","Suitable reinforcing fibres include: glass, carbon, Kevlar, ultra high molecular weight polyethylene, ultra high molecular weight polypropylene and steel.","Techniques for combining reinforcing fibres and thermoplastic matrix materials, in both continuous and discontinuous fibre lengths, which are applicable for use with the above process include: melt impregnation, film stacking, solvent impregnation, commingling, powder impregnating using both aqueous slurries and electrostatic methods of coating, powder impregnated fibres with an unmelted sheath around the powder impregnated bundle for textile processing, standard fabrics impregnated by powder to form a semifinished product, the utilization of existing textile processing techniques applied to the warp knitting of split polymer films with weft insertion of reinforcement fibres, weft-inserted multi-axial warp knitting of reinforcing fibres and thermoplastic matrix ribbons, co-weaving yarns of the two fibres into a form possessing good drape characteristics, using a polymer fibre woven around a bundle of reinforcement fibres in the plied matrix technique, reinforcing and matrix yarns inter-dispersed on line during fabric formation to produce drapable stitch bonded fabrics, heating of fibres impregnated with monomers that polymerize in-situ to form the composite, wire coating processes adapted to coating reinforcing fibres or bundles of fibres with matrix, pultrusion of fibres and matrix materials together and any other such process that achieves the required combination of fibre and matrix in a form between fully consolidated and unconsolidated forms.","Commingled materials, which are a preferred material for this moulding process, can be formed either directly as described in U.S. Pat.","No.","5,011,523 and U.S. Pat.","No.","5,316,561 where the reinforcing fibers are drawn from the molten state through a die where the reinforcing glass fibers are combined with a drawing head where the thermoplastic matrix is extruded and drawn mechanically onto the glass fibres or indirectly by separating fibre and matrix fibre yarns (U.S. Pat.","No.","5,241,731) into open ribbons where the matrix fibres are electrically charged and then drawn into a flat open bundle via a ribboning bar to the commingling bar where the Carbon fibre yarn is opened with an air curtain into an open flat ribbon that is then combined with the PEEK ribbon at the commingling bar or in the example shown herein of the material used to illustrate this invention where stretch broken Carbon fibres (80 mm average length) are blended with polyamide 12 (PA12) staple fibres using a textile spinning technique (U.S. Pat.","No.","4,825,635) with the addition of a wrapping filament (U.S. Pat.","No.","5,910,361).","BRIEF DESCRIPTION OF THE DRAWINGS The invention, both as to organisation and method of operation, may be best understood by reference to the following description taken in conjunction with the claims and the accompanying drawings of which: FIG.","1 Pictorial view of a liner/mandrel employed in practising a first inventive embodiment; FIG.","2 Cross-sectional view taken along the lines A-A of FIG.","1 with optional inserts; FIG.","3 Enlarged cross-sectional view of FIG.","2 showing an optional incorporated insert; FIG.","4 A pictorial view of the liner or mandrel being over-wrapped with a mixture of thermoplastic resin and reinforcing material; FIG.","5 A pictorial view of a continuous process where the liner or mandrel are being over-wrapped with a mixture of thermoplastic resin and reinforcing material; FIG.","6 A pictorial view of a device for consolidating the over-wrapped composite onto inserts for attaching exterior features; FIG.","7 A pictorial view of a device for separating the over-wrapped inserts with composite consolidated in place over the inserts; FIG.","8 A cross-sectional view of the liner over-wrapped with composite with an insert in place including an enlarged fragmentary cross-sectional view; FIG.","9 Pictorial view of the liner after being over-laid with the layer(s) of composite; FIG.","10 A cross-sectional sequential view of the conventional isothermal processing sequence used to produce hollow composite structures or vessels; FIG.","11 A cross-sectional sequential view of the non-isothermal processing sequence employed in practising a first inventive embodiment; FIG.","12 A cross-sectional view of the over-wrapped and preheated liner located in the mould just prior to closure of the mould; FIG.","13 A cross-sectional view of the over-wrapped and preheated liner located in the mould during internal pressurization such that the over-wrapped liner is formed into the shape defined by the inner surface of the mould; FIG.","14 A cross-sectional sequential view illustrating temperature gradients, including four non-exclusive possibilities, through the thickness of the liner and over-wrapped composite for retaining integrity of the bladder and maximising surface consolidation of the over-wrapped composite, including an enlarged cross-sectional view and corresponding temperature (T) distance (d) graph.","FIG.","15 A cross-sectional sequential view of a high speed connection device to attach a pressurization source; FIG.","16 Temperature and pressure histories of a typical moulding cycle; FIG.","17 A cross-sectional view of the completed hollow composite structure or pressure vessel prepared by practising the first and second inventive embodiments.","DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The first embodiment uses a hollow thermoplastic liner or bladder, produced by processes such as extrusion blow moulding or cast rotational moulding, such as shown in FIG.","1.In the example shown the liner 1 or bladder is generally an elongated structure of geometry such as can be produced via extrusion blow moulding, terminating with either one or more openings, located either on the central axis of the component or at any other suitable location.","Hence bladder geometries are not restricted to shapes of revolution, but are applicable to any hollow thermoplastic shape that can be made by processes such as extrusion blow moulding or cast rotational moulding, processes which are well known in the art.","In addition to using a thermoplastic liner 1 or bladder, short fibre reinforced or discontinuous fibre reinforced thermoplastic materials may also be used such that the melt viscosity or melt strength of the polymer and fibre mixture is increased both during the extrusion blow moulding process and during the preheat, transfer and moulding stages for the non-isothermal bladder inflation moulding process.","The addition of short glass fibre also increases the mechanical properties of the liner or bladder such that it becomes part of the load bearing material in a hollow composite structure or vessel.","Over the hollow liner or bladder, metallic, ceramic or polymeric inserts 2 can optionally be added before the over-wrapping or over-braiding of thermoplastic matrix reinforced material.","The inner insert 2 must have an operating temperature such that the critical dimensions, for example thread geometries, are not distorted by heat during the final non-isothermal moulding process.","Such inserts can be made via a variety of processes well known in the art including injection moulding, compression moulding, casting and machining.","Alternatively, inserts may be placed into the extrusion blow moulding tool and be directly over moulded during the process of forming the bladder, relying either on mechanical interlocking with non-compatible insert materials and/or fusion bonding with compatible insert materials.","Inserts 2 located on the liner or bladder are shown in FIGS.","2 and 3.The bladder 1 with positioned inserts 2, held either via a locating device on the liner or bladder or by an external locating fixture, is used as a mandrel for an over-wrapping or over-braiding procedure whereby reinforcing fibres, such as the ones mentioned above, intimately mixed with a thermoplastic resin are placed over the liner or bladder, as shown in FIG.","4.Suitable processes for over-wrapping or over-braiding are textile based processes where yarns or tows 3-6 of reinforced thermoplastic matrix based material are wound around the liner or mandrel to provide an even coverage of material over the liner or bladder surface, including processes such as: braiding, either cold or with the addition of heating, tailored braiding where the fibre orientation is controlled across the geometry of the liner or bladder, 3-D braiding processes where interlocking occurs between layers of the applied reinforced thermoplastic matrix based material, over-wrapping of woven tapes or fabrics of reinforced thermoplastic matrix based materials such that a greater area of coverage occurs decreasing the cycle time and optimising system economics and filament winding, either cold or with the addition of heating.","Pressure is not directly applied during the over-wrapping or over-braiding process, thereby increasing winding speeds and decreasing cycle times.","In addition to the over-wrapping or over-braiding of mixtures of thermoplastic matrix and reinforcing fibres, a final layer of material can be added, depending on the application, consisting of either a higher matrix fraction in the thermoplastic and reinforcing fibre mixture or solely of thermoplastic matrix fibres or wrapping films, including extrudates, to provide a thermoplastic matrix rich surface to both improve surface quality, sealing the reinforcing fibres in the component, to give a surface that is more readily painted or coated with additional materials or processes after moulding and to ensure full surface consolidation.","This higher matrix fraction can be achieved with either the same matrix polymer as the matrix for the reinforced material or with a compatible but different polymer to optimise barrier and surface performance properties.","One or more liners or bladders, with inserts attached, may optionally be placed on the same over-wrapping or braiding line such as to form a continuous over-braiding or over-wrapping process, as shown in FIG.","5.To locate the inserts onto the liner or bladder, where the inserts are not directly incorporated, and to ensure that good contact and final consolidation occurs between the insert, liner or bladder and the reinforced thermoplastic matrix based material, where either over-moulded or located inserts are used, the over wrapped or over braided material is optionally consolidated onto the insert, as shown in FIG.","6.A hot clamping device 7 is used that rapidly heats the reinforced thermoplastic matrix based material above the matrix melt temperature and applies a force 9 to impregnate the reinforcing fibres with the thermoplastic matrix, followed by cooling of the clamping device 7 while clamping pressure 9 is maintained such that consolidation pressure is applied while the reinforced thermoplastic matrix based material cools.","Alternatively, a heating source 8 is used to rapidly heat the reinforced thermoplastic matrix based material above the matrix melt temperature followed by a clamping device 7, maintained at a temperature below the melting point of the polymer, that applies consolidation pressure while the reinforced thermoplastic matrix based material cools.","Either built into or separate from the heating and cooling device that consolidates the over wrapped or over braided material onto the insert, a cutting device 10 separates the preform assembly of liner or bladder 1, reinforced thermoplastic matrix based material, and inserts 2 from the adjacent preform assembly, where reinforced thermoplastic matrix based material crosses over between liners or bladders where a continuous process is used, as shown in FIG.","7.Cutting can either occur on consolidated and rigid reinforced thermoplastic matrix based material or on the placed reinforced thermoplastic matrix based material that has not been fully consolidated.","As a further step to reduce cycle times during the eventual non-isothermal bladder inflation moulding process, heat may be applied during any of the over-wrapping or over-braiding processes such that the matrix in the reinforced thermoplastic matrix based material is heated above the melting temperature of the matrix enabling spontaneous wetting of the reinforcement via capillary action and partial impregnation via the tension forces induced in the over-wrapping or over-braiding material as it is placed over the liner or bladder.","As such a partially impregnated material results with a lower bulk than material which has not been partially impregnated, resulting in a material that has less trapped gas during the heating and consolidation phases of the non-isothermal bladder inflation moulding process.","Alternatively, the assembled preform of liner or bladder, over-wrapped or over braided reinforced thermoplastic matrix based material and inserts may be flash heated using a high flux infra red heating or similar heating source to melt the over-wrapped or over-braided reinforced thermoplastic matrix based material without affecting the integrity of the liner or bladder, thereby providing a preform with increased stability for any transport to a different location where necessary before the final moulding operation.","A liner 1 or bladder 12 over-wrapped with composite material 11 is thereby obtained, with the optional location of inserts 2 for attaching external features to the hollow composite part or vessel, as shown in FIGS.","8 and 9.Processes according to the state of the art have completed the final consolidation of thermoplastic composite materials using either rubber-like bladders or with in-situ extruded thermoplastic bladder material.","Such materials are heated to bring the thermoplastic composite matrix above its melting temperature, either through latent heat of a directly produced extrudate, cycling of the mould temperature above and below the matrix melting temperature, or via heating of the bladder internally via hot fluid circulation or embedded electrical elements.","Such processes either produce components of limited quality, in the case of heating via the latent heated of extruded polymeric material, restrict geometries and have limited bladder life for embedded heaters in rubber-like bladders, or require complex internal heating circuits.","Where the mould temperature is cycled, an isothermal process results, such as shown in FIG.","10.The liner 1 and over-wrapped composite 11 (FIG.","10a) are placed into a mould 13 below the melting temperature of the liner 1 or bladder and/or over-wrapped composite 11 (FIG.","10b).","The mould is then held closed while rapid heating 15 is applied to heat the mould above the melting temperature of the liner 1 or bladder and/or over-wrapped composite 11 (FIG.","10c).","After the required impregnation time, the rapid heating source 15 is replaced by a rapid cooling source 18, while the mould remains closed 14, to reduce the temperature below the melting temperature of the liner 1 or bladder and/or over-wrapped composite 11 (FIG.","10d).","After the required consolidation and cooling time, the mould 13 is opened and the part obtained (FIG.","10e).","The proposed process is a non-isothermal bladder inflation process that does not require cycling of the mould temperature or heating of the bladder internally whilst in the closed mould.","To melt the thermoplastic matrix and hence reduce the viscosity, enabling impregnation of the reinforcing fibres, the assembly of a thermoplastic liner 1 or bladder and over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres 11 (FIG.","11a) are heated in an oven 15 outside of the closed final moulding tool 13 (FIG.","11b).","After the heating cycle, the over-wrapped liner is located in the moulding tool 13 (FIG.","11c), FIG.","12), where the moulding tool 13 is closed and an internal pressure 21 applied inside the liner 1 or bladder, so as to force the over-wrapped liner or bladder to conform to the shape of the mould geometry (FIG.","13).","The moulding tool 13 is maintained below the melting temperature of the liner or bladder and/or over-wrapped composite 11, while the internal pressure 21 is maintained for the time required to consolidate and cool the hollow composite part or vessel.","The mould is then opened and the part removed (FIG.","11d).","During the heating cycle, the oven parameters are adjusted such that the outer over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres are heated sufficiently above the matrix melting temperature for good consolidation to occur and a good surface finish to result from the final moulding process, while not reaching excessive temperatures where degradation of the matrix occurs.","For an example of a polyamide 12 matrix in the reinforcing material, an upper temperature is chosen such that degradation of the polymer does not initiate to an extent such that the short term or long term mechanical properties are not adversely affected, odour problems do not occur beyond acceptable limits and surface quality is not unduly influenced, which is typically around 280° C. Also during the heating process, the oven parameters are adjusted such that the inner liner or bladder temperature is optimised to be extendable under the applied internal pressure 21 during the final moulding cycle, thereby enabling the internal pressure 21 in the liner 1 or bladder to be transferred to the hot over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres giving the driving force for concurrent consolidation and cooling in the moulding tool 20.Additionally, the bladder temperature is restricted such that the viscosity of the bladder matrix does not become too low such that the liner or bladder looses integrity, excessive diffusion of the pressurizing fluid is permitted and the liner or bladder is thereby unable to resist the application of an internal pressure during the final moulding cycle.","A suitable temperature, for an example of the present embodiments using polyamide 12, consists of heating the bladder temperature to 220° C. Preferred materials for liners or bladders are high molecular weight grades of the same polymer as used in the matrix of the over-wrapped or over-braided mixtures of thermoplastic and reinforcing fibres, where a higher molecular weight results in higher melt viscosities, thereby maintaining the integrity of the liner or bladder material to a higher temperature.","Other preferred materials are thermoplastic polymers that have been partially cross-linked to give rubbery behaviour above the melt temperature with a delayed loss of integrity, such as cross-linked polyethylene.","A temperature gradient therefore results during the heating cycle of the assembled liner or bladder with over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres, such that the outer layers of over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres are heated to around 280° C., for the example of a polyamide 12 matrix, and the inner liner or bladder material is heated to only around 220° C. Such temperature profiles, as described above for the over-wrapped composite and liner or bladder materials, are illustrated in FIG.","14, where four different non-exclusive heating profiles are shown.","Suitable oven systems include: hot gas ovens, forced convection hot gas ovens, infra-red ovens, and combinations of infra-red and hot gas or forced hot gas ovens, using either electrical or gas powered heating elements or emitters.","A preferred oven combination for this heating cycle is a combined forced hot gas and infra red oven, where the preset and controlled temperature of the forced hot gas penetrates to the inner liner or bladder and heats the liner or bladder to the required temperature, followed by infra red heating that quickly heats the outer layers of over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres while not penetrating to the inner liner or bladder, whereby the flux level from the infra red emitters are tailored to give the desired temperature gradient through the material.","During the heating process, the assembled liner or bladder and over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres are maintained to the desired geometry using a frame that clamps the assembled preform.","This clamp system is then transferred after the heating cycle to the moulding tool 20, where the frame fits integrally with the moulding tool to locate the preheated assembly rapidly in the correct location inside the moulding tool.","A fast transfer from the oven to the moulding tool 20 is preferable to avoid cooling of the preheated assembly of liner 1 or bladder and over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres.","Typically the internal pressure 21 must be applied to the liner 1 or bladder and the mould 20 held closed within 20 seconds of the finish of the preheat cycle, and faster times improve part quality notably, both in terms of reducing void contents, hence increasing mechanical properties, and improving the surface quality or degree of surface impregnation.","Additionally, a low internal pressure of up to 2 bar can be applied to the inside of the liner or mandrel during the heating cycle, to help maintain the shape of the bladder or liner against gravitational forces.","Where the preferred method of assembling preforms with previously incorporated inserts and locally preconsolidated, at the insert region, over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres is used, the following mechanism is a preferred method of holding the over-wrapped or over braiding liner 1 or mandrel during the heating, transfer and moulding processes: a carrier frame, which is designed to interface with the mould, consists of side members to rigidly connect the end devices, which are used to hold the inserts and connect the internal pressure system.","Spring loaded split blocks are clamped over the inserts 2 such that they maintain the shape of the over-wrapped or over-braided liner 1 or bladder during the heating process, and these blocks are transferred with the heated material into the mould.","Also as part of the end device assembly, a cone 23 with an axial hole is used such that when pushed into the liner, a seal is created against the polymeric material, as shown in FIG.","15.The cone and deformed bladder interface with a block containing a profiled radius on an internal axial hole such that the hole restrains the movement of the cone thereby ensuring that a seal is made.","The cone is pushed into the deformable liner or bladder (FIG.","15c) as the internal pressure system is connected in the moulding tool by an integral pneumatic cylinder, actuated by initiation of the internal pressure, coinciding with closure of the mould, such that cooling of the assembled liner or bladder with over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres is minimised before the internal pressure 21 is applied.","After the moulding cycle is completed, the pneumatic cylinder is reversed and the cone 23 removed to enable release of the part.","The polymeric liner or bladder material that protrudes from the finished component, typically 25 mm by 15 mm diameter, is then trimmed and can be recycled in the production of liners where required.","The preheated assembly of liner or bladder with over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres is transported to a mould that is generally composed of two main pieces, with movable cores where appropriate to create detail and concave features, where the assembly is held between the two principle mould halves such that contact of both mould halves occurs substantially simultaneously, thereby ensuring substantially balanced heat transfer and cooling from the hot liner or bladder and over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres with the mould, which is maintained sufficiently below the matrix melting temperature to ensure a fast cooling rate and a reduced cycle time, but sufficiently above the glass transition temperature to achieve the desired surface impregnation, consolidation and hence surface quality (FIG.","12).","Internal pressure 21 is then applied to the liner 1 or bladder such that the liner 1 or bladder deforms against the over-braided mixtures of thermoplastic matrix and reinforcing fibres, impregnating and consolidating the over-braided mixtures of thermoplastic matrix and reinforcing fibres against the geometry of the mould wall where concurrent cooling of the assembled liner 1 or bladder and over-braided mixtures of thermoplastic matrix and reinforcing fibres occurs against the mould 20, as shown in FIG.","13, where the temperature of the mould is maintained using a heating circuit and also a cooling circuit, where needed, as commonly used with other thermoplastic composite non-isothermal moulding processes.","The internal pressure 21 in the liner 1 or bladder is maintained while the mould 20 is closed via a hydraulic or mechanical clamping device.","Typical internal pressures using an over-wrapping or over-braiding of carbon fiber reinforced polyamide 12 are 10 bar, with the pressure depending on the degree of intimacy achieved between the reinforcing fibre and matrix polymer.","Such low moulding pressures reduce the size and expense of both the hydraulic or mechanical mould clamping equipment and the mould itself.","The internal pressure is maintained for a time period that ensures a good consolidation of the over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres, typically between 30 seconds and 180 seconds depending on the final void content required for the application and the wall thickness of the component.","The internal pressure 21 is then released and the mould 20 opened to give the hollow composite part or vessel.","FIG.","16 summarises the principles of the temperature and pressure histories of the disclosed invention, which define the process as non-isothermal.","The solid line indicates the pressure inside the liner, the dashed lines indicate the outer composite temperature (short dashes) and the liner temperature (long dashes).","Where the preferred method of assembling and preparing the liner 1 or mandrel with over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres with integral inserts 2 has been used, a net shape finished component results (FIG.","17) with only minor finishing operations such as removing polymeric flash from the tool interface area, thereby avoiding the high additional cost of machining or trimming operations.","In addition, the fibre reinforcement is sealed in the polymeric matrix and the lack of additional machining or trimming processes ensures good environmental resistance.","The hollow composite parts or vessels can be used for many applications, as typical diameters of these vessels could be 25 cm or larger.","These applications include for example the use as containers for compressed air e.g.","for brake systems of cars and trucks, breathing apparatus, water storage, hollow automotive or aerospace space frame structures, and generally everywhere where lightweight is of advantage.","List of Signs and Reference Numerals 1 liner 2 insert 3 fabrics 4 knits 5 tape 6 yarns/tows 7 forming clamps 8 heat 9 pressure 10 separating device 11 over-wrapped composite 12 liner over-wrapped with composite material with integral inserts 13 cool mould 14 clamping force 15 rapid heat source 16 pressurizing fluid 17 hot mould 18 rapid cooling source 19 cool mould 20 moulding tool 21 internal pressure 22 liner, over-wrapped composite not shown 23 conical or profiled plunger activated by 16 24 pre-heat phase of composite over-wrapped liner 25 transfer to mould 26 application of internal pressure to liner, impregnation, formation to mould geometry, consolidation, cooling 27 consolidated composite TMOLD mould temperature, less than melting temperature of thermoplastic liner and thermoplastic composite matrix Tm melting temperature of thermoplastic liner and/or thermoplastic composite matrix Tdeg degradation onset temperature"]],"string":"[\n [\n \"Non-isothermal method for fabricating hollow composite parts\",\n \"A process for making hollow composite structures or vessels which includes the steps of: A) heating a mixture of thermoplastic matrix and reinforcing fibres wrapped over a rigid or semi-rigid thermoplastic liner or bladder above the melting point of the thermoplastic composite matrix outside of a moulding tool; B) transferring the heated assembly to a mould that is maintained below the melting temperature of the thermoplastic matrix of the composite; C) closure of the mould and application of internal fluid pressure to the liner or bladder to apply pressure to the thermoplastic matrix and reinforcing fibres; D) optionally the use of a special coupling system for rapid connection of the internal pressure; E) cooling of the liner or bladder and thermoplastic matrix and reinforcing fibre assembly in contact with the cold or warm mould while consolidation of the assembly occurs; F) opening of the mould and removal of the finished assembly.\",\n \"Suitable thermoplastic materials for the liner/bladder and thermoplastic composite matrix material include: polypropylene, polyamide, polyethylene, cross-linked polyethylene, polybutylene terephthalate, polyethylene terephthalate, polyoxymethylene, polyphenylene sulfide and polyetheretherketone.\"\n ],\n [\n \"1.A non-isothermal process for fabricating a hollow composite structure, comprising the steps of: preforming at least one thermoplastic liner; over-wrapping the at least one thermoplastic liner with thermoplastic composite material to form at least one over-wrapped liner; heating the at least one over-wrapped liner in an oven to a first temperature above a melting temperature (Tm) of the thermoplastic composite material to form a heated over-wrapped liner; placing the heated over-wrapped liner into a moulding tool that is maintained below the melting temperature (Tm) of the thermoplastic composite material; applying an internal pressure to an interior of the heated over-wrapped liner while the moulding tool is closed to consolidate the composite material and a bladder against a geometry defined by the mould; maintaining the internal pressure to the interior of over-wrapped liner until a second temperature of the thermoplastic composite material is below the thermoplastic composite melting temperature (Tm); and removing the over-wrapped liner from the moulding tool.\",\n \"2.The process according to claim 1, wherein a plurality of over-wrapping phase inserts are positioned on the thermoplastic liner after the step of preforming the thermoplastic liner, and the over-wrapping phase inserts are consolidated with the over-wrapped thermoplastic comnposite material before the step of heating the over-wrapped thermoplastic liner.\",\n \"3.The process according to claim 2, wherein the over-wrapping phase inserts have an operating temperature such that critical dimensions of the over-wrapping phase inserts are not distorted by the first temperature during the step of heating.\",\n \"4.The process according to claim 2, wherein the thermoplastic composite material is locally consolidated onto the over-wrapping phase inserts.\",\n \"5.The process according to claim 1, wherein a plurality of the thermoplastic liners are included during the step of overwrapping the at least one thermoplastic liner to form a continuous process.\",\n \"6.The process according to claim 1, wherein the step of applying an internal pressure includes a rapid coupling system.\",\n \"7.The process according to claim 6, wherein the rapid coupling system includes a rapid gas connection system having an integral pneumatic cylinder comprising a conical member having an axial hole that is pushed into the heated over-wrapped liner against a block having an internally profiled axial hole to create a seal against the heated over-wrapped liner.\",\n \"8.The process according to claim 1, wherein at least one of the thermoplastic liner and the thermoplastic composite material is selected from a group comprising polypropylene, polyamide, particularly polyamide 12, polyethylene, cross-linked polyethylene, polybutylene terephthalate, polyethylene terephthalate, polyoxymethylene, polyphenylene sulfide, and polyetheretherketone.\",\n \"9.A The process according to claim 1, wherein the step of preforming includes at least one of extrusion blow moulding and rotary casting.\",\n \"10.The process according to claim 1, wherein the step of preforming includes high molecular weight polymers to increase a melt strength of the thermoplastic liner.\",\n \"11.The process according to claim 1, wherein the step of preforming includes discontinuous reinforcing fibres to increase a melt strength of the thermoplastic liner.\",\n \"12.The process according to claim 1, wherein the step of preforming includes discontinuous reinforcing fibres to perform a load bearing function of the hollow composite structure.\",\n \"13.The process according to claim 1, wherein the step of over-wrapping includes one of filament winding, braiding, tailored braiding, 3-D braiding, and over-wrapping of one of fabrics and wide tapes.\",\n \"14.The process according to claim 1, wherein the step of over-wrapping includes a final layer of material to tailor surface properties.\",\n \"15.The process according to claim 1, further comprising a step of heating the at least one over-wrapped liner to a third temperature above the first temperature before the step of heating the at least one over-wrapped liner to the first temperature.\",\n \"16.The process according to claim 1, wherein the step of heating the at least one over-wrapped liner includes inducing a temperature gradient such that a third temperature of an outer layer of the thermoplastic composite material is above the melting temperature (Tm) of the thermoplastic composite material and the over-wrapped liner is maintained at a fourth temperature above the melting point of the thermoplastic liner.\",\n \"17.The process according to claim 1, wherein the step of heating the at least one over-wrapped liner includes an oven system comprising a combined forced hot gas oven and an infra red oven.\",\n \"18.The process according to claim 1, wherein the internal pressure is about 2 bar to maintain a shape of the at least one over-wrapped liner.\",\n \"19.The process according to claim 1, wherein the step of heating the at least one over-wrapped liner includes maintaining the liner at a geometry using a frame that enables heat transfer into the moulding tool.\",\n \"20.The process according to claim 1, wherein during the step of applying an internal pressure the heated over-wrapped liner is located between two mould halves of the moulding tool such that contact of both mould halves occurs substantially simultaneously.\",\n \"21.The process according to claim 1, wherein during the step of placing the heated over-wrapped liner the over-wrapped liner is heated to a third temperature above the melting temperature (Tm) of the thermoplastic composite material such that partial impregnation of the thermoplastic liner occurs and the bulk of the thermoplastic composite material is reduced.\",\n \"22.The process according to claim 1, further comprising at least one step of connecting external features to the hollow composite structure.\",\n \"23.The process according to claim 1, wherein the hollow composite structure includes an integral thermoplastic liner.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>Hollow composite structures, or pressure vessels, here referred to as ‘vessels’, such as used to store fluids and solids, particularly under pressure, such as pressurized gas tanks, or more generally in space frame or tubular, load bearing, assemblies have traditionally been fabricated from metals such as steel or aluminium.\",\n \"However, in recent years, the use of composite vessels has become more prevalent.\",\n \"Such vessels are manufactured by a variety of processes, which include filament winding, resin transfer moulding and bladder-assisted moulding.\",\n \"The technology of filament winding is the process of impregnating dry reinforcing fibres, such as fibreglass or carbon strands, with an activated resin prior to application to a mandrel.\",\n \"Preimpregnated materials may also be used.\",\n \"The mixture of reinforcing fibres and matrix is then applied to the mandrel.\",\n \"In the case of thermoset based matrices, a cure cycle follows, while for thermoplastic matrices the polymer is softened (melted) before application to the mandrel where fusion bonding then occurs between layers of the wound material that forms the structure.\",\n \"Either the cure cycle with thermosets or cooling of the material as it is wound with thermoplastics produces a hollow structure or shell that is then removed from the mandrel to give the component.\",\n \"To produce hollow structures or vessels, the technology of resin transfer moulding uses a mandrel and an outer mould, where a mixture of activated matrix resin is injected in the cavity between the mandrel and the outer mould where reinforcing fibres of typically glass, carbon or Kevlar have been placed.\",\n \"Impregnation of the reinforcing fibres then occurs, with cross-linking of thermoset polymers or polymerisation of thermoplastic monomer forming the matrix polymer and the finished shell-like or hollow component.\",\n \"The mandrel could then be removed.\",\n \"The technology of bladder-assisted moulding, for the case of thermoplastic matrices, uses an outer mould and inner bladder to define the shape of the part.\",\n \"To melt the thermoplastic matrix and hence impregnate the reinforcing fibres, of typically glass, carbon or Kevlar, the thermoplastic is heated above its melting temperature and a pressure is then applied to the bladder to consolidate the material.\",\n \"The thermoplastic material must then be cooled, while under pressure, to form the hard shell-like or hollow composite component.\",\n \"Bladder materials comprising rubber-like materials, typically silicon rubber, are used to apply the compaction force, whereby the bladder stretches or elongates under the influence of the applied internal pressure.\",\n \"Alternate bladders consist of in-situ extrusion blow moulded polymeric materials.\",\n \"The thermoplastic matrix is typically heated by heating the outer mould, often metallic, above the matrix melting temperature before the pressure is applied for the required period, after which the mould is cooled sufficiently below the matrix melting temperature for solidification and crystallisation to occur.\",\n \"Alternately, additional or separate heating may be applied by passing hot oil through the bladder or by using embedded electric heaters in the bladder, to heat the composite from the inside, thus eliminating the need to cycle the mould temperature.\",\n \"Conductive reinforcing materials, such as carbon fibre based composites or composites containing a proportion of conductive fibres such as carbon or steel, may be heated in the mould through induction heating or electrical resistance heating.\",\n \"In-situ extrusion blow moulding has been previously claimed to provide heat to aid the melting of the thermoplastic matrix composite, but in practice additional temperature cycling of the mould above and below the matrix melting temperature is also needed.\",\n \"The technology of bladder-assisted moulding, for the case of thermoset based matrices, uses an outer mould and inner bladder to define the shape of the part.\",\n \"A preimpregnated mixture of reinforcing fibres and activated matrix are placed over the bladder and the bladder with the surrounding preimpregnated material is placed into the outer mould.\",\n \"The mould temperature must be high enough to induce cure of the thermosetting matrix and after cure, cooled to allow part release.\",\n \"The bladder may also be heated, either through the passage of a hot fluid, or by embedded electrical heaters, to apply the heat necessary for cure of the impregnated material.\",\n \"The technology also consists of bladder-assisted resin transfer moulding where reinforcement substantially without matrix is placed over the bladder and the bladder coated with reinforcement is then placed into the mould.\",\n \"Activated resin is then applied that impregnates the reinforcing fibres with the application of internal pressure via the bladder, thereby aiding impregnation.\",\n \"Such techniques for fabricating hollow structures or vessels, particularly if a rapid cycle time is required (which is the case for many applications of hollow composite structures or vessels as it reduces the cost of the component) have well known drawbacks which include: for thermoplastic filament winding based processes, the thermoplastic matrix based reinforcement must be heated prior to the application to a mandrel to ensure the thermal conditions necessary for fusion bonding.\",\n \"Hence high processing speeds, notably the application of the thermoplastic matrix based reinforcement to the mandrel, creates a limit on the speed of the process and hence the cycle time.\",\n \"High drag or friction forces induced by the placement of thermoplastic matrix based reinforcement create further quality problems whereby segregation of the thermoplastic matrix and reinforcing fibres may occur, creating an unfavourable microstructure.\",\n \"Additionally, the geometry of the component is limited where there is a need to remove the mandrel after processing.\",\n \"Where the mandrel remains in the component after processing, the mandrel must be compatible with the thermoplastic matrix based reinforcement and be able to sustain the high radial crush forces induced by hot fibre and matrix mixtures applied under high tension forces.\",\n \"The sequential application of composite layers to the mandrel is known to create high internal stresses in the component, affecting dimensional stability and the eventual durability of the component.\",\n \"The filament winding process also places restrictions on geometries that can be produced, where concave curvature requires in-situ bonding of the thermoplastic based composite material to the mandrel or preceding layer of composite; for thermoset based filament winding processes, the thermoset matrix must be cured onto the mandrel requiring a heating cycle either directly as the thermoset matrix based reinforcement is placed, or subsequent heating of the placed material and the mandrel.\",\n \"The process cycle time depends on the winding speed and hence this creates a limit on the speed of the process and thereby the cycle time.\",\n \"High drag or friction forces induced by the placement of thermoset matrix based reinforcement create further quality problems whereby segregation of the thermosetting matrix and reinforcing fibres may occur, creating an unfavourable microstructure.\",\n \"Additionally, the geometry of the component is limited where there is a need to remove the mandrel after processing.\",\n \"Where the mandrel remains in the component after processing, the mandrel must be compatible with the thermoset matrix based reinforcement and be able to sustaining the high radial crush forces induced by the fibre and matrix mixtures applied under high tension forces; for the resin transfer moulding based process, as used with thermoset based reinforced material, a preform must be assembled over the mandrel and resin injected between the mould and the mandrel.\",\n \"The cycle time is limited by the need to inject the resin into this space, where pressures are limited where a permanent hollow mandrel is used such that the pressure of the incoming fluid, here the activated resin, does not distort the geometry of the mandrel, hence limiting processes to lower rate and pressure injection rather than the shorter cycle time, high pressure process of structural reaction injection moulding.\",\n \"Furthermore, the cure reaction of the thermoset matrix either limits the cycle time or, where tailored to occur at a higher speed, induces high stresses in the composite.\",\n \"Unless complex and expensive preforms of reinforcement are prefabricated, parts of a constant wall thickness must be produced unless a local decrease in the reinforcing fibre fraction can be tolerated.\",\n \"Where the mandrel needs to be removed, expensive technologies, such as low melting temperature alloys, must be used where part geometries are not to be impaired.\",\n \"for the resin transfer moulding based process, for the case of thermoplastic based reinforced material, a preform must be assembled over the mandrel and activated monomer injected between the mould and the mandrel.\",\n \"The cycle time is limited by the need to inject the resin into this space, where pressures are limited where a permanent hollow mandrel is used such that the pressure of the incoming fluid, here the activated resin, does not distort the geometry of the mandrel, hence limiting processes to lower rate and pressure injection rather than the shorter cycle time, high pressure process of structural reaction injection moulding.\",\n \"Furthermore, the polymerisation reaction of the monomer either limits the cycle time or, where tailored to occur at a higher speed, places limitations on bladder materials.\",\n \"Thermal cycling above the monomer melting temperature for polymerisation to occur most rapidly and cooling after polymerisation to below the polymer melting temperature are also often required, requiring cycling of the mould temperature and/or mandrel temperatures for certain thermoplastic resin transfer moulding material systems.\",\n \"Unless complex and expensive preforms of reinforcement are prefabricated, parts of a constant wall thickness must be produced unless a local decrease in the reinforcing fibre fraction can be tolerated.\",\n \"Where the mandrel needs to be removed, expensive technologies, such as low melting temperature alloys, must be used where part geometries are not to be impaired.\",\n \"for the bladder assisted moulding process, for the case of thermoset based reinforced material, the need to cure the thermoset matrix material creates problems with chemical attack with rubber-like bladder materials, typically styrene with silicon rubbers, and hence bladder life is reduced.\",\n \"Bladder materials are generally removable due to the difficulty of achieving a bladder that is flexible enough to enable compaction of the impregnated reinforcing fibres prior and during cure while forming a reliable bond between the bladder and cured composite with the lack of high internal stresses in the finished component.\",\n \"Heating of the impregnated reinforcement is also required that is accomplished either through cycling of the mould temperature or heating the bladder material.\",\n \"Cycling the mould temperature is energy inefficient and time consuming, limiting the potential to reduce cycle times, and the fabrication of heated bladder systems is uneconomic for components made in large quantities while also being fragile and of limited life span.\",\n \"Additional internal inspection is also required in the case of removable bladders to ensure that small particles of the bladder material do not remain on the inside wall of the hollow composite or vessel and that the bladder integrity has been maintained such that it is safely usable for further moulding trials.\",\n \"for the bladder assisted moulding process, for the case of thermoplastic based reinforced material, the thermoplastic composite must be heated above the melt temperature of the matrix and cooled below the melt temperature before demoulding.\",\n \"This occurs either by heating the outer mould temperature above the matrix melting temperature and then cooling the mould below the matrix melting temperature, whereby the thermoplastic matrix composite is also heated above and below the thermoplastic composite matrix melting temperature during which time a compaction force is applied by the bladder, or by heating the bladder material such that the thermoplastic matrix is heated sufficiently above the melting temperature of the matrix for adequate impregnation to occur, followed by cooling of the composite structure sufficiently below the matrix melting temperature for stable component release from the mould.\",\n \"The currently available techniques outlined above have inherent limitations that reduce the potential of the process for applications where a minimum cycle time is required.\",\n \"Heating the (often metallic) outer mould is both time consuming and requires large amounts of energy.\",\n \"The use of embedded electrical heaters in a silicon rubber bladder creates a bladder system that is both fragile and difficult to extract from the cooled hollow composite structure or vessel, thereby limiting the geometry of applications.\",\n \"Furthermore, at the higher processing temperatures required for certain thermoplastics, for example PA12 and PET, the life span of the often costly silicon rubber bladders is limited.\",\n \"Previously disclosed inventions have claimed that in-situ extrusion blow moulding can form a bladder material that renders the thermoplastic composite matrix fluid, but where additional heating of the mould is required to ensure full melting.\",\n \"Where the outer layers of the thermoplastic composite are not heated sufficiently above the melting temperature of the thermoplastic matrix, inadequate impregnation occurs, and hence an unfavourable set of mechanical properties results and surface quality is diminished.\",\n \"Thermosetting materials have additional disadvantages compared with thermoplastic based materials, notably for higher rate production, whereby a limited temperature capability results, unsatisfactory finished product aesthetics, lack of extended durability, lack of appropriateness for recycling and manufacturing related issues such as downtime due to clean-up and material handling costs and sub-ambient storage.\",\n \"Further, there are environmental concerns arising from worker exposure to vapour, overspray, emissions, etc., encountered during the fabrication process.\",\n \"Some engineered thermoset resins improve material performance through higher temperature capacity, but unacceptable material costs are associated with them.\",\n \"Thermoplastic based materials have overcome many of the above problems but the processing techniques available are limited in cycle time due to complex heating and cooling cycles, either of the mould or of expensive and fragile bladder materials and bladders based assemblies with a heating ability.\",\n \"Additionally, high energy costs are associated with processes that require a cycling of the mould temperature above and below the matrix melting temperature or complex bladder based heating systems.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>It is therefore the object of the present invention to provide an improved process for the fabrication of hollow composite parts and vessels.\",\n \"This object is achieved by a process as outlined in claim 1 .\",\n \"This non-isothermal process for fabricating hollow composite structures or vessels comprises the steps of: a) preforming of a thermoplastic liner; b) over-wrapping the thermoplastic liner with thermoplastic composite material; c) heating the over-wrapped liner in an oven above the melting temperature of the thermoplastic composite material; d) placing the heated over-wrapped liner into a mould that is maintained below the melting temperature of the thermoplastic composite material; e) applying an internal pressure inside the heated over-wrapped liner while the moulding tool is closed to consolidate the composite material and bladder against a geometry defined by the mould; f) maintaining the internal pressure inside the cooling over-wrapped liner until the thermoplastic composite material is sufficiently below the thermoplastic composite melting temperature; and g) removing the formed hollow composite structure or vessel from the mould.\",\n \"Such an improved process enjoys advantages including, as opposed to prior art processes of fabricating hollow composite parts and vessels: reduced cycle times due to the method of assembling the fibre, matrix and bladder prior to insertion into the mould, reduced cycle times due to the method of assembling the fibre, matrix and bladder in the mould, a reduction in the number of manufacturing steps, faster changeover times, faster start-ups, potential labour saving due to less material handling, floor space reduction, adaptability to automation, simplification of material storage and handling, a safer environment for employees, lower training costs, recyclable scrap materials, reduced cycle times from the thermal conditions required for moulding, reduced energy usage from the thermal conditions required for moulding, better distributions of fibers and matrix in the composite, the ability to mould in metallic or polymeric based inserts for connection of additional items to the vessel, the ability to have an integral liner of the same or different polymer to the matrix material of the composite, the ability to tailor barrier properties at the inside of the vessel, the ability to tailor the reinforcing fibre content through the wall thickness of the hollow or vessel component, etc.\",\n \"The resulting hollow composite parts or vessels can be used for many applications, as typical diameters of these vessels could be 25 cm or larger.\",\n \"These applications include for example the use as containers for compressed air e.g.\",\n \"for brake systems of cars and trucks, breathing apparatus, water storage, hollow automotive or aerospace space frame structures, and generally everywhere where lightweight is of advantage.\",\n \"According to another preferential mode of the invention, after step a) before the over-wrapping phase inserts are positioned on the liner, and before step c) the over-wrapped composite is consolidated onto the positioned inserts.\",\n \"Even more preferentially, it is possible to choose the inserts to have an operating temperature such that the critical dimensions are not distorted by the temperature of the heating phase during step c).\",\n \"Additionally, the over-wrapped composite can be locally consolidated onto the positioned inserts.\",\n \"According to a further preferential mode of the invention, during step b) one or more liners are placed on the same over-wrapping line to form a continuous process and/or in step e) a rapid coupling device is used for rapid connection of the internal pressure.\",\n \"Additional modes of realization are described in the dependent claims.\",\n \"Material forms that may be considered for this invention combine either or both discontinuous and continuous reinforcing fibres with a thermoplastic matrix material with different reinforcing fibre architectures including both random and different degrees of reinforcing fibre alignment.\",\n \"Suitable arrangements of materials with a suitable mingling quality of reinforcement and matrix that may be used include tows or yarns, tapes, braids, weaves, knits, fabrics and other textile processes suitable for manipulating or combining reinforcing fibre and matrix.\",\n \"Materials may be used in either or both an unconsolidated state, a partially consolidated state or a fully consolidated state where the material becomes flexible for the moulding process when the combination of matrix and reinforcing fibre material is heated using the principle of this invention to a forming temperature.\",\n \"Suitable thermoplastic materials include: polypropylene, polyamide, in particular polyamide 12, polyethylene, cross-linked polyethylene, polybutylene terephthalate, polyethylene terephthalate and polyoxymethylene.\",\n \"Suitable reinforcing fibres include: glass, carbon, Kevlar, ultra high molecular weight polyethylene, ultra high molecular weight polypropylene and steel.\",\n \"Techniques for combining reinforcing fibres and thermoplastic matrix materials, in both continuous and discontinuous fibre lengths, which are applicable for use with the above process include: melt impregnation, film stacking, solvent impregnation, commingling, powder impregnating using both aqueous slurries and electrostatic methods of coating, powder impregnated fibres with an unmelted sheath around the powder impregnated bundle for textile processing, standard fabrics impregnated by powder to form a semifinished product, the utilization of existing textile processing techniques applied to the warp knitting of split polymer films with weft insertion of reinforcement fibres, weft-inserted multi-axial warp knitting of reinforcing fibres and thermoplastic matrix ribbons, co-weaving yarns of the two fibres into a form possessing good drape characteristics, using a polymer fibre woven around a bundle of reinforcement fibres in the plied matrix technique, reinforcing and matrix yarns inter-dispersed on line during fabric formation to produce drapable stitch bonded fabrics, heating of fibres impregnated with monomers that polymerize in-situ to form the composite, wire coating processes adapted to coating reinforcing fibres or bundles of fibres with matrix, pultrusion of fibres and matrix materials together and any other such process that achieves the required combination of fibre and matrix in a form between fully consolidated and unconsolidated forms.\",\n \"Commingled materials, which are a preferred material for this moulding process, can be formed either directly as described in U.S. Pat.\",\n \"No.\",\n \"5,011,523 and U.S. Pat.\",\n \"No.\",\n \"5,316,561 where the reinforcing fibers are drawn from the molten state through a die where the reinforcing glass fibers are combined with a drawing head where the thermoplastic matrix is extruded and drawn mechanically onto the glass fibres or indirectly by separating fibre and matrix fibre yarns (U.S. Pat.\",\n \"No.\",\n \"5,241,731) into open ribbons where the matrix fibres are electrically charged and then drawn into a flat open bundle via a ribboning bar to the commingling bar where the Carbon fibre yarn is opened with an air curtain into an open flat ribbon that is then combined with the PEEK ribbon at the commingling bar or in the example shown herein of the material used to illustrate this invention where stretch broken Carbon fibres (80 mm average length) are blended with polyamide 12 (PA12) staple fibres using a textile spinning technique (U.S. Pat.\",\n \"No.\",\n \"4,825,635) with the addition of a wrapping filament (U.S. Pat.\",\n \"No.\",\n \"5,910,361).\"\n ],\n [\n \"FIELD OF THE INVENTION This invention relates to the field of fabricating hollow composite parts, especially space frame structures or vessels, and more particularly to improved methods for fabricating space frame structures or vessels and to composite vessels made in accordance with the improved methods.\",\n \"BACKGROUND OF THE INVENTION Hollow composite structures, or pressure vessels, here referred to as ‘vessels’, such as used to store fluids and solids, particularly under pressure, such as pressurized gas tanks, or more generally in space frame or tubular, load bearing, assemblies have traditionally been fabricated from metals such as steel or aluminium.\",\n \"However, in recent years, the use of composite vessels has become more prevalent.\",\n \"Such vessels are manufactured by a variety of processes, which include filament winding, resin transfer moulding and bladder-assisted moulding.\",\n \"The technology of filament winding is the process of impregnating dry reinforcing fibres, such as fibreglass or carbon strands, with an activated resin prior to application to a mandrel.\",\n \"Preimpregnated materials may also be used.\",\n \"The mixture of reinforcing fibres and matrix is then applied to the mandrel.\",\n \"In the case of thermoset based matrices, a cure cycle follows, while for thermoplastic matrices the polymer is softened (melted) before application to the mandrel where fusion bonding then occurs between layers of the wound material that forms the structure.\",\n \"Either the cure cycle with thermosets or cooling of the material as it is wound with thermoplastics produces a hollow structure or shell that is then removed from the mandrel to give the component.\",\n \"To produce hollow structures or vessels, the technology of resin transfer moulding uses a mandrel and an outer mould, where a mixture of activated matrix resin is injected in the cavity between the mandrel and the outer mould where reinforcing fibres of typically glass, carbon or Kevlar have been placed.\",\n \"Impregnation of the reinforcing fibres then occurs, with cross-linking of thermoset polymers or polymerisation of thermoplastic monomer forming the matrix polymer and the finished shell-like or hollow component.\",\n \"The mandrel could then be removed.\",\n \"The technology of bladder-assisted moulding, for the case of thermoplastic matrices, uses an outer mould and inner bladder to define the shape of the part.\",\n \"To melt the thermoplastic matrix and hence impregnate the reinforcing fibres, of typically glass, carbon or Kevlar, the thermoplastic is heated above its melting temperature and a pressure is then applied to the bladder to consolidate the material.\",\n \"The thermoplastic material must then be cooled, while under pressure, to form the hard shell-like or hollow composite component.\",\n \"Bladder materials comprising rubber-like materials, typically silicon rubber, are used to apply the compaction force, whereby the bladder stretches or elongates under the influence of the applied internal pressure.\",\n \"Alternate bladders consist of in-situ extrusion blow moulded polymeric materials.\",\n \"The thermoplastic matrix is typically heated by heating the outer mould, often metallic, above the matrix melting temperature before the pressure is applied for the required period, after which the mould is cooled sufficiently below the matrix melting temperature for solidification and crystallisation to occur.\",\n \"Alternately, additional or separate heating may be applied by passing hot oil through the bladder or by using embedded electric heaters in the bladder, to heat the composite from the inside, thus eliminating the need to cycle the mould temperature.\",\n \"Conductive reinforcing materials, such as carbon fibre based composites or composites containing a proportion of conductive fibres such as carbon or steel, may be heated in the mould through induction heating or electrical resistance heating.\",\n \"In-situ extrusion blow moulding has been previously claimed to provide heat to aid the melting of the thermoplastic matrix composite, but in practice additional temperature cycling of the mould above and below the matrix melting temperature is also needed.\",\n \"The technology of bladder-assisted moulding, for the case of thermoset based matrices, uses an outer mould and inner bladder to define the shape of the part.\",\n \"A preimpregnated mixture of reinforcing fibres and activated matrix are placed over the bladder and the bladder with the surrounding preimpregnated material is placed into the outer mould.\",\n \"The mould temperature must be high enough to induce cure of the thermosetting matrix and after cure, cooled to allow part release.\",\n \"The bladder may also be heated, either through the passage of a hot fluid, or by embedded electrical heaters, to apply the heat necessary for cure of the impregnated material.\",\n \"The technology also consists of bladder-assisted resin transfer moulding where reinforcement substantially without matrix is placed over the bladder and the bladder coated with reinforcement is then placed into the mould.\",\n \"Activated resin is then applied that impregnates the reinforcing fibres with the application of internal pressure via the bladder, thereby aiding impregnation.\",\n \"Such techniques for fabricating hollow structures or vessels, particularly if a rapid cycle time is required (which is the case for many applications of hollow composite structures or vessels as it reduces the cost of the component) have well known drawbacks which include: for thermoplastic filament winding based processes, the thermoplastic matrix based reinforcement must be heated prior to the application to a mandrel to ensure the thermal conditions necessary for fusion bonding.\",\n \"Hence high processing speeds, notably the application of the thermoplastic matrix based reinforcement to the mandrel, creates a limit on the speed of the process and hence the cycle time.\",\n \"High drag or friction forces induced by the placement of thermoplastic matrix based reinforcement create further quality problems whereby segregation of the thermoplastic matrix and reinforcing fibres may occur, creating an unfavourable microstructure.\",\n \"Additionally, the geometry of the component is limited where there is a need to remove the mandrel after processing.\",\n \"Where the mandrel remains in the component after processing, the mandrel must be compatible with the thermoplastic matrix based reinforcement and be able to sustain the high radial crush forces induced by hot fibre and matrix mixtures applied under high tension forces.\",\n \"The sequential application of composite layers to the mandrel is known to create high internal stresses in the component, affecting dimensional stability and the eventual durability of the component.\",\n \"The filament winding process also places restrictions on geometries that can be produced, where concave curvature requires in-situ bonding of the thermoplastic based composite material to the mandrel or preceding layer of composite; for thermoset based filament winding processes, the thermoset matrix must be cured onto the mandrel requiring a heating cycle either directly as the thermoset matrix based reinforcement is placed, or subsequent heating of the placed material and the mandrel.\",\n \"The process cycle time depends on the winding speed and hence this creates a limit on the speed of the process and thereby the cycle time.\",\n \"High drag or friction forces induced by the placement of thermoset matrix based reinforcement create further quality problems whereby segregation of the thermosetting matrix and reinforcing fibres may occur, creating an unfavourable microstructure.\",\n \"Additionally, the geometry of the component is limited where there is a need to remove the mandrel after processing.\",\n \"Where the mandrel remains in the component after processing, the mandrel must be compatible with the thermoset matrix based reinforcement and be able to sustaining the high radial crush forces induced by the fibre and matrix mixtures applied under high tension forces; for the resin transfer moulding based process, as used with thermoset based reinforced material, a preform must be assembled over the mandrel and resin injected between the mould and the mandrel.\",\n \"The cycle time is limited by the need to inject the resin into this space, where pressures are limited where a permanent hollow mandrel is used such that the pressure of the incoming fluid, here the activated resin, does not distort the geometry of the mandrel, hence limiting processes to lower rate and pressure injection rather than the shorter cycle time, high pressure process of structural reaction injection moulding.\",\n \"Furthermore, the cure reaction of the thermoset matrix either limits the cycle time or, where tailored to occur at a higher speed, induces high stresses in the composite.\",\n \"Unless complex and expensive preforms of reinforcement are prefabricated, parts of a constant wall thickness must be produced unless a local decrease in the reinforcing fibre fraction can be tolerated.\",\n \"Where the mandrel needs to be removed, expensive technologies, such as low melting temperature alloys, must be used where part geometries are not to be impaired.\",\n \"for the resin transfer moulding based process, for the case of thermoplastic based reinforced material, a preform must be assembled over the mandrel and activated monomer injected between the mould and the mandrel.\",\n \"The cycle time is limited by the need to inject the resin into this space, where pressures are limited where a permanent hollow mandrel is used such that the pressure of the incoming fluid, here the activated resin, does not distort the geometry of the mandrel, hence limiting processes to lower rate and pressure injection rather than the shorter cycle time, high pressure process of structural reaction injection moulding.\",\n \"Furthermore, the polymerisation reaction of the monomer either limits the cycle time or, where tailored to occur at a higher speed, places limitations on bladder materials.\",\n \"Thermal cycling above the monomer melting temperature for polymerisation to occur most rapidly and cooling after polymerisation to below the polymer melting temperature are also often required, requiring cycling of the mould temperature and/or mandrel temperatures for certain thermoplastic resin transfer moulding material systems.\",\n \"Unless complex and expensive preforms of reinforcement are prefabricated, parts of a constant wall thickness must be produced unless a local decrease in the reinforcing fibre fraction can be tolerated.\",\n \"Where the mandrel needs to be removed, expensive technologies, such as low melting temperature alloys, must be used where part geometries are not to be impaired.\",\n \"for the bladder assisted moulding process, for the case of thermoset based reinforced material, the need to cure the thermoset matrix material creates problems with chemical attack with rubber-like bladder materials, typically styrene with silicon rubbers, and hence bladder life is reduced.\",\n \"Bladder materials are generally removable due to the difficulty of achieving a bladder that is flexible enough to enable compaction of the impregnated reinforcing fibres prior and during cure while forming a reliable bond between the bladder and cured composite with the lack of high internal stresses in the finished component.\",\n \"Heating of the impregnated reinforcement is also required that is accomplished either through cycling of the mould temperature or heating the bladder material.\",\n \"Cycling the mould temperature is energy inefficient and time consuming, limiting the potential to reduce cycle times, and the fabrication of heated bladder systems is uneconomic for components made in large quantities while also being fragile and of limited life span.\",\n \"Additional internal inspection is also required in the case of removable bladders to ensure that small particles of the bladder material do not remain on the inside wall of the hollow composite or vessel and that the bladder integrity has been maintained such that it is safely usable for further moulding trials.\",\n \"for the bladder assisted moulding process, for the case of thermoplastic based reinforced material, the thermoplastic composite must be heated above the melt temperature of the matrix and cooled below the melt temperature before demoulding.\",\n \"This occurs either by heating the outer mould temperature above the matrix melting temperature and then cooling the mould below the matrix melting temperature, whereby the thermoplastic matrix composite is also heated above and below the thermoplastic composite matrix melting temperature during which time a compaction force is applied by the bladder, or by heating the bladder material such that the thermoplastic matrix is heated sufficiently above the melting temperature of the matrix for adequate impregnation to occur, followed by cooling of the composite structure sufficiently below the matrix melting temperature for stable component release from the mould.\",\n \"The currently available techniques outlined above have inherent limitations that reduce the potential of the process for applications where a minimum cycle time is required.\",\n \"Heating the (often metallic) outer mould is both time consuming and requires large amounts of energy.\",\n \"The use of embedded electrical heaters in a silicon rubber bladder creates a bladder system that is both fragile and difficult to extract from the cooled hollow composite structure or vessel, thereby limiting the geometry of applications.\",\n \"Furthermore, at the higher processing temperatures required for certain thermoplastics, for example PA12 and PET, the life span of the often costly silicon rubber bladders is limited.\",\n \"Previously disclosed inventions have claimed that in-situ extrusion blow moulding can form a bladder material that renders the thermoplastic composite matrix fluid, but where additional heating of the mould is required to ensure full melting.\",\n \"Where the outer layers of the thermoplastic composite are not heated sufficiently above the melting temperature of the thermoplastic matrix, inadequate impregnation occurs, and hence an unfavourable set of mechanical properties results and surface quality is diminished.\",\n \"Thermosetting materials have additional disadvantages compared with thermoplastic based materials, notably for higher rate production, whereby a limited temperature capability results, unsatisfactory finished product aesthetics, lack of extended durability, lack of appropriateness for recycling and manufacturing related issues such as downtime due to clean-up and material handling costs and sub-ambient storage.\",\n \"Further, there are environmental concerns arising from worker exposure to vapour, overspray, emissions, etc., encountered during the fabrication process.\",\n \"Some engineered thermoset resins improve material performance through higher temperature capacity, but unacceptable material costs are associated with them.\",\n \"Thermoplastic based materials have overcome many of the above problems but the processing techniques available are limited in cycle time due to complex heating and cooling cycles, either of the mould or of expensive and fragile bladder materials and bladders based assemblies with a heating ability.\",\n \"Additionally, high energy costs are associated with processes that require a cycling of the mould temperature above and below the matrix melting temperature or complex bladder based heating systems.\",\n \"SUMMARY OF THE INVENTION It is therefore the object of the present invention to provide an improved process for the fabrication of hollow composite parts and vessels.\",\n \"This object is achieved by a process as outlined in claim 1.This non-isothermal process for fabricating hollow composite structures or vessels comprises the steps of: a) preforming of a thermoplastic liner; b) over-wrapping the thermoplastic liner with thermoplastic composite material; c) heating the over-wrapped liner in an oven above the melting temperature of the thermoplastic composite material; d) placing the heated over-wrapped liner into a mould that is maintained below the melting temperature of the thermoplastic composite material; e) applying an internal pressure inside the heated over-wrapped liner while the moulding tool is closed to consolidate the composite material and bladder against a geometry defined by the mould; f) maintaining the internal pressure inside the cooling over-wrapped liner until the thermoplastic composite material is sufficiently below the thermoplastic composite melting temperature; and g) removing the formed hollow composite structure or vessel from the mould.\",\n \"Such an improved process enjoys advantages including, as opposed to prior art processes of fabricating hollow composite parts and vessels: reduced cycle times due to the method of assembling the fibre, matrix and bladder prior to insertion into the mould, reduced cycle times due to the method of assembling the fibre, matrix and bladder in the mould, a reduction in the number of manufacturing steps, faster changeover times, faster start-ups, potential labour saving due to less material handling, floor space reduction, adaptability to automation, simplification of material storage and handling, a safer environment for employees, lower training costs, recyclable scrap materials, reduced cycle times from the thermal conditions required for moulding, reduced energy usage from the thermal conditions required for moulding, better distributions of fibers and matrix in the composite, the ability to mould in metallic or polymeric based inserts for connection of additional items to the vessel, the ability to have an integral liner of the same or different polymer to the matrix material of the composite, the ability to tailor barrier properties at the inside of the vessel, the ability to tailor the reinforcing fibre content through the wall thickness of the hollow or vessel component, etc.\",\n \"The resulting hollow composite parts or vessels can be used for many applications, as typical diameters of these vessels could be 25 cm or larger.\",\n \"These applications include for example the use as containers for compressed air e.g.\",\n \"for brake systems of cars and trucks, breathing apparatus, water storage, hollow automotive or aerospace space frame structures, and generally everywhere where lightweight is of advantage.\",\n \"According to another preferential mode of the invention, after step a) before the over-wrapping phase inserts are positioned on the liner, and before step c) the over-wrapped composite is consolidated onto the positioned inserts.\",\n \"Even more preferentially, it is possible to choose the inserts to have an operating temperature such that the critical dimensions are not distorted by the temperature of the heating phase during step c).\",\n \"Additionally, the over-wrapped composite can be locally consolidated onto the positioned inserts.\",\n \"According to a further preferential mode of the invention, during step b) one or more liners are placed on the same over-wrapping line to form a continuous process and/or in step e) a rapid coupling device is used for rapid connection of the internal pressure.\",\n \"Additional modes of realization are described in the dependent claims.\",\n \"Material forms that may be considered for this invention combine either or both discontinuous and continuous reinforcing fibres with a thermoplastic matrix material with different reinforcing fibre architectures including both random and different degrees of reinforcing fibre alignment.\",\n \"Suitable arrangements of materials with a suitable mingling quality of reinforcement and matrix that may be used include tows or yarns, tapes, braids, weaves, knits, fabrics and other textile processes suitable for manipulating or combining reinforcing fibre and matrix.\",\n \"Materials may be used in either or both an unconsolidated state, a partially consolidated state or a fully consolidated state where the material becomes flexible for the moulding process when the combination of matrix and reinforcing fibre material is heated using the principle of this invention to a forming temperature.\",\n \"Suitable thermoplastic materials include: polypropylene, polyamide, in particular polyamide 12, polyethylene, cross-linked polyethylene, polybutylene terephthalate, polyethylene terephthalate and polyoxymethylene.\",\n \"Suitable reinforcing fibres include: glass, carbon, Kevlar, ultra high molecular weight polyethylene, ultra high molecular weight polypropylene and steel.\",\n \"Techniques for combining reinforcing fibres and thermoplastic matrix materials, in both continuous and discontinuous fibre lengths, which are applicable for use with the above process include: melt impregnation, film stacking, solvent impregnation, commingling, powder impregnating using both aqueous slurries and electrostatic methods of coating, powder impregnated fibres with an unmelted sheath around the powder impregnated bundle for textile processing, standard fabrics impregnated by powder to form a semifinished product, the utilization of existing textile processing techniques applied to the warp knitting of split polymer films with weft insertion of reinforcement fibres, weft-inserted multi-axial warp knitting of reinforcing fibres and thermoplastic matrix ribbons, co-weaving yarns of the two fibres into a form possessing good drape characteristics, using a polymer fibre woven around a bundle of reinforcement fibres in the plied matrix technique, reinforcing and matrix yarns inter-dispersed on line during fabric formation to produce drapable stitch bonded fabrics, heating of fibres impregnated with monomers that polymerize in-situ to form the composite, wire coating processes adapted to coating reinforcing fibres or bundles of fibres with matrix, pultrusion of fibres and matrix materials together and any other such process that achieves the required combination of fibre and matrix in a form between fully consolidated and unconsolidated forms.\",\n \"Commingled materials, which are a preferred material for this moulding process, can be formed either directly as described in U.S. Pat.\",\n \"No.\",\n \"5,011,523 and U.S. Pat.\",\n \"No.\",\n \"5,316,561 where the reinforcing fibers are drawn from the molten state through a die where the reinforcing glass fibers are combined with a drawing head where the thermoplastic matrix is extruded and drawn mechanically onto the glass fibres or indirectly by separating fibre and matrix fibre yarns (U.S. Pat.\",\n \"No.\",\n \"5,241,731) into open ribbons where the matrix fibres are electrically charged and then drawn into a flat open bundle via a ribboning bar to the commingling bar where the Carbon fibre yarn is opened with an air curtain into an open flat ribbon that is then combined with the PEEK ribbon at the commingling bar or in the example shown herein of the material used to illustrate this invention where stretch broken Carbon fibres (80 mm average length) are blended with polyamide 12 (PA12) staple fibres using a textile spinning technique (U.S. Pat.\",\n \"No.\",\n \"4,825,635) with the addition of a wrapping filament (U.S. Pat.\",\n \"No.\",\n \"5,910,361).\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS The invention, both as to organisation and method of operation, may be best understood by reference to the following description taken in conjunction with the claims and the accompanying drawings of which: FIG.\",\n \"1 Pictorial view of a liner/mandrel employed in practising a first inventive embodiment; FIG.\",\n \"2 Cross-sectional view taken along the lines A-A of FIG.\",\n \"1 with optional inserts; FIG.\",\n \"3 Enlarged cross-sectional view of FIG.\",\n \"2 showing an optional incorporated insert; FIG.\",\n \"4 A pictorial view of the liner or mandrel being over-wrapped with a mixture of thermoplastic resin and reinforcing material; FIG.\",\n \"5 A pictorial view of a continuous process where the liner or mandrel are being over-wrapped with a mixture of thermoplastic resin and reinforcing material; FIG.\",\n \"6 A pictorial view of a device for consolidating the over-wrapped composite onto inserts for attaching exterior features; FIG.\",\n \"7 A pictorial view of a device for separating the over-wrapped inserts with composite consolidated in place over the inserts; FIG.\",\n \"8 A cross-sectional view of the liner over-wrapped with composite with an insert in place including an enlarged fragmentary cross-sectional view; FIG.\",\n \"9 Pictorial view of the liner after being over-laid with the layer(s) of composite; FIG.\",\n \"10 A cross-sectional sequential view of the conventional isothermal processing sequence used to produce hollow composite structures or vessels; FIG.\",\n \"11 A cross-sectional sequential view of the non-isothermal processing sequence employed in practising a first inventive embodiment; FIG.\",\n \"12 A cross-sectional view of the over-wrapped and preheated liner located in the mould just prior to closure of the mould; FIG.\",\n \"13 A cross-sectional view of the over-wrapped and preheated liner located in the mould during internal pressurization such that the over-wrapped liner is formed into the shape defined by the inner surface of the mould; FIG.\",\n \"14 A cross-sectional sequential view illustrating temperature gradients, including four non-exclusive possibilities, through the thickness of the liner and over-wrapped composite for retaining integrity of the bladder and maximising surface consolidation of the over-wrapped composite, including an enlarged cross-sectional view and corresponding temperature (T) distance (d) graph.\",\n \"FIG.\",\n \"15 A cross-sectional sequential view of a high speed connection device to attach a pressurization source; FIG.\",\n \"16 Temperature and pressure histories of a typical moulding cycle; FIG.\",\n \"17 A cross-sectional view of the completed hollow composite structure or pressure vessel prepared by practising the first and second inventive embodiments.\",\n \"DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The first embodiment uses a hollow thermoplastic liner or bladder, produced by processes such as extrusion blow moulding or cast rotational moulding, such as shown in FIG.\",\n \"1.In the example shown the liner 1 or bladder is generally an elongated structure of geometry such as can be produced via extrusion blow moulding, terminating with either one or more openings, located either on the central axis of the component or at any other suitable location.\",\n \"Hence bladder geometries are not restricted to shapes of revolution, but are applicable to any hollow thermoplastic shape that can be made by processes such as extrusion blow moulding or cast rotational moulding, processes which are well known in the art.\",\n \"In addition to using a thermoplastic liner 1 or bladder, short fibre reinforced or discontinuous fibre reinforced thermoplastic materials may also be used such that the melt viscosity or melt strength of the polymer and fibre mixture is increased both during the extrusion blow moulding process and during the preheat, transfer and moulding stages for the non-isothermal bladder inflation moulding process.\",\n \"The addition of short glass fibre also increases the mechanical properties of the liner or bladder such that it becomes part of the load bearing material in a hollow composite structure or vessel.\",\n \"Over the hollow liner or bladder, metallic, ceramic or polymeric inserts 2 can optionally be added before the over-wrapping or over-braiding of thermoplastic matrix reinforced material.\",\n \"The inner insert 2 must have an operating temperature such that the critical dimensions, for example thread geometries, are not distorted by heat during the final non-isothermal moulding process.\",\n \"Such inserts can be made via a variety of processes well known in the art including injection moulding, compression moulding, casting and machining.\",\n \"Alternatively, inserts may be placed into the extrusion blow moulding tool and be directly over moulded during the process of forming the bladder, relying either on mechanical interlocking with non-compatible insert materials and/or fusion bonding with compatible insert materials.\",\n \"Inserts 2 located on the liner or bladder are shown in FIGS.\",\n \"2 and 3.The bladder 1 with positioned inserts 2, held either via a locating device on the liner or bladder or by an external locating fixture, is used as a mandrel for an over-wrapping or over-braiding procedure whereby reinforcing fibres, such as the ones mentioned above, intimately mixed with a thermoplastic resin are placed over the liner or bladder, as shown in FIG.\",\n \"4.Suitable processes for over-wrapping or over-braiding are textile based processes where yarns or tows 3-6 of reinforced thermoplastic matrix based material are wound around the liner or mandrel to provide an even coverage of material over the liner or bladder surface, including processes such as: braiding, either cold or with the addition of heating, tailored braiding where the fibre orientation is controlled across the geometry of the liner or bladder, 3-D braiding processes where interlocking occurs between layers of the applied reinforced thermoplastic matrix based material, over-wrapping of woven tapes or fabrics of reinforced thermoplastic matrix based materials such that a greater area of coverage occurs decreasing the cycle time and optimising system economics and filament winding, either cold or with the addition of heating.\",\n \"Pressure is not directly applied during the over-wrapping or over-braiding process, thereby increasing winding speeds and decreasing cycle times.\",\n \"In addition to the over-wrapping or over-braiding of mixtures of thermoplastic matrix and reinforcing fibres, a final layer of material can be added, depending on the application, consisting of either a higher matrix fraction in the thermoplastic and reinforcing fibre mixture or solely of thermoplastic matrix fibres or wrapping films, including extrudates, to provide a thermoplastic matrix rich surface to both improve surface quality, sealing the reinforcing fibres in the component, to give a surface that is more readily painted or coated with additional materials or processes after moulding and to ensure full surface consolidation.\",\n \"This higher matrix fraction can be achieved with either the same matrix polymer as the matrix for the reinforced material or with a compatible but different polymer to optimise barrier and surface performance properties.\",\n \"One or more liners or bladders, with inserts attached, may optionally be placed on the same over-wrapping or braiding line such as to form a continuous over-braiding or over-wrapping process, as shown in FIG.\",\n \"5.To locate the inserts onto the liner or bladder, where the inserts are not directly incorporated, and to ensure that good contact and final consolidation occurs between the insert, liner or bladder and the reinforced thermoplastic matrix based material, where either over-moulded or located inserts are used, the over wrapped or over braided material is optionally consolidated onto the insert, as shown in FIG.\",\n \"6.A hot clamping device 7 is used that rapidly heats the reinforced thermoplastic matrix based material above the matrix melt temperature and applies a force 9 to impregnate the reinforcing fibres with the thermoplastic matrix, followed by cooling of the clamping device 7 while clamping pressure 9 is maintained such that consolidation pressure is applied while the reinforced thermoplastic matrix based material cools.\",\n \"Alternatively, a heating source 8 is used to rapidly heat the reinforced thermoplastic matrix based material above the matrix melt temperature followed by a clamping device 7, maintained at a temperature below the melting point of the polymer, that applies consolidation pressure while the reinforced thermoplastic matrix based material cools.\",\n \"Either built into or separate from the heating and cooling device that consolidates the over wrapped or over braided material onto the insert, a cutting device 10 separates the preform assembly of liner or bladder 1, reinforced thermoplastic matrix based material, and inserts 2 from the adjacent preform assembly, where reinforced thermoplastic matrix based material crosses over between liners or bladders where a continuous process is used, as shown in FIG.\",\n \"7.Cutting can either occur on consolidated and rigid reinforced thermoplastic matrix based material or on the placed reinforced thermoplastic matrix based material that has not been fully consolidated.\",\n \"As a further step to reduce cycle times during the eventual non-isothermal bladder inflation moulding process, heat may be applied during any of the over-wrapping or over-braiding processes such that the matrix in the reinforced thermoplastic matrix based material is heated above the melting temperature of the matrix enabling spontaneous wetting of the reinforcement via capillary action and partial impregnation via the tension forces induced in the over-wrapping or over-braiding material as it is placed over the liner or bladder.\",\n \"As such a partially impregnated material results with a lower bulk than material which has not been partially impregnated, resulting in a material that has less trapped gas during the heating and consolidation phases of the non-isothermal bladder inflation moulding process.\",\n \"Alternatively, the assembled preform of liner or bladder, over-wrapped or over braided reinforced thermoplastic matrix based material and inserts may be flash heated using a high flux infra red heating or similar heating source to melt the over-wrapped or over-braided reinforced thermoplastic matrix based material without affecting the integrity of the liner or bladder, thereby providing a preform with increased stability for any transport to a different location where necessary before the final moulding operation.\",\n \"A liner 1 or bladder 12 over-wrapped with composite material 11 is thereby obtained, with the optional location of inserts 2 for attaching external features to the hollow composite part or vessel, as shown in FIGS.\",\n \"8 and 9.Processes according to the state of the art have completed the final consolidation of thermoplastic composite materials using either rubber-like bladders or with in-situ extruded thermoplastic bladder material.\",\n \"Such materials are heated to bring the thermoplastic composite matrix above its melting temperature, either through latent heat of a directly produced extrudate, cycling of the mould temperature above and below the matrix melting temperature, or via heating of the bladder internally via hot fluid circulation or embedded electrical elements.\",\n \"Such processes either produce components of limited quality, in the case of heating via the latent heated of extruded polymeric material, restrict geometries and have limited bladder life for embedded heaters in rubber-like bladders, or require complex internal heating circuits.\",\n \"Where the mould temperature is cycled, an isothermal process results, such as shown in FIG.\",\n \"10.The liner 1 and over-wrapped composite 11 (FIG.\",\n \"10a) are placed into a mould 13 below the melting temperature of the liner 1 or bladder and/or over-wrapped composite 11 (FIG.\",\n \"10b).\",\n \"The mould is then held closed while rapid heating 15 is applied to heat the mould above the melting temperature of the liner 1 or bladder and/or over-wrapped composite 11 (FIG.\",\n \"10c).\",\n \"After the required impregnation time, the rapid heating source 15 is replaced by a rapid cooling source 18, while the mould remains closed 14, to reduce the temperature below the melting temperature of the liner 1 or bladder and/or over-wrapped composite 11 (FIG.\",\n \"10d).\",\n \"After the required consolidation and cooling time, the mould 13 is opened and the part obtained (FIG.\",\n \"10e).\",\n \"The proposed process is a non-isothermal bladder inflation process that does not require cycling of the mould temperature or heating of the bladder internally whilst in the closed mould.\",\n \"To melt the thermoplastic matrix and hence reduce the viscosity, enabling impregnation of the reinforcing fibres, the assembly of a thermoplastic liner 1 or bladder and over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres 11 (FIG.\",\n \"11a) are heated in an oven 15 outside of the closed final moulding tool 13 (FIG.\",\n \"11b).\",\n \"After the heating cycle, the over-wrapped liner is located in the moulding tool 13 (FIG.\",\n \"11c), FIG.\",\n \"12), where the moulding tool 13 is closed and an internal pressure 21 applied inside the liner 1 or bladder, so as to force the over-wrapped liner or bladder to conform to the shape of the mould geometry (FIG.\",\n \"13).\",\n \"The moulding tool 13 is maintained below the melting temperature of the liner or bladder and/or over-wrapped composite 11, while the internal pressure 21 is maintained for the time required to consolidate and cool the hollow composite part or vessel.\",\n \"The mould is then opened and the part removed (FIG.\",\n \"11d).\",\n \"During the heating cycle, the oven parameters are adjusted such that the outer over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres are heated sufficiently above the matrix melting temperature for good consolidation to occur and a good surface finish to result from the final moulding process, while not reaching excessive temperatures where degradation of the matrix occurs.\",\n \"For an example of a polyamide 12 matrix in the reinforcing material, an upper temperature is chosen such that degradation of the polymer does not initiate to an extent such that the short term or long term mechanical properties are not adversely affected, odour problems do not occur beyond acceptable limits and surface quality is not unduly influenced, which is typically around 280° C. Also during the heating process, the oven parameters are adjusted such that the inner liner or bladder temperature is optimised to be extendable under the applied internal pressure 21 during the final moulding cycle, thereby enabling the internal pressure 21 in the liner 1 or bladder to be transferred to the hot over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres giving the driving force for concurrent consolidation and cooling in the moulding tool 20.Additionally, the bladder temperature is restricted such that the viscosity of the bladder matrix does not become too low such that the liner or bladder looses integrity, excessive diffusion of the pressurizing fluid is permitted and the liner or bladder is thereby unable to resist the application of an internal pressure during the final moulding cycle.\",\n \"A suitable temperature, for an example of the present embodiments using polyamide 12, consists of heating the bladder temperature to 220° C. Preferred materials for liners or bladders are high molecular weight grades of the same polymer as used in the matrix of the over-wrapped or over-braided mixtures of thermoplastic and reinforcing fibres, where a higher molecular weight results in higher melt viscosities, thereby maintaining the integrity of the liner or bladder material to a higher temperature.\",\n \"Other preferred materials are thermoplastic polymers that have been partially cross-linked to give rubbery behaviour above the melt temperature with a delayed loss of integrity, such as cross-linked polyethylene.\",\n \"A temperature gradient therefore results during the heating cycle of the assembled liner or bladder with over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres, such that the outer layers of over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres are heated to around 280° C., for the example of a polyamide 12 matrix, and the inner liner or bladder material is heated to only around 220° C. Such temperature profiles, as described above for the over-wrapped composite and liner or bladder materials, are illustrated in FIG.\",\n \"14, where four different non-exclusive heating profiles are shown.\",\n \"Suitable oven systems include: hot gas ovens, forced convection hot gas ovens, infra-red ovens, and combinations of infra-red and hot gas or forced hot gas ovens, using either electrical or gas powered heating elements or emitters.\",\n \"A preferred oven combination for this heating cycle is a combined forced hot gas and infra red oven, where the preset and controlled temperature of the forced hot gas penetrates to the inner liner or bladder and heats the liner or bladder to the required temperature, followed by infra red heating that quickly heats the outer layers of over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres while not penetrating to the inner liner or bladder, whereby the flux level from the infra red emitters are tailored to give the desired temperature gradient through the material.\",\n \"During the heating process, the assembled liner or bladder and over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres are maintained to the desired geometry using a frame that clamps the assembled preform.\",\n \"This clamp system is then transferred after the heating cycle to the moulding tool 20, where the frame fits integrally with the moulding tool to locate the preheated assembly rapidly in the correct location inside the moulding tool.\",\n \"A fast transfer from the oven to the moulding tool 20 is preferable to avoid cooling of the preheated assembly of liner 1 or bladder and over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres.\",\n \"Typically the internal pressure 21 must be applied to the liner 1 or bladder and the mould 20 held closed within 20 seconds of the finish of the preheat cycle, and faster times improve part quality notably, both in terms of reducing void contents, hence increasing mechanical properties, and improving the surface quality or degree of surface impregnation.\",\n \"Additionally, a low internal pressure of up to 2 bar can be applied to the inside of the liner or mandrel during the heating cycle, to help maintain the shape of the bladder or liner against gravitational forces.\",\n \"Where the preferred method of assembling preforms with previously incorporated inserts and locally preconsolidated, at the insert region, over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres is used, the following mechanism is a preferred method of holding the over-wrapped or over braiding liner 1 or mandrel during the heating, transfer and moulding processes: a carrier frame, which is designed to interface with the mould, consists of side members to rigidly connect the end devices, which are used to hold the inserts and connect the internal pressure system.\",\n \"Spring loaded split blocks are clamped over the inserts 2 such that they maintain the shape of the over-wrapped or over-braided liner 1 or bladder during the heating process, and these blocks are transferred with the heated material into the mould.\",\n \"Also as part of the end device assembly, a cone 23 with an axial hole is used such that when pushed into the liner, a seal is created against the polymeric material, as shown in FIG.\",\n \"15.The cone and deformed bladder interface with a block containing a profiled radius on an internal axial hole such that the hole restrains the movement of the cone thereby ensuring that a seal is made.\",\n \"The cone is pushed into the deformable liner or bladder (FIG.\",\n \"15c) as the internal pressure system is connected in the moulding tool by an integral pneumatic cylinder, actuated by initiation of the internal pressure, coinciding with closure of the mould, such that cooling of the assembled liner or bladder with over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres is minimised before the internal pressure 21 is applied.\",\n \"After the moulding cycle is completed, the pneumatic cylinder is reversed and the cone 23 removed to enable release of the part.\",\n \"The polymeric liner or bladder material that protrudes from the finished component, typically 25 mm by 15 mm diameter, is then trimmed and can be recycled in the production of liners where required.\",\n \"The preheated assembly of liner or bladder with over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres is transported to a mould that is generally composed of two main pieces, with movable cores where appropriate to create detail and concave features, where the assembly is held between the two principle mould halves such that contact of both mould halves occurs substantially simultaneously, thereby ensuring substantially balanced heat transfer and cooling from the hot liner or bladder and over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres with the mould, which is maintained sufficiently below the matrix melting temperature to ensure a fast cooling rate and a reduced cycle time, but sufficiently above the glass transition temperature to achieve the desired surface impregnation, consolidation and hence surface quality (FIG.\",\n \"12).\",\n \"Internal pressure 21 is then applied to the liner 1 or bladder such that the liner 1 or bladder deforms against the over-braided mixtures of thermoplastic matrix and reinforcing fibres, impregnating and consolidating the over-braided mixtures of thermoplastic matrix and reinforcing fibres against the geometry of the mould wall where concurrent cooling of the assembled liner 1 or bladder and over-braided mixtures of thermoplastic matrix and reinforcing fibres occurs against the mould 20, as shown in FIG.\",\n \"13, where the temperature of the mould is maintained using a heating circuit and also a cooling circuit, where needed, as commonly used with other thermoplastic composite non-isothermal moulding processes.\",\n \"The internal pressure 21 in the liner 1 or bladder is maintained while the mould 20 is closed via a hydraulic or mechanical clamping device.\",\n \"Typical internal pressures using an over-wrapping or over-braiding of carbon fiber reinforced polyamide 12 are 10 bar, with the pressure depending on the degree of intimacy achieved between the reinforcing fibre and matrix polymer.\",\n \"Such low moulding pressures reduce the size and expense of both the hydraulic or mechanical mould clamping equipment and the mould itself.\",\n \"The internal pressure is maintained for a time period that ensures a good consolidation of the over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres, typically between 30 seconds and 180 seconds depending on the final void content required for the application and the wall thickness of the component.\",\n \"The internal pressure 21 is then released and the mould 20 opened to give the hollow composite part or vessel.\",\n \"FIG.\",\n \"16 summarises the principles of the temperature and pressure histories of the disclosed invention, which define the process as non-isothermal.\",\n \"The solid line indicates the pressure inside the liner, the dashed lines indicate the outer composite temperature (short dashes) and the liner temperature (long dashes).\",\n \"Where the preferred method of assembling and preparing the liner 1 or mandrel with over-wrapped or over-braided mixtures of thermoplastic matrix and reinforcing fibres with integral inserts 2 has been used, a net shape finished component results (FIG.\",\n \"17) with only minor finishing operations such as removing polymeric flash from the tool interface area, thereby avoiding the high additional cost of machining or trimming operations.\",\n \"In addition, the fibre reinforcement is sealed in the polymeric matrix and the lack of additional machining or trimming processes ensures good environmental resistance.\",\n \"The hollow composite parts or vessels can be used for many applications, as typical diameters of these vessels could be 25 cm or larger.\",\n \"These applications include for example the use as containers for compressed air e.g.\",\n \"for brake systems of cars and trucks, breathing apparatus, water storage, hollow automotive or aerospace space frame structures, and generally everywhere where lightweight is of advantage.\",\n \"List of Signs and Reference Numerals 1 liner 2 insert 3 fabrics 4 knits 5 tape 6 yarns/tows 7 forming clamps 8 heat 9 pressure 10 separating device 11 over-wrapped composite 12 liner over-wrapped with composite material with integral inserts 13 cool mould 14 clamping force 15 rapid heat source 16 pressurizing fluid 17 hot mould 18 rapid cooling source 19 cool mould 20 moulding tool 21 internal pressure 22 liner, over-wrapped composite not shown 23 conical or profiled plunger activated by 16 24 pre-heat phase of composite over-wrapped liner 25 transfer to mould 26 application of internal pressure to liner, impregnation, formation to mould geometry, consolidation, cooling 27 consolidated composite TMOLD mould temperature, less than melting temperature of thermoplastic liner and thermoplastic composite matrix Tm melting temperature of thermoplastic liner and/or thermoplastic composite matrix Tdeg degradation onset temperature\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471054"}}},{"rowIdx":569,"cells":{"text":{"kind":"list like","value":[["Methods and devices for treating and/or processing data","At the inputs and/or outputs, memories are assigned to a reconfigurable module to achieve decoupling of internal data processing and in particular decoupling of the reconfiguration cycles from the external data streams (to/from peripherals, memories, etc.",")."],["1-9.","(canceled) 10.A method for decoupling data streams in a reconfigurable module, comprising: decoupling module-internal data transfers from external data transfers via at least one FIFO memory.","11.A method for decoupling data streams in a reconfigurable module, comprising: providing multiple independent address generators.","12.The method as recited in claim 11, further comprising: configuring the address generators independently of data processing.","13.A method for programming a reconfigurable module, comprising: breaking down, by a compiler, address computations into multiple configurations.","14.A device for data processing, comprising: a field of reconfigurable modules; and an interface module configured for an exchange of data to be processed between the field of reconfigurable modules and an external circuit configuration, wherein the interface module includes at least one FIFO memory in which data may be stored and from which data may be retrieved.","15.The device as recited in claim 14, further comprising: a circuit configuration to determine data which will be needed in a future configuration and to preloading the determined data into the interface configuration.","16.The device according to claim 15, wherein the circuit configuration includes a memory area addressable rapidly via the interface module.","17.The device as recited in claim 16 wherein the rapidly addressable memory area includes at least one of a cache memory, an external RAM, a FIFO memory, and a register.","18.The device as recited in claim 14, further comprising: means for backing up data of a configuration which has previously been executed and is at least partially no longer active at the time of data retrieval, the configuration data being stored in an interface memory, the configuration data already canceled by reconfiguration of participating field elements.","19.The device as recited in claim 14, further comprising: means for backing up data of a configuration which has previously been executed and is at least partially no longer active at the time of data retrieval, for at least one of preventing deletion, and overwriting of data already sent once but not yet known to have been satisfactorily received.","20.The device as recited in claim 14, further comprising: at least one of multiple field elements and field elements grouped into configurations of addressable memories to store data at least one of non-locally and temporarily.","21.The device as recited in claim 20, wherein the addressable memory are RAM cells."],["<SOH> BACKGROUND INFORMATION <EOH>The present invention relates to reconfigurable components in general, and in particular but not exclusively the decoupling of data processing within the reconfigurable component and/or within parts of the reconfigurable component and data streams, specifically both within the reconfigurable component and also to and from peripherals, mass memories, host processors, and the like (see, e.g., German Patent Application Nos.","DE 101 10 530.4 and DE 102 02 044.2).","Memories are assigned to a reconfigurable module (VPU) at the inputs and/or outputs to achieve decoupling of internal data processing, the reconfiguration cycles in particular, from the external data streams (to/from peripherals, memories, etc.).","Reconfigurable architecture includes modules (VPUs) having a configurable function and/or interconnection, in particular integrated modules having a plurality of unidimensionally or multidimensionally positioned arithmetic and/or logic and/or analog and/or storage and/or internally/externally interconnecting modules, which are interconnected directly or via a bus system.","These generic modules include in particular systolic arrays, neural networks, multiprocessor systems, processors having a plurality of arithmetic units and/or logic cells and/or communication/peripheral cells (IO), interconnecting and networking modules such as crossbar switches, as well as conventional modules including FPGA, DPGA, Chameleon, XPUTER, etc.","Reference is also made in particular in this context to the following patents and patent applications of the same applicant: P 44 16 881.0-53, DE 197 81 412.3, DE 197 81 483.2, DE 196 54 846.2-53, DE 196 54 593.5-53, DE 197 04 044.6-53, DE 198 80 129.7, DE 198 61 088.2-53, DE 199 80 312.9, PCT/DE 00/01869, DE 100 36 627.9-33, DE 100 28 397.7, DE 101 10 530.4, DE 101 11 014.6, PCT/EP 00/10516, EP 01 102 674.7, DE 196 51 075.9, DE 196 54 846.2, DE 196 54 593.5, DE 197 04 728.9, DE 198 07 872.2, DE 101 39 170.6, DE 199 26 538.0, DE 101 42 904.5, DE 101 10 530.4, DE 102 02 044.2, DE 102 06 857.7, DE 101 35 210.7, EP 02 001 331.4, EP 01 129 923.7 as well as the particular parallel patent applications thereto.","The entire disclosure of these documents are incorporated herein by reference.","The above-mentioned architecture is used as an example to illustrate the present invention and is referred to hereinafter as VPU.","The architecture includes an arbitrary number of arithmetic, logic (including memory) and/or memory cells and/or networking cells and/or communication/peripheral (IO) cells (PAEs—Processing Array Elements), which may be positioned to form a unidimensional or multidimensional matrix (PA); the matrix may have different cells of any desired configuration.","Bus systems are also understood here as cells.","A configuration unit (CT) which affects the interconnection and function of the PA is assigned to the entire matrix or parts thereof.","Memory access methods for reconfigurable modules which operate according to a DMA principle are described in German Patent No.","P 44 16 881.0, where one or more DMAs are formed by configuration.","In German Patent Application No.","196 54 595.1, DMAs are fixedly implemented in the interface modules and may be triggered by the PA or the CT. German Patent Application No.","DE 196 54 846.2 describes how internal memories are written by external data streams and data is read out of the memory back into external units.","German Patent Application No.","DE 199 26 538.0 describes expanded memory concepts according to DE 196 54 846.2 for achieving more efficient and easier-to-program data transmission.","U.S. Pat.","No.","6,347,346 describes a memory system which corresponds in all essential points to German Patent Application No.","DE 196 54 846.2, having an explicit bus (global system port) to a global memory.","U.S. Pat.","No.","6,341,318 describes a method for decoupling external data streams from internal data processing by using a double-buffer method, in which one buffer records/reads out the external data while another buffer records/reads out the internal data; as soon as the buffers are full/empty, depending on their function, the buffers are switched, i.e., the buffer formerly responsible for the internal data now sends its data to the periphery (or reads new data from the periphery) and the buffer formerly responsible for the external data now sends its data to the PA (reads new data from the PA).","These double buffers are used in the application to buffer a cohesive data area.","Such double-buffer configurations have enormous disadvantages in the data-stream area in particular, i.e., in data streaming, in which large volumes of data streaming successively into a processor field or the like must always be processed in the same way."],["<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.","1 shows an example reconfigurable processor.","FIG.","2 a shows a direct FIFO to PA coupling.","FIG.","2 b shows IO connected via RAM-PAEs.","FIG.","2 c shows FIFOs connected upstream from the IOs.","FIGS.","3 a - 3 e show an example data processing method in a VPU.","FIGS.","4 a - 4 e show another example data processing method in a VPU.","FIG.","5 shows an example embodiment of a PAE.","FIG.","6 shows an example of a wiring connection of ALU-PAEs and RAM-PAEs via a bus system.","FIG.","7 a shows a circuit for writing data.","FIG.","7 b shows a circuit for reading data.","FIG.","8 shows an example connection between interface modules and/or PAEs to numerous and/or other data streams.","FIG.","9 shows an example sequence of a data read transfer via the circuit of FIG.","8 .","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["BACKGROUND INFORMATION The present invention relates to reconfigurable components in general, and in particular but not exclusively the decoupling of data processing within the reconfigurable component and/or within parts of the reconfigurable component and data streams, specifically both within the reconfigurable component and also to and from peripherals, mass memories, host processors, and the like (see, e.g., German Patent Application Nos.","DE 101 10 530.4 and DE 102 02 044.2).","Memories are assigned to a reconfigurable module (VPU) at the inputs and/or outputs to achieve decoupling of internal data processing, the reconfiguration cycles in particular, from the external data streams (to/from peripherals, memories, etc.).","Reconfigurable architecture includes modules (VPUs) having a configurable function and/or interconnection, in particular integrated modules having a plurality of unidimensionally or multidimensionally positioned arithmetic and/or logic and/or analog and/or storage and/or internally/externally interconnecting modules, which are interconnected directly or via a bus system.","These generic modules include in particular systolic arrays, neural networks, multiprocessor systems, processors having a plurality of arithmetic units and/or logic cells and/or communication/peripheral cells (IO), interconnecting and networking modules such as crossbar switches, as well as conventional modules including FPGA, DPGA, Chameleon, XPUTER, etc.","Reference is also made in particular in this context to the following patents and patent applications of the same applicant: P 44 16 881.0-53, DE 197 81 412.3, DE 197 81 483.2, DE 196 54 846.2-53, DE 196 54 593.5-53, DE 197 04 044.6-53, DE 198 80 129.7, DE 198 61 088.2-53, DE 199 80 312.9, PCT/DE 00/01869, DE 100 36 627.9-33, DE 100 28 397.7, DE 101 10 530.4, DE 101 11 014.6, PCT/EP 00/10516, EP 01 102 674.7, DE 196 51 075.9, DE 196 54 846.2, DE 196 54 593.5, DE 197 04 728.9, DE 198 07 872.2, DE 101 39 170.6, DE 199 26 538.0, DE 101 42 904.5, DE 101 10 530.4, DE 102 02 044.2, DE 102 06 857.7, DE 101 35 210.7, EP 02 001 331.4, EP 01 129 923.7 as well as the particular parallel patent applications thereto.","The entire disclosure of these documents are incorporated herein by reference.","The above-mentioned architecture is used as an example to illustrate the present invention and is referred to hereinafter as VPU.","The architecture includes an arbitrary number of arithmetic, logic (including memory) and/or memory cells and/or networking cells and/or communication/peripheral (IO) cells (PAEs—Processing Array Elements), which may be positioned to form a unidimensional or multidimensional matrix (PA); the matrix may have different cells of any desired configuration.","Bus systems are also understood here as cells.","A configuration unit (CT) which affects the interconnection and function of the PA is assigned to the entire matrix or parts thereof.","Memory access methods for reconfigurable modules which operate according to a DMA principle are described in German Patent No.","P 44 16 881.0, where one or more DMAs are formed by configuration.","In German Patent Application No.","196 54 595.1, DMAs are fixedly implemented in the interface modules and may be triggered by the PA or the CT. German Patent Application No.","DE 196 54 846.2 describes how internal memories are written by external data streams and data is read out of the memory back into external units.","German Patent Application No.","DE 199 26 538.0 describes expanded memory concepts according to DE 196 54 846.2 for achieving more efficient and easier-to-program data transmission.","U.S. Pat.","No.","6,347,346 describes a memory system which corresponds in all essential points to German Patent Application No.","DE 196 54 846.2, having an explicit bus (global system port) to a global memory.","U.S. Pat.","No.","6,341,318 describes a method for decoupling external data streams from internal data processing by using a double-buffer method, in which one buffer records/reads out the external data while another buffer records/reads out the internal data; as soon as the buffers are full/empty, depending on their function, the buffers are switched, i.e., the buffer formerly responsible for the internal data now sends its data to the periphery (or reads new data from the periphery) and the buffer formerly responsible for the external data now sends its data to the PA (reads new data from the PA).","These double buffers are used in the application to buffer a cohesive data area.","Such double-buffer configurations have enormous disadvantages in the data-stream area in particular, i.e., in data streaming, in which large volumes of data streaming successively into a processor field or the like must always be processed in the same way.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 shows an example reconfigurable processor.","FIG.","2a shows a direct FIFO to PA coupling.","FIG.","2b shows IO connected via RAM-PAEs.","FIG.","2c shows FIFOs connected upstream from the IOs.","FIGS.","3a-3e show an example data processing method in a VPU.","FIGS.","4a-4e show another example data processing method in a VPU.","FIG.","5 shows an example embodiment of a PAE.","FIG.","6 shows an example of a wiring connection of ALU-PAEs and RAM-PAEs via a bus system.","FIG.","7a shows a circuit for writing data.","FIG.","7b shows a circuit for reading data.","FIG.","8 shows an example connection between interface modules and/or PAEs to numerous and/or other data streams.","FIG.","9 shows an example sequence of a data read transfer via the circuit of FIG.","8.DETAILED DESCRIPTION An object of the present invention is to provide a novel approach for commercial use.","A method according to an example embodiment of the present invention, in contrast to the previously known related art, allows a significantly simpler means of controlling the buffers, i.e., memories, connected in between; the related art is disadvantageous in the core area of typical applications of reconfigurable processors in particular.","External and internal bus systems may be operated at different transfer rates and/or clock frequencies with no problem due to the memory devices connected in between because data is stored temporarily by the buffers.","In comparison with inferior designs from the related art, this method requires fewer memory devices, typically only half as many buffers, i.e., data transfer interface memory devices, thus greatly reducing the hardware costs.","The estimated reduction in hardware costs amounts to 25% to 50%.","It is also simpler to generate addresses and to program the configuration because the buffers are transparent for the programmer.","Hardware is simpler to write and to debug.","A paging method which buffers various data areas in particular for different configurations may be integrated.","It should first be pointed out that various memory systems are known as interfaces to the IO.","Reference is made to German Patent No.","and German Patent Application Nos.","P 44 16 881.0, DE 196 54 595.1, and DE 199 26 538.0.In addition, a method is described in German Patent Applciation No.","DE 196 54 846.2 in which data is first loaded from the IO, (1) data is stored within a VPU after being computed, (2) the array (PA) is reconfigured, (3) data is read out from the internal memory and written back to another internal memory, (4) this is continued until the fully computed result is sent to the IO.","Reconfiguration means, for example, that a function executed by a part of the field of reconfigurable units or the entire field and/or the data network and/or data and/or constants which are necessary in data processing is/are determined anew.","Depending on the application and/or embodiment, VPUs are reconfigured only completely or also partially, for example.","Different reconfiguration methods are implementable, e.g., complete reconfiguration by switching memory areas (see, e.g., German Patent Application Nos.","DE 196 51 075.9, DE 196 54 846.2) and/or wave reconfiguration (see, e.g., German Patent Application-Nos.","DE 198 07 872.2, DE 199 26 538.0, DE 100 28 397.7, DE 102 06 857.7) and/or simple configuring of addressable configuration memories (see, e.g., German Patent Application Nos.","DE 196 51 075.9, DE 196 54 846.2, DE 196 54 593.5).","The entire disclosure of each of the particular patent specifications is expressly incorporated herewith.","In one example embodiment, a VPU is entirely or partially configurable by wave reconfiguration or by directly setting addressable configuration memories.","Thus, one of the main operating principles of VPU modules is to copy data back and forth between multiple memories, with additional and optionally the same operations (e.g., long FIR filter) and/or other operations (e.g., FFT followed by Viterbi) being performed with the same data during each copying operation.","Depending on the particular application, data is read out from one or more memories and written into one or more memories.","For storing data streams and/or states (triggers, see, e.g., German Patent Application Nos.","DE 197 04 728.9, DE 199 26 538.0), internal/external memories (e.g., as FIFOs) are used and corresponding address generators are utilized.","Any appropriate memory architecture may be fixedly implemented specifically in the algorithm and/or flexibly configured.","For performance reasons, the internal memories of the VPU are preferably used, but basically external memories may also be used.","Assuming this, the following comments shall now be made regarding the basic design: Interface modules which communicate data between the bus systems of the PA and external units are assigned to an array (PA) (see, e.g., German Patent No.","P 44 16 881.0, and German Patent Application No.","DE 196 54 595.1).","Interface modules connect address buses and data buses in such a way as to form a fixed allocation between addresses and data.","Interface modules may preferably generate addresses or parts of addresses independently.","Interface modules are assigned to FIFOs which decouple internal data processing from external data transmission.","A FIFO here is a data-streamable buffer, i.e., input/output data memory, which need not be switched for data processing, in particular during execution of one and the same configuration.","If other data-streamable buffers are known in addition to FIFO memories, they will subsequently also be covered by the term where applicable.","In particular, ring memories having one or more pointers, in particular at least one write memory and one read memory, should also be mentioned.","Thus, for example, during multiple reconfiguration cycles for processing an application, the external data stream may be maintained as largely constant, regardless of internal processing cycles.","FIFOs are able to store incoming/outgoing data and/or addresses.","FIFOs may be integrated into an interface module or assigned to one or more of them.","Depending on the design, FIFOs may also be integrated into the interface modules, and at the same time additional FIFOs may be implemented separately.","It is also possible to use data-streamable buffers integrated into the module, e.g., by integration of FIFO groups into a chip which forms a reconfigurable processor array.","In one example embodiment, multiplexers for free allocation of interface modules and FIFOs may also be present between the FIFOs (including those that are separate) and the interface modules.","In one configuration, the connection of FIFOs to external modules or internal parts of the processor field performed by a multiplexer may be specified based on the processor field, e.g., by the PAE sending and/or receiving data, but it may also be determined, if desired, by a unit at a higher level of the hierarchy, such as a host processor in the case of division of data processing into a highly parallel part of the task and a poorly parallelizable part of the task and/or the multiplexer circuit may be determined by external specifications, which may be appropriate if, for example, it is indicated with the data which type of data is involved and how it is to be processed.","With regard to the external connection, units for protocol conversion between the internal and external bus protocols (e.g., RAMBUS, AMBA, PCI, etc.)","are also provided.","A plurality of different protocol converters may also be used within one embodiment.","The protocol converters may be designed separately or integrated into the FIFOs or interface modules.","In one possible embodiment, multiplexers for free assignment of interface modules/FIFOs and protocol converters may be provided between the (separate) protocol converters and the interface modules/FIFOs.","Downstream from the protocol converters there may be another multiplexer stage, so that a plurality of AMBA bus interfaces may be connected to the same AMBA bus, for example.","This multiplexer stage may also be formed, for example, by the property of an external bus of being able to address a plurality of units.","In one example embodiment, the circuit operates in master and slave operating modes.","In the master mode, addresses and bus accesses are generated by the circuit and/or the assigned PA; in slave mode, external units access the circuit, i.e., the PA.","In other embodiments, additional buffer memories or data collectors may be provided within the circuit, depending on the application, for exchanging data between interface modules.","These buffer memories preferably operate in a random access mode and/or an MMU paging mode and/or a stack mode and may have their own address generators.","The buffer memories are preferably designed as multi-port memories to permit simultaneous access of a plurality of interface modules.","It is possible to access the buffer memories from a higher-level data processing unit, in particular from processors such as DSPs, CPUs, microcontrollers, etc., assigned to the reconfigurable module (VPU).","Now the decoupling of external data streams in particular will be described.","According to one aspect of the present invention, the external data streams are decoupled by FIFOs (input/output FIFO, combined as IO-FIFO) which are used between protocol converters and interface modules.","The data processing method functions as follows: Through one or more input FIFOs, incoming data is decoupled from data processing in the array (PA).","Data processing may be performed in the following steps: 1.The input FIFO(s) is (are) read out, processed by the array (PA) and/or written into one or more (other) memories (RAM banki) assigned locally to the array and/or preferably connected laterally to the array.","The lateral connection has the advantage that the chip architecture and/or its design is/are simplified.","2.The array (PA) is reconfigured.","The memories (e.g., RAM bankl) are read out, data is processed and written into one or more memories (e.g., RAM bank2 and/or RAM bank1) or, as an alternative, data may already be written to the output FIFOs according to step 4.3.The array (PA) is reconfigured again and data is again written into a memory.","4.This is continued until the result is sent to one or more output FIFOs for output.","5.Then new data is again read out from the input FIFO(s) and processed accordingly, i.e., data processing is continued in step 1.With the preferred design of the input/output FIFOs (IO-FIFOs) as multi-ported FIFOs, data processing may be performed by protocol converters simultaneously with writing into and/or reading out from the particular FIFOs.","The method described above yields a time decoupling which permits “quasi-steady-state” processing of constant data streams in such a way that there is only a latency but no interruption in the data stream when the first data packets have passed through.","In an expanded embodiment, the IO-FIFOs may be designed so that the number of IO-FIFOs and their depth may be selected according to the application.","In other words, IO-FIFOs may be distributed or combined (e.g., via a transmission gate, multiplexer/demultiplexer, etc.)","so that there are more IO-FIFOs or they are deeper.","For example, 8 FIFOs of 1,024 words each may be implemented and configured so that 8 FIFOs of 1,024 words or 2 FIFOs of 4,096 words are configured or, for example, 1 FIFO may be configured with 4,096 words and 4 with 1,024 words.","Modifications of the data processing method described here are possible, depending on the design of the system and the requirements of the algorithms.","In an expanded embodiment, the FIFOs function in such a way that in the case of output FIFOs the addresses belonging to the data inputs are also stored in the FIFOs and/or input FIFOs are designed so that there is one FIFO for the reading addresses to be sent out/already sent out and one FIFO for the incoming data words assigned to the addresses.","Below is a discussion of how a FIFO-RAM bank coupling, which is possible according to the present invention, may be implemented in a particularly preferred variant of the present invention.","Depending on the application, it is possible to conduct the data transfer with the IO-FIFOs via one or more additional memory stages (RAM bank) which are assigned locally to the array or are preferably coupled laterally to the array and only then relay data to the data processing PAEs (e.g., ALU-PAEs described in, e.g., German Patent Appliation No.","DE 196 51 075.9).","In a preferred embodiment, RAM-PAEs have a plurality of data interfaces and address interfaces, they are thus designed as multi-port arrays.","Designability of a data interface and/or address interface as a global system port should also be mentioned as a possibility.","Additional memory stage(s) (RAM banks) may be implemented, for example, by memory modules corresponding to the RAM-PAEs, as described in, for example, German Patent Application No.","DE 196 54 846.2 and/or German Patent Application No.","DE 199 26 538.0 and/or International Patent Application No.","PCT/EP 00/10516.In other words, a RAM-PAE may constitute a passive memory which is limited (essentially) to the memory function (see, e.g., German Patent Application No.","DE 196 54 846.2) or an active memory which automatically generates and controls functions such as address computation and/or bus accesses (see, e.g., German Patent Application No.","DE 199 26 538.0).","In particular, in one possible embodiment, active address generation functions and/or data transfer functions may also be implemented for a “global system port.” Depending on the design, active memories may actively manage one or more data interfaces and address interfaces (active interfaces).","Active interfaces may be implemented, for example, by additional modules such as sequencers/state machines and/or ALUs and/or registers, etc., within a RAM-PAE and/or by suitable hardwiring of an active interface to other PAEs whose function and networking are configured in one or more RAM-PAEs in accordance with the functions to be implemented.","Different RAM-PAEs may be assigned to different other PAEs.","RAM-PAEs preferably have one or more of the following functions, i.e., modes of operation: random access, FIFO, stack, cache, MMU paging.","In a preferred embodiment, RAM-PAEs are connected via a bus to a higher-level configuration unit (CT) and may be configured by it in their function and/or interconnection and/or memory depth and/or mode of operation.","In addition, there is preferably also the possibility of preloading and reading out the memory contents by the CT, for example, to set constants and/or lookup tables (cos/sin).","Due to the use of multi-ported memories for the RAM-PAEs, writing and/or reading out of data into/from the IO-FIFOs and data access by the array (PA) may take place simultaneously, so that the RAM-PAEs may in turn again have a buffer property, as described in German Patent Application No.","DE 196 54 846.2, for example.","RAM-PAEs may be combined (as discussed in International Patent Application No.","PCT/EP 00/10516, for example) in such a way that larger memory blocks are formed and/or the RAM-PAEs operate so that the function of a larger memory is obtained (e.g., one 1,024-word RAM-PAE from two 512-word RAM-PAEs).","In an example embodiment, the units may be combined so that the same address is sent to multiple memories.","The address is subdivided so that one portion addresses the entries in the memories and another portion indicates the number of the memory selected (SEL).","Each memory has a unique number and may be selected unambiguously by comparing it with SEL.","In a preferred embodiment, the number for each memory is configurable.","In another and/or additional example embodiment, an address is relayed from one memory to the next.","This address is subdivided so that one portion addresses the entries in the memories and another portion indicates the number (SEL) of the memory selected.","This is modified each time data is relayed; for example, a 1 may be subtracted from this each time data is relayed.","The memory in which this address part has a certain value (e.g., zero) is activated.","In an example embodiment, the units may be combined so that the same address is sent to a plurality of memories.","The address is subdivided so that one part addresses the entries in the memories and another part indicates the number (SEL) of the memory selected.","A bus runs between memories, namely from one memory to the next, which has a reference address such that the address has a certain value (e.g., zero) in the first memory and this value is modified each time data is relayed (e.g., incremented by 1).","Therefore, each memory has a different unique reference address.","The portion of the address having the number of the selected memory is compared with the reference address in each case.","If they are identical, the particular memory is selected.","Depending on the design, the reference bus may be constructed using the ordinary data bus system or a separated bus.","In an example embodiment, there may be an area check of the address part SEL to rule out faulty addressing.","It should now be pointed out that RAM-PAEs may be used as FIFOs.","This may be preferred in particular when a comparatively large memory capacity is provided by RAM-PAEs.","Thus, in particular when using multi-ported memories for the RAM-PAEs, this yields the design option of dispensing with explicit IO-FIFOs and/or configuring a corresponding number of RAM-PAEs as FIFOs in addition to the IO-FIFOs and sending data from the IO to the corresponding memory ports.","This embodiment may be regarded as particularly cost efficient because no additional memories need be provided, but instead the memories of the VPU architecture, which are configurable in their function and/or interconnection (see, e.g., German Patent Application No.","DE 196 54 846.2, DE 199 26 538.0 and International Patent Application No.","PCT/EP 00/10516), are configured corresponding to the character of configurable processors.","It is also possible to provide a multiplexer/demultiplexer upstream and/or downstream from the FIFO.","Incoming and/or outgoing data streams may be formed from one or more data records.","For example, the following function uses two incoming data streams (a and b) and one outgoing data stream (x): function example (a, b : integer) -> x : integer for i:= 1 to 100 for j:= 1 to 100 x[i]:= a[i] * b[j].","This requirement may be met by using two approaches, for example: a) The number of IO channels implemented is exactly equal to the number of data streams required (see, e.g., German Patent No.","P 44 16 881.0; German Patent Application No.","DE 196 54 595.1); in the stated function, for example, three I/O channels would thus be necessary; or b) By using internal memories for decoupling data streams, more or less as a register set (see, e.g., German Patent Application Nos.","DE 199 26 538.0, DE 196 54 846.2).","The different data streams are exchanged between one or more memories and the IO (e.g., memory, peripheral, etc.)","by a time multiplex method, for example.","Data may then be exchanged internally in parallel with a plurality of memories, if necessary, if the IO data is sorted (split) accordingly during the transfer between these memories and the IO.","Approach a) is supported according to the present invention by making available a sufficient number of IO channels and IO-FIFOs.","However, this simple approach is unsatisfactory because an algorithm-dependent and very expensive number of IO channels, which cannot be determined precisely, must be made available.","Therefore, approach b) or a suitable combination of a) and b) may be preferred, e.g., two IO channels, one input and one output, data streams being multiplexed on each channel if necessary.","It should be pointed out that the interfaces should be capable of processing data streams, i.e., a sufficiently high clock frequency and/or sufficiently short latencies should be provided on the internal and/or external buses.","This may be the reason why a combination of the two variants may be particularly preferred, because by providing a plurality of parallel IO channels, the required clocking of external and/or internal buses may be reduced accordingly.","For approach b) or approaches based at least partially on approach b), it may be necessary to provide multiplexers and/or demultiplexers and to separate the data streams of one data channel (e.g., a) and b) should be separated from the input channel) or to combine a plurality of result channels on one output channel.","One or more multiplexers/demultiplexers (MuxDemux stage) may be located at different positions, depending on the technical hardware implementation and/or the functions to be executed.","For example, a) a MuxDemux stage may be connected between the input/output interface (e.g., described in German Patent Application No.","DE 196 54 595.1) and the FIFO stage (IO-FIFO and/or RAM-PAE as FIFO), b) a MuxDemux stage may be connected downstream from the FIFO stage (IO-FIFO and/or RAM-PAE as FIFO), i.e., between the FIFO stage and the PA, c) a MuxDemux stage may be connected between the IO-FIFO and the RAM-PAEs.","The MuxDemux stage may in turn either be fixedly implemented in the hardware and/or formed by a suitable configuration of any PAEs designed accordingly.","The position of the multiplexers/demultiplexers of the MuxDemux stage is determined by the configuration by a CT and/or the array (PA) and/or the IO itself, which may also be dynamically influenced, e.g., on the basis of the degree of filling of the FIFO(s) and/or on the basis of pending data transfers (arbitration).","In an example embodiment, the multiplexer/demultiplexer structure is formed by a configurable bus system (e.g., according to or resembling the bus system between the RAM/ALU/etc.—PAEs), whereby the bus system may in particular also be physically the same which is also used either by resource sharing or by a time multiplex method which may be implemented through a suitable reconfiguration.","It may be particularly preferred if addresses are generated in a particular manner, as is evident from the following discussion.","Addresses for internal or external memories may be computed by address generators.","For example, groups of PAEs may be configured accordingly and/or explicit address generators, implemented separately and specially, if necessary (e.g., DMAs such as those described in German Patent No.","DE 44 16 881) or within interface cells (such as those described in German Patent Application No.","DE 196 54 595.1) may be used.","In other words, either fixedly implemented address generators, which are integrated into a VPU or are implemented externally, may be used and/or the addresses may be calculated by a configuration of PAEs according to the requirements of an algorithm.","Simple address generators are preferably fixedly implemented in the interface modules and/or active memories (e.g., RAM-PAES).","For generation of complex address sequences (e.g., nonlinear, multidimensional, etc.","), PAEs may be configured accordingly and connected to the interface cells.","Such methods having the corresponding configurations are described in International Patent Application No.","PCT/EP 00/10516.Configured address generators may belong to another configuration (ConfigID, see, e.g., German Patent Application Nos.","DE 198 07 872.2, DE 199 26 538.0 and DE 100 28 397.7) other than data processing.","This makes a decoupling of address generation from data processing possible, so that in a preferred method, for example, addresses may already be generated and the corresponding data already loaded before or during the time when the data processing configuration is being configured.","It should be pointed out that such data preloading and/or address pregeneration is particularly preferred for increasing processor performance, in particular by reducing latency and/or the wait clock cycle.","Accordingly, the result data and its addresses may still be processed during or after removal of the data processing/generating configuration.","In particular, it is possible through the use of memories and/or buffers such as the FIFOs described here, for example, to further decouple data processing from memory access and/or IO access.","In a preferred procedure, it may be particularly effective to combine fixedly implemented address generators (HARD-AG) (see, e.g., German Patent Application No.","DE 196 54 595.1) and configurable address generators in the PA (SOFT-AG) in such a way that HARD-AGs are used for implementation of simple addressing schemes, while complex addressing sequences are computed by the SOFT-AG and then sent to the HARD-AG.","In other words, individual address generators may overload and reset one another.","Interface modules for reconfigurable components are described in German Patent Application No.","DE 196 54 595.1.The interface modules disclosed therein and their operation could still be improved further to increase processor efficiency and/or performance.","Therefore, within the scope of the present invention, a particular embodiment of interface modules is proposed below such as that disclosed in particular in German Patent Application No.","DE 196 54 595.1.Each interface module may have its own unique identifier (IOID) which is transmitted from/to a protocol converter and is used for assigning data transfers to a certain interface module or for addressing a certain interface module.","The IOID is preferably CT-configurable.","For example, the IOID may be used to select a certain interface module for a data transfer in the case of accesses by an external master.","In addition, the IOID may be used to assign the correct interface module to incoming read data.","To do so, the IOID is, for example, transmitted with the address of a data-read access to the IO-FIFOs and either stored there and/or relayed further to the external bus.","IO-FIFOs assign the IOIDs of the addresses sent out to the incoming read data and/or the IOIDs are also transmitted via the external bus and assigned by external devices or memories to the read data sent back.","IOIDs may then address the multiplexers (e.g., upstream from the interface modules) so that they direct the incoming read data to the correct interface module.","Interface modules and/or protocol converters conventionally operate as bus masters.","In a special embodiment, it is now proposed that interface modules and/or protocol converters shall function alternatively and/or fixedly and/or temporarily as bus slaves, in particular in a selectable manner, e.g., in response to certain events, states of state machines in PAEs, requirements of a central configuration administration unit (CT), etc.","In an additional embodiment, the interface modules are expanded so that generated addresses, in particular addresses generated in SOFT-AGs, are assigned a certain data packet.","A preferred embodiment of an interface module is described below: A preferred coupling of an interface module is accomplished by connecting any PAEs (RAM, ALU, etc.)","and/or the array (PA) via a bus (preferably configurable) to interface modules which are either connected to the protocol converters or have the protocol converters integrated into them.","In a variant embodiment, IO-FIFOs are integrated into the interface modules.","For write access (the VPU sends data to external IOs, e.g., memories/peripherals, etc.)","it is advantageous to link the address output to the data output, i.e., a data transfer takes place with the IO precisely when a valid address word and a valid data word are applied at the interface module, the two words may be originating from different sources.","Validity may be identified by a handshake protocol (RDY/ACK) according to German Patent Application Nos.","DE 196 51 075.9 or DE 101 10 530.4, for example.","Through suitable logic gating (e.g., AND) of RDY signals of address word and data word, the presence of two valid words is detectable, and IO access may be executed.","On execution of the IO access, the data words and the address words may be acknowledged by generating a corresponding ACK for the two transfers.","The IO access including the address and data, as well as the associated status signals, if necessary, may be decoupled in output FIFOs according to the present invention.","Bus control signals are preferably generated in the protocol converters.","For read access (the VPU receives data from external IOs, e.g., memories/peripherals, etc.","), the addresses for the access are first generated by an address generator (HARD-AG and/or SOFT-AG) and the address transfer is executed.","Read data may arrive in the same clock cycle or, at high frequencies, may arrive pipelined one or more clock cycles later.","Both addresses and data may be decoupled through IO-FIFOs.","The conventional RDY/ACK protocol may be used for acknowledgment of the data, and it may also be pipelined (see, e.g., German Patent Application Nos.","DE 196 54 595.1, DE 197 04 742.4, DE 199 26 538.0, DE 100 28 397.7 and DE 101 10 530.4).","The conventional RDY/ACK protocol may also be used for acknowledgment of the addresses.","However, acknowledgment of the addresses by the receiver results in a very long latency, which may have a negative effect on the performance of VPUs.","The latency may be bypassed in that the interface module acknowledges receipt of the address and synchronizes the incoming data assigned to the address with the address.","Acknowledgment and synchronization may be performed by any suitable acknowledgment circuit.","Two possible embodiments are explained in greater detail below, although in a non-limiting fashion: a) FIFO A FIFO stores the outgoing address cycles of the external bus transfers.","With each incoming data word as a response to an external-bus access, the FIFO is instructed accordingly.","Due to the FIFO character, the sequence of outgoing addresses corresponds to the sequence of outgoing data words.","The depth of the FIFO (i.e., the number of possible entries) is preferably adapted to the latency of the external system, so that any outgoing address may be acknowledged without latency and optimum data throughput is achieved.","Incoming data words are acknowledged according to the FIFO entry of the assigned address.","If the FIFO is full, the external system is no longer able to accept any additional addresses and the current outgoing address is not acknowledged and is thus held until data words of a preceding bus transfer have been received and one FIFO entry has been removed.","If the FIFO is empty, no valid bus transfer is executed and possibly incoming data words are not acknowledged.","b) Credit Counter Each outgoing address of external bus transfers is acknowledged and added to a counter (credit counter).","Incoming data words as a response to an external bus transfer are subtracted from the counter.","If the counter reaches a defined maximum value, the external system can no longer accept any more addresses and the current outgoing address is not acknowledged and is thus held until data words of a preceding bus transfer have been received and the counter has been decremented.","If the counter content is zero, no valid bus transfer is executed and incoming data words are not acknowledged.","To optimally support burst transfers, the method using a) (FIFO) is particularly preferred, and in particular FIFOs may be used like the FIFOs described below for handling burst accesses and the assignment of IOIDs to the read data.","The IO-FIFOs described here may be integrated into the interface modules.","In particular, an IO-FIFO may also be used for embodiment variant a).","The optional possibility of providing protocol converters is discussed above.","With regard to particularly advantageous possible embodiments of protocol converters, the following comments should be made: A protocol converter is responsible for managing and controlling an external bus.","The detailed structure and functioning of a protocol converter depend on the design of the external bus.","For example, an AMBA bus requires a protocol converter different from a RAMBUS.","Different protocol converters are connectable to the interface modules, and within one embodiment of a VPU, a plurality of, in particular, different protocol converters may be implemented.","In one preferred embodiment, the protocol converters are integrated into the IO-FIFOs of the present invention.","It is possible according to the present invention to provide burst bus access.","Modern bus systems and SoC bus systems transmit large volumes of data via burst sequences.","An address is first transmitted and data is then transmitted exclusively for a number of cycles (see AMBA Specification 2.0, ARM Limited).","For correctly executing burst accesses, several tasks are to be carried out: 1) Recognizing Burst Cycles Linear bus accesses, which may be converted into bursts, must be recognized to trigger burst transfers on the external bus.","For recognizing linear address sequences, a counter (TCOUNTER) may be used; it is first loaded with a first address of a first access and counts linearly up/down after each access.","If the subsequent address corresponds to the counter content, there is a linear and burst-capable sequence.","2) Aborting at Boundaries Some bus systems (e.g., AMBA) allow bursts (a) only up to a certain length and/or (b) only up to certain address limits (e.g., 1024 address blocks).","For (a), a simple counter may be implemented according to the present invention, which counts from the first desired or necessary bus access the number of data transmissions and at a certain-value which corresponds to the maximum length of the burst transfer, signals the boundary limits using a comparator, for example.","For L(b), the corresponding bit (e.g., the 10th bit for 1024 address limits) which represents the boundary limit may be compared between TCOUNTER and the current address (e.g., by an XOR function).","If the bit in the TCOUNTER is not equal to the bit in the current address, there has been a transfer beyond a boundary limit which is signaled accordingly.","3) Defining the Length If the external bus system does not require any information regarding the length of a burst cycle, it is possible and preferable according to the present invention to perform burst transfers of an indefinite length (cf.","AMBA).","If length information is expected and/or certain burst lengths are predetermined, the following procedure may be used according to the present invention.","Data and addresses to be transmitted are written into a FIFO, preferably with the joint use of the IO-FIFO, and are known on the basis of the number of addresses in the (IO-)FIFO.","For the addresses, an address FIFO is used, transmitting in master mode the addresses from the interface modules to the external bus and/or operating conversely in slave mode.","Data is written into a data FIFO, which transmits data according to the transmission (read/write).","In particular, a different FIFO may be used for write transfers and for read transfers.","The bus transfers may then be subdivided into fixed burst lengths, so that they are known before the individual burst transfers and may be stated on initiation of the burst, burst transfers of the maximum burst length preferably being formed first and if the number of remaining (IO-)FIFO entries is smaller than the current burst length, a next smaller burst length is used in each case.","For example, ten (IO-;)FIFO entries may be transmitted at a maximum burst length of 4 with 4, 4, 2 burst transfers.","4) Error Recovery Many external bus systems (cf.","AMBA) provide methods for error elimination in which failed bus transfers are repeated, for example.","The information as to whether a bus transfer has failed is transmitted at the end of a bus transfer, more or less as an acknowledgment for the bus transfer.","To repeat a bus transfer, it is now necessary for all the addresses to be available, and in the case of write access, the data to be written away must also be available.","According to the present invention, the address FIFOs (preferably the address FIFOs of the IO-FIFOs) are modified so that the read pointer is stored before each burst transfer.","Thus, a FIFO read pointer position memory means is provided, in particular an address FIFO read pointer position memory means.","This may form an integral part of the address FIFO in which, for example, a flag is provided, indicating that information stored in the FIFO represents a read pointer position or it may be provided separately from the FIFO.","As an alternative, a status indicating deletability could also be assigned to data stored in the FIFO, this status also being stored and reset to “deletable” if successful data transmission has been acknowledged.","If an error has occurred, the read pointer is reset at the position stored previously and the burst transfer is repeated.","If no error has occurred, the next burst transfer is executed and the read pointer is restored accordingly.","To prevent the write pointer from arriving at a current burst transfer and thus overwriting values which might still be needed in a repeat of the burst transfer, the full status of the FIFOs is determined by comparing the stored read pointer with the write pointer.","IO-FIFOs and/or FIFOs for managing burst transfers may preferably be expanded to incoming read data using the function of address assignment, which is known from the interface modules.","Incoming read data may also be assigned the IOID which is preferably stored in the FIFOs together with the addresses.","Through the assignment of the IOID to incoming read data, the assignment of the read data to the corresponding interface modules is possible by switching the multiplexers according to the IOIDs, for example.","According to the present invention, it is possible to use certain bus systems and/or to design bus systems in different ways.","This is described in further detail below.","Depending on the design, different bus systems may be used between the individual units, in particular the interface modules, the IO-FIFOs, the protocol converters, and a different bus system may be implemented between each of two units.","Different designs are implementable, the functions of a plurality of designs being combinable within one design.","A few design options are described below.","The simplest possible design is a direct connection of two units.","In an expanded embodiment, multiplexers are provided between the units, which may have different designs.","This example embodiment is preferred in particular when using a plurality of the particular units.","A multiplex function may be obtained using a configurable bus, which is configurable by a higher-level configuration unit (CT), specifically for a period of time for the connection of certain units.","In an example embodiment, the connections are defined by selectors which decode a portion of an address and/or an IOID, for example, by triggering the multiplexers for the interconnection of the units.","In a particularly preferred embodiment, the selectors are designed in such a way that a plurality of units may select a different unit at the same time, each of the units being arbitrated for selection in chronological sequence.","An example of a suitable bus system is described in, e.g., German Patent Application No.","DE 199 26 538.0.Additional states may be used for arbitration.","For example, data transfers between the interface modules and the IO-FIFOs may be optimized as follows: In each case one block of a defined size of data to be transmitted is combined within the FIFO stages.","As soon as a block is full/empty, a bus access is signaled to the arbiter for transmitting the data.","Data is transmitted in a type of burst transfer, i.e., the entire data block is transmitted by the arbiter during a bus allocation phase.","In other words, a bus allocation may take place in a manner determined by FIFO states of the connected FIFOs, data blocks being used for the determination of state within a FIFO.","If a FIFO is full, it may arbitrate the bus for emptying; if a FIFO is empty, it may arbitrate the bus for filling.","Additional states may be provided, e.g., in flush, which is used for emptying only partially full FIFOs and/or for filling only partially empty FIFOs.","For example, flush may be used in a change of configuration (reconfiguration).","In a preferred embodiment, the bus systems are designed as pipelines in order to achieve high data transfer rates and clock rates by using suitable register stages and may also function as FIFOs themselves, for example.","In a preferred embodiment, the multiplexer stage may also be designed as a pipeline.","According to the present invention, it is possible to connect a plurality of modules to one IO and to provide communication among the modules.","In this regard, the following should be pointed out: configuration modules which include a certain function and are reusable and/or relocatable within the PA are described in, for example, German Patent Application Nos.","DE 198 07 872.2, DE 199 26 538.0, and DE 100 28 397.7.A plurality of these configuration modules may be configured simultaneously into the PA, dependently and/or independently of one another.","The configuration modules must be hardwired to a limited IO, which is typically provided in particular only at certain locations and is therefore not relocatable, in such a way that the configuration modules are able to use the IOs simultaneously and data is assigned to the correct modules.","In addition, configuration modules that belong together (dependent) must be hardwired together in such a way that free relocation of the configuration modules is possible among one another in the PA.","Such a flexible design is in most cases not possible through the conventional networks (see, e.g., German Patent Nos.","P 44 16 881.0, 02, 03, 08), because this network must usually be explicitly allocated and routed through a router.","German Patent Application No.","DE 197 04 742.4 describes a method of constructing flexible data channels within a PAE matrix according to the algorithms to be executed so that a direct connection through and in accordance with a data transmission is created and subsequently dismantled again.","Data to be transmitted may be precisely assigned to one source and/or one destination.","In addition and/or as an alternative to German Patent Application No.","DE 197 04 742.4 and the procedures and configurations described therein, additional possibilities are now provided through the present invention, and methods (hereinafter referred to jointly as GlobalTrack) that permit flexible allocation and interconnection during run time may be used, e.g., serial buses, parallel buses and fiber optics, each with suitable protocols (e.g., Ethernet, Firewire, USB).","Reference is made here explicitly to transmission by light using a light-conducting substrate, in particular with appropriate modulation for decoupling of the channels.","Another particular feature of the present invention with respect to memory addressing, in particular paging and MMU options, is described below.","Data channels of one or multiple GlobalTracks may be connected via mediating nodes to an ordinary network, e.g., according to German Patent Nos.","P 44 16 881.0, 02, 03, 08.Depending on the implementation, the mediating nodes may be configured differently in the PA, e.g., assigned to each PAE, to a group and/or hierarchy of PAEs, and/or to every nth PAE.","In a particularly preferred embodiment, all PAEs, interface modules, etc., have a dedicated connection to a GlobalTrack.","A configuration module is designed in such a way that it has access to one or a plurality of these mediating nodes.","A plurality of configuration modules among one another and/or configuration modules and IOs may now be connected via the GlobalTrack.","With proper implementation (e.g., German Patent Application No.","DE 197 04 742.4) a plurality of connections may now be established and used simultaneously.","The connection between transmitters and receivers may be established in an addressed manner to permit individual data transfer.","In other words, transmitters and receivers are identifiable via GlobalTrack.","An unambiguous assignment of transmitted data is thus possible.","Using an expanded IO, which also transmits the transmitter address and receiver address—as is described in German Patent Application No.","DE 101 10 530.4, for example—and the multiplexing methods described in German Patent Application No.","DE 196 54 595.1, data for different modules may be transmitted via the IO and may also be assigned unambiguously.","In a preferred embodiment, data transfer is synchronized by handshake signals, for example.","In addition, data transfer may also be pipelined, i.e., via a plurality of registers implemented in the GlobalTrack or assigned to it.","In a very complex design for large-scale VPUs or for their interconnection, a GlobalTrack may be designed in a network topology using switches and routers; for example, Ethernet could be used.","It should be pointed out that different media may be used for GlobalTrack topologies, e.g., the method described in German Patent Application No.","DE 197 04 742.4 for VPU-internal connections and Ethernet for connections among VPUs.","Memories (e.g., RAM-PAEs) may be equipped with an MMU-like paging method.","For example, a large external memory could then be broken down into segments (pages), which in the case of data access within a segment would be loaded into one of the internal memories and, at a later point in time, after termination of data access, would be written back into the external memory.","In a preferred embodiment, addresses sent to a (internal) memory are broken down into an address area, which is within the internal memory (MEMADR) (e.g., the lower 10 bits in a 1,024-entry memory) and a page address (the bits above the lower 10).","The size of a page is thus determined by MEMADR.","The page address is compared with a register (page register) assigned to the internal memory.","The register stores the value of the page address last transferred from a higher-level external (main) memory into the internal memory.","If the page address matches the page register, free access to the internal memory may take place.","If the address does not match (page fault), the current page content is written, preferably linearly, into the external (main) memory at the location indicated by the page register.","The memory area in the external (main) memory (page) which begins at the location of the current new page address is written into the internal memory.","In a particularly preferred embodiment, it is possible to specify by configuration whether or not, in the event of a page fault, the new page is to be transferred from the external (main) memory into the internal memory.","In a particularly preferred embodiment, it is possible to specify by configuration whether or not, in the event of a page fault, the old page is to be transferred from the internal memory into the external (main) memory.","The comparison of the page address with the page register preferably takes place within the particular memory.","Data transfer control in the event of page faults may be configured accordingly by any PAEs and/or may take place via DMAs (e.g., in the interface modules or external DMAs).","In a particularly preferred embodiment, the internal memories are designed as active memories having integrated data transfer control (see, e.g., German Patent Application No.","DE 199 26 538.0).","In another possible embodiment, an internal memory may have a plurality (p) of pages, the size of a page then preferably being equal to the size of the memory divided by p. A translation table (translation look-aside buffer=TLB) which is preferably designed like a fully associative cache replaces the page register and translates page addresses to addresses in the internal memory; in other words, a virtual address may be translated into a physical address.","If a page is not included in the translation table (TLB), a page fault occurs.","If the translation table has no room for new additional pages, pages may be transferred from the internal memory into the external (main) memory and removed from the translation table so that free space is again available in the internal memory.","It should be pointed out explicitly that a detailed discussion is not necessary because a plurality of conventional MMU methods may be used and may be used with only minor and obvious modifications.","The possibility of providing a collector memory, as it is known, has been mentioned above.","In this regard, the following details should also be mentioned.","A collector memory (collector) capable of storing larger volumes of data may be connected between the interface modules and IO-FIFOs.","The collector may be used for exchanging data between the interface modules, i.e., between memories assigned to the array (e.g., RAM-PAEs).","The collector may be used as a buffer between data within a reconfigurable module and external data.","A collector may function as a buffer for data between different reconfiguration steps; for example, it may store data of different configurations while different configurations are active and are being configured.","At deactivation of configurations, the collector stores their data, and data of the newly configured and active configurations is transmitted to the PA, e.g., to memories assigned to the array (RAM-PAEs).","A plurality of interface modules may have access to the collector and may manage data in separate and/or jointly accessible memory areas.","In a preferred embodiment, the collector may have multiple terminals for interface modules, which may be accessed simultaneously (i.e., it is designed as a multi-port collector device).","The collector has one or more terminals to an external memory and/or external peripherals.","These terminals may be connected to the IO-FIFOs in particular.","In an expanded embodiment, processors assigned to the VPU, such as DSPs, CPUs and microcontrollers, may access the collector.","This is preferably accomplished via another multi-port interface.","In a preferred embodiment, an address translation table is assigned to the collector.","Each interface may have its own address translation table or all the interfaces may share one address translation table.","The address translation table may be managed by the PA and/or a CT and/or an external unit.","The address translation table is used to assign collector memory areas to any addresses and it operates like an MMU system.","If an address area (page) is not present within the collector (pagemiss), this address area may be loaded into the collector from an external memory.","In addition, address areas (pages) may be written from the collector into the external memory.","For data transfer to or between the external memory, a DMA is preferably used.","A memory area within the collector may be indicated to the DMA for reading or writing transmission; the corresponding addresses in the external memory may be indicated separately or preferably removed by the DMA from the address translation table.","A collector and its address generators (e.g., DMAs) may preferably operate according to or like MMU systems, which are conventional for processors according to the related art.","Addresses may be translated by using translation tables (TLB) for access to the collector.","According to the present invention, all MMU embodiments and methods described for internal memories may also be used on a collector.","The operational specifics will not be discussed further here because they correspond to or closely resemble the related art.","In an expanded or preferred embodiment, a plurality of collectors may be implemented.","According to the present invention, it is possible to optimize access to memory.","The following should be pointed out in this regard: One basic property of the preferred reconfigurable VPU architecture PACT-XPP is the possibility of superimposing reconfiguration and data processing (see, e.g., German Patent No.","P 44 16 881.0, and German Patent Application Nos.","DE 196 51 075.9, DE 196 54 846.2, DE 196 54 593.5, DE 198 07 872.2, DE 199 26 538.0, DE 100 28 397.7, DE 102 06 857.7).","In other words, for example: a) the next configuration may already be preloaded during data processing; and/or b) data processing in other already-configured elements may already begin while a number of configurable elements or certain configurations are not yet configured or are in the process of being configured; and/or c) the configuration of various activities is superimposed or decoupled in such a way that they run with a mutual time offset at optimum performance (see 8.1 address generation).","Modern memory protocols (e.g., SDRAM, DDRAM, RAMBUS) usually have the following sequence or a sequence having a similar effect, but steps 2 and 3 may possibly also occur in the opposite order: 1.Initializing access with the address given; 2.A long latency; 3.Rapid transmission of data blocks, usually as a burst.","This property may be utilized in a performance-efficient manner in VPU technology.","For example, it is possible to separate the steps of computation of the address(es), initialization of memory access, data transfer and data processing in the array (PA) in such a way that different (chronological) configurations occur, so that largely optimum superpositioning of the memory cycles and data processing cycles may be achieved.","Multiple steps may also be combined, depending on the application.","For example, the following method corresponds to this principle: The application AP, which includes a plurality of configurations (ap=1, 2, .",".",".",", z), is to be executed.","Furthermore, additional applications/configurations which are combined under WA are to be executed on the VPU: 1.Read addresses are first computed (in an ap configuration of AP) and the data transfers and IO-FIFOs are initialized; 2.Data transmitted for AP and now present in IO-FIFOs is processed (in an (ap+1) configuration) and, if necessary, stored in FIFOs, buffers or intermediate memories, etc.","; 2a.","Computation of results may require a plurality of configuration cycles (n) at the end of which the results are stored in an IO-FIFO, and 3.The addresses of the results are computed and the data transfer is initialized; this may take place in parallel or later in the same configuration or in an (ap+n+2) configuration; at the same time or with a time offset, data is then written from the IO-FIFOs into the memories.","Between the steps, any configuration from WA may be executed, e.g., when a waiting time is necessary between steps, because data is not yet available.","Likewise, in parallel with the processing of AP, configurations from WA may be executed during the steps, e.g., if AP does not use the resources required for WA.","It will be self-evident to those skilled in the art that variously modified embodiments of this method are also possible.","In one possible embodiment, the processing method may take place as shown below (Z marks a configuration cycle, i.e., a unit of time): Other configurations Z Configuration AP (WA) 1 Compute read addresses, Any other initialize access configurations 2 Input of data and/or data 3 + k Process data processing, read/ (if necessary in a plurality of write processes (k) configuration cycles) using IO-FIFOs 4 + k Compute write addresses, and/or RAM-PAEs in initialize access other resources or 5 + k Output of data time-multiplexed resources via configuration cycles This sequence may be utilized efficiently by the data processing method described in, for example, German Patent Application No.","DE 102 02 044.2 in particular.","The methods and devices described above are preferably operated using special compilers, which are expanded in particular in comparison with traditional compilers.","The following should be pointed out in this regard: For generating configurations, compilers that run on any computer system are used.","Typical compilers include, for example, C-compilers and/or even NML compilers for VPU technology, for example.","Particularly suitable compiler methods are described in German Patent Application Nos.","DE 101 39 170.6, and DE 101 29 237.6, and European Patent No.","EP 02 001 331.4, for example.","The compiler, at least partially, preferably takes into account the following particular factors: Separation of addressing into 1.external addressing, i.e., data transfers with external modules, 2.internal addressing, i.e., data transfers among PAEs, in particular between RAM-PAEs and ALU-PAEs, 3.in addition, time decoupling also deserves special attention.","Bus transfers are broken down into internal and external transfers.","bt1) External read accesses are separated and, in one possible embodiment, they are also translated into a separate configuration.","Data is transmitted from an external memory to an internal memory.","bt2) Internal accesses are coupled to data processing, i.e., internal memories are read and/or written for data processing.","bt3) External write accesses are separated and, in one possible embodiment, they are also translated into a separate configuration.","Data is transmitted from an internal memory into an external memory.","bt1, bt2, and bt3 may be translated into different configurations which may, if necessary, be executed at a different point in time.","This method will now be illustrated on the basis of the following example: function example (a, b:integer)→x:integer for i := 1 to 100 for j := 1 to 100 x[i] := a[i] * b[j].","This function is transformed by the compiler into three parts, i.e., configurations (subconfig): example#dload: Loads data from externally (memories, peripherals, etc.)","and writes it into internal memories.","Internal memories are indicated by r# and the name of the original variable.","example#process: Corresponds to the actual data processing.","This reads data out of internal operands and writes the results back into internal memories.","example#dstore: Writes the results from the internal memory into externally (memories, peripherals, etc.).","function example# (a, b : integer) -> x : integer subconfig example#dload for i := 1 to 100 r#a[i] := a[i] for j := 1 to 100 r#b[j] := b[j] subconfig example#process for i := 1 to 100 for j := 1 to 100 r#x[i] := r#a[i] * r#b[j] subconfig example#dstore for i := 1 to 100 x[i] := r#x[i].","An effect of the example method is that instead of i*j=100*100=10,000 external accesses, only i+j=100+100=200 external accesses are performed for reading the operands.","These accesses are also completely linear, which greatly accelerates the transfer rate in modern bus systems (burst) and/or memories (SDRAM, DDRAM, RAMBUS, etc.).","Internal memory accesses take place in parallel, because different memories have been assigned to the operands.","For writing the results, i=100 external accesses are necessary and may again be performed linearly at maximum performance.","If the number of data transfers is not known in advance (e.g., WHILE loop) or is very large, a method may be used which reloads the operands as necessary through subprogram call instructions and/or writes the results externally.","In a preferred embodiment, the states of the FIFOs may (also) be queried: “empty” if the FIFO is empty and “full” if the FIFO is full.","The program flow responds according to the states.","It should be pointed out that certain variables (e.g., ai, bi, xi) are defined globally.","For performance optimization, a scheduler may execute the configurations example#dloada, example#dloadb before calling up example#process according to the methods already described, so that data is already preloaded.","Likewise, example#dstore(n) may still be called up after termination of example#process in order to empty r#x.","subconfig example#dloada(n) while !full(r#a) AND ai <= n r#a[ai] := a[ai] ai++ subconfig example#dloadb(n) while !full(r#b) AND bi <= n r#b[bi] := b[bi] bi++ subconfig example#dstore (n) while !empty(r#x) AND xi <= n x[xi] := r#x[xi] xi++ subconfig example#process for i := 1 to n for j := 1 to m if empty(r#a) then example#dloada(n) if empty(r#b) then example#dloadb(m) if full(r#x) then example#dstore(n) r#x[i] := r#a[i] + r#b[j] bj := 1.The subprogram call instructions and managing of the global variables are comparatively complex for reconfigurable architectures.","Therefore, in a preferred embodiment, the following optimization may be performed; in this optimized method, all configurations are run largely independently and are terminated after being completely processed (terminate).","Since data b[j] is required repeatedly, example#dloadb must accordingly be run through repeatedly.","To do so, for example, two alternatives will be described: Alternative 1: example#dloadb terminates after each run-through and is reconfigured for each new start by example#process.","Alternative 2: example#dloadb runs infinitely and is terminated by example#process.","While “idle,” a configuration is inactive (waiting).","subconfig example#dloada(n) for i := 1 to n while full(r#a) idle r#a[i] :=a[i] terminate subconfig example#dloadb(n) while 1 // ALTERNATIVE 2 for i := 1 to n while full(r#b) idle r#b[i] := a[i] terminate subconfig example#dstore(n) for i := 1 to n while empty(r#b) idle x[i] := r#x[i] terminate subconfig example#process for i := 1 to n for j := 1 to m while empty(r#a) or empty(r#b) or full(r#x) idle r#x[i] := r#a[i] * r#b[j] config example#dloadb(n) // ALTERNATIVE 1 terminate example#dloadb(n) // ALTERNATIVE 2 terminate To avoid waiting cycles, configurations may also be terminated as soon as they are temporarily no longer able to continue fulfilling their function.","The corresponding configuration is removed from the reconfigurable module but remains in the scheduler.","Therefore, the “reenter” instruction is used for this below.","The relevant variables are saved before termination and are restored when configuration is repeated: subconfig example#dloada(n) for ai := 1 to n if full(r#a) reenter r#a[ai] := a[ai] terminate subconfig example#dloadb(n) while 1 // ALTERNATIVE 2 for bi := 1 to n if full(r#b) reenter r#b[bi] := a[bi] terminate subconfig example#dstore(n) for xi := 1 to n if empty(r#b) reenter x[xi] := r#x[xi] terminate subconfig example#process for i := 1 to n for j := 1 to m if empty(r#a) or empty(r#b) or full(r#x) reenter r#x[i] := r#a[i] * r#b[j] config example#dloadb(n) // ALTERNATIVE 1 terminate example#dloadb(n) // ALTERNATIVE 2 terminate With regard to the preceding discussion and to the following, the possibility of using a ‘context switch’ according to the present invention should also be pointed out.","In this regard, the following should be noted: Repeated start of configurations, e.g., “reenter,” requires that local data (e.g., ai, bi, xi) be backed up and restored.","Known related-art methods provide explicit interfaces to memories or to a CT to transmit data.","All of these methods may be inconsistent and/or may require additional hardware.","The context switch according to the present invention is implemented in such a way that a first configuration is removed; data to be backed up remains in the corresponding memories (REGs) (memories, registers, counters, etc.).","A second configuration is loaded; this connects the REGs in a suitable manner and in a defined sequence to one or multiple global memory (memories).","The configuration may use address generators, for example, to access the global memory (memories).","The configuration may use address generators, for example, to access REGs designed as memories.","According to the configured connection between the REGs, the contents of the REGs are written into the global memory in a defined sequence, the particular addresses being predetermined by address generators.","The address generator generates the addresses for the global memory (memories) in such a way that the memory areas (PUSHAREA) that have been written are unambiguously assigned to the first configuration removed.","In other words, different address areas are preferably provided for different configurations.","The configuration corresponds to a PUSH of ordinary processors.","Other configurations subsequently use the resources.","The first configuration is to be started again, but first a third configuration which connects the REGs of the first configuration in a defined sequence is started.","The configuration may use address generators, for example, to access the global memory or memories.","The configuration may use address generators, for example, to access REGs designed as memories.","An address generator generates addresses, so that correct access to the PUSHAREA assigned to the first configuration takes place.","The generated addresses and the configured sequence of the REGs are such that data of the REGs is written from the memories into the REGs in the original order.","The configuration corresponds to a POP of ordinary processors.","The first configuration is restarted.","In summary, a context switch is implemented in such a way that data to be backed up is exchanged with a global memory by loading particular configurations which operate like processor architectures known from PUSH/POP.","There is also the possibility of providing a special task switch and/or multiconfiguration handling.","In a preferred mode of operation, different data blocks of different configurations may be partitioned.","These partitions may be accessed in a time-optimized manner by preloading a portion of the operands of a subsequent configuration P from external (main) memories and/or other (peripheral) data streams into the internal memories, e.g., during execution of a configuration Q, and during the execution of P, the results of Q as a portion of the total result from the internal memories are written into external (main) memories and/or other (peripheral) data streams.","The functioning here differs considerably from that described in, for example, U.S. Pat.","No.","6,341,318.A data stream or data block is preferably decoupled by a FIFO structure (e.g., IO-FIFO).","Different data streams or data blocks of different configurations in particular are preferably decoupled by different memories and/or FIFO areas and/or assignment marks in the FIFOs.","The optional MMU methods described above may be used for decoupling and buffering external data.","In one type of application, a large external data block may be broken down into a plurality of segments, each may be processed within a VPU.","In an additional preferred mode of operation, different data blocks of different configurations may be broken down into partitions according to the method described above, these partitions now being defined as pages for an MMU.","In this way, time-optimized access is possible by preloading the operands of a subsequent configuration P as a page from external (main) memories and/or other (peripheral) data streams into the internal memories, e.g., during execution of a configuration Q in the PA, and during the execution of P, the results of Q as a page from the internal memories are written into external (main) memories and/or other (peripheral) data streams.","For the methods described above, preferably internal memories capable of managing a plurality of partitions and/or pages are used.","These methods may be used for RAM-PAEs and/or collector memories.","Memories having a plurality of bus interfaces (multi-port) are preferably used to permit simultaneous access of MMUs and/or the PA and/or additional address generators/data transfer devices.","In one embodiment, identifiers are also transmitted in the data transfers, permitting an assignment of data to a resource and/or an application.","For example, the method described in German Patent Application No.","DE 101 10 530.4 may be used.","Different identifiers may also be used simnultaneously.","In a particularly preferred embodiment, an application identifier (APID) is also transmitted in each data transfer along with the addresses and/or data.","An application includes a plurality of configurations.","On the basis of the APID, the transmitted data is assigned to an application and/or to the memories or other resources (e.g., PAEs, buses, etc.)","intended for an application.","To this end, the APIDs may be used in different ways.","Interface modules, for example, may be selected by APIDs accordingly.","Memories, for example, may be selected by APIDs accordingly.","PAEs, for example, may be selected by APIDs accordingly.","For example, memory segments in internal memories (e.g., RAM-PAEs, collector(s)) may be assigned by APIDs.","To do so, the APIDS, like an address part, may be entered into a TLB assigned to an internal memory so that a certain memory area (page) is assigned and selected as a function of an APID.","This method yields the possibility of efficiently managing and accessing data of different applications within a VPU.","There is the option of explicitly deleting data of certain APIDs (APID-DEL) and/or writing into external (main) memories and/or other (peripheral) data streams (APID-FLUSH).","This may take place whenever an application is terminated.","APID-DEL and/or APID-FLUSH may be triggered by a configuration and/or by a higher-level loading unit (CT) and/or externally.","The following processing example is presented to illustrate the method.","An application Q (e.g., APID=Q) may include a configuration for reading operands (e.g., ConfigID=j), a configuration for processing operands (e.g., ConfigID=w), and a configuration for writing results (e.g., ConfigID=s).","Configuration j is executed first to read the operands chronologically optimally decoupled.","Configurations of other applications may be executed simultaneously.","The operands are written from external (main) memories and/or (peripheral) data streams into certain internal memories and/or memory areas according to the APID identifier.","Configuration w is executed to process the stored operands.","To do so, the corresponding operands in the internal memories and/or memory areas are accessed by citation of APIDs.","Results are written into internal memories and/or memory areas accordingly by citation of APIDs.","Configurations of other applications may be executed simultaneously.","In conclusion, configuration s writes the stored results from the internal memories and/or memory areas into external (main) memories and/or other (peripheral) data streams.","Configurations of other applications may be executed simultaneously.","To this extent, the basic sequence of the method corresponds to that described above for optimization of memory access.","If data for a certain APID is not present in the memories or if there is no longer any free memory space for this data, a page fault may be triggered for transmission of the data.","While a module was initially assumed in which a field of reconfigurable elements is provided having little additional wiring, such as memories, FIFOs, and the like, it is also possible to use the ideas according to the present invention for systems known as “systems on a chip” (SoC).","For SoCs the terms “internal” and “external” are not completely applicable in the traditional terminology, e.g., when a VPU is linked to other modules (e.g., peripherals, other processors, and in particular memories) on a single chip.","The following definition of terms may then apply; this should not be interpreted as restricting the scope of the invention but instead is given only as an example of how the ideas of the present invention may be applied with no problem to constructs which traditionally use a different terminology: internal: within a VPU architecture and/or areas belonging to the VPU architecture and IP, external: outside of a VPU architecture, i.e., all other modules, e.g., peripherals, other processors, and in particular memories on a SoC and/or outside the chip in which the VPU architecture is located.","A preferred embodiment will now be described.","In a particularly preferred embodiment, data processing PAEs are located and connected locally in the PA (e.g., ALUs, logic, etc.).","RAM-PAEs may be incorporated locally into the PA, but in a particularly preferred embodiment they are remote from the PA or are placed at its edges (see, e.g., German Patent Application No.","DE 100 50 442.6).","This takes place so as not to interfere with the homogeneity of the PA in the case of large RAM-PAE memories, where the space required is much greater than with ALU-PAEs and because of a gate/transistor layout (e.g., GDS2) of memory cells, which usually varies greatly.","If the RAM-PAEs have dedicated connections to an external bus system (e.g., global bus), they are preferably located at the edges of a PA for reasons of layout, floor plan, and manufacturing.","The configurable bus system of the PA is typically used for the physical connection.","In an expanded embodiment, PAEs and interface modules, as well as additional configurable modules, if necessary, have a dedicated connection to a dedicated global bus, e.g., a GlobalTrack.","Interface modules and in particular protocol converters are preferably remote from the PA and are placed outside of its configuration.","This takes place so as not to interfere with the homogeneity of the PA and because of a gate/transistor layout (e.g., GDS2) of the interface modules/protocol converters, which usually varies greatly.","In addition, the connections to external units are preferably placed at the edges of a PA for reasons of layout, floor plan, and manufacturing.","The interface modules are preferably connected to the PA by the configurable bus system of the PA, the interface modules being connected to its outer edges.","The bus system allows data exchange to take place configurably between interface modules and any PAEs within the PA.","In other words, within one or different configurations, some interface modules may be connected to RAM-PAEs, for example, while other interface modules may be connected to ALU-PAEs, for example.","The IO-FIFOs are preferably integrated into the protocol converter.","To permit a greater flexibility in the assignment of the internal data streams to the external data streams, the interface modules and protocol converters are designed separately and are connected via a configurable bus system.","The present invention is explained in greater detail below only as an example and in a nonrestrictive manner with reference to the drawings.","FIG.","1 shows a particularly preferred design of a reconfigurable processor which includes a core (array PA) (0103) including, for example, a configuration of ALU-PAEs (0101) (for performing computations) and RAM-PAEs (0102) (for saving data) and thus corresponds to the basic principle described in, for example, German Patent Application No.","DE 196 54 846.2.The RAM-PAEs are preferably not integrated locally into the core, but instead are remote from the ALU-PAEs at the edges of or outside the core.","This takes place so as not to interfere with the homogeneity of the PA in the case of large RAM-PAE memories where the space requirement is far greater than that of ALU-PAEs and because of a gate/transistor layout (e.g., GDS2) of memory cells which usually varies greatly.","If the RAM-PAEs have dedicated connections to an external bus system (e.g., dedicated global bus; GlobalTrack; etc.","), then they are preferably placed at the edges of a PA for reasons of layout, floor plan, and manufacturing.","The individual units are interlinked via bus systems (0104).","Interface modules (interface modules and protocol converters, if necessary) (0105) are located at the edges of the core and are connected to external buses (IO), as similarly described in German Patent Application No.","DE 196 54 595.1.The interface modules may have different designs, depending on the implementation, and may fulfill one or more of the following functions, for example: 1.Combining and synchronizing a plurality of bus systems to synchronize addresses and data for example, 2.Address generators and/or DMAs, 3.FIFO stages for decoupling data and/or addresses, 4.Interface controllers (e.g., for AMBA bus, RAMBUS, RapidIO, USB, DDRRAM, etc.).","FIG.","2 shows a different embodiment of the architecture according to the present invention, depicting a configuration 0201 of ALU-PAEs (PA) linked to a plurality of RAM-PAEs (0202).","External buses (IOs) (0204) are connected via FIFOs (0203).","FIG.","2a shows a direct FIFO to PA coupling.","FIG.","2b shows the IO (0204) connected to 0201 via the RAM-PAEs (0202).","The connection occurs typically via the configurable bus system 0104 or a dedicated bus system.","Multiplexers/demultiplexers (0205) switch a plurality of buses (0104) to the IOs (0204).","The multiplexers are triggered by a configuration logic and/or address selector logic and/or an arbiter (0206).","The multiplexers may also be triggered through the PA.","FIG.","2c corresponds to FIG.","2b, but FIFOs (0203) have been connected upstream from the IOs.","The diagrams in FIG.","3 correspond to those in FIG.","2, which is why the same reference numbers are used.","FIG.","3 illustrates the preferred data processing method in a VPU.","FIG.","3a: data passes through the IO (0204) into an input FIFO (0303 corresponding to 0203) and is loaded from this into the PA (0201) and/or beforehand into memory 0202.FIGS.","3b-e show the data execution in which data is transmitted between the memories.","During this period of time, the FIFOs may still transmit input data (0301) and/or output data (0302).","In FIG.","3f, data is loaded from the PA and/or from the memories into the output FIFO (0304).","It should be pointed out again that input of data from the input FIFO into the RAM-PAEs or 0201 and writing of data from 0201 or the RAM-PAEs may take place simultaneously.","It should likewise be pointed out that the input/output FIFOs are able to receive and/or send external data continuously during steps a-f.","FIG.","4 shows the same method in a slightly modified version in which multiplexers/demultiplexers (0401) are connected between the FIFOs and 0201 for simple data distribution.","The multiplexers are triggered by a configuration logic and/or address selector logic and/or an arbiter (0402).","Multiple configurations take place for data processing (a-e).","The data may be read into memories and/or directly (0403) into the PA from the FIFOs (input FIFOs).","During the input operation, data may be written from the PA and/or memories into FIFOs (output FIFOS) (0404).","For data output, data may be written from the memories and/or directly (0405) from the PA into the FIFOs.","Meanwhile, new data may be written from the input FIFOs into memories and/or the PA (0406).","New data (0407) may already be entered during a last configuration, for example.","During the entire processing, data may be transmitted from externally into the input FIFOs (0408) and/or from the output FIFOs to externally (0409).","FIG.","5 shows a possible embodiment of a PAE.","A first bus system (0104a) is connected to a data processing unit (0501), the results of which are transmitted to a second bus system (0104b).","The vertical data transfer is carried over two register/multiplexer stages (FREG 0502, BREG 0503), each with a different transfer direction.","Preferably simple ALUs, e.g., for addition, subtraction, and multiplex operations, may be integrated into the FREG/BREG.","The unit is configured in its function and interconnection by a configuration unit (CT) via an additional interface (0504).","In a preferred embodiment, there is the possibility of setting constants in registers and/or memories for data processing.","In another embodiment, a configuration unit (CT) may read out data from the working registers and/or memories.","In an expanded embodiment, a PAE may additionally have a connection to a dedicated global bus (0505) (e.g., a GlobalTrack) and may thus communicate directly with a global, and if necessary also an external memory and/or peripheral unit, for example.","In addition, a global bus may be designed so that different PAEs may communicate directly with one another via this bus, and in a preferred embodiment they may also communicate with modules for an external connection (e.g., interface modules).","A bus system such as that described in German Patent Application No.","DE 197 04 742.4, for example, may be used for such purposes.","The data processing unit (0501) may be designed for ALU-PAEs as an arithmetic logic unit (ALU), for example.","Different ALU-PAEs may use different ALUs and bus connection systems.","One ALU may have more than two bus connections to 0104a and/or 0104b, for example.","The data processing unit (0501) may be designed as a memory for RAM-PAEs, for example.","Different RAM-PAEs may use different memories and bus connection systems.","For example, a memory may have a plurality, in particular, more than two bus connections to 0104a and/or 0104b to allow access of a plurality of senders/receivers to one memory, for example.","Accesses may preferably also take place simultaneously (multi-port).","The function of the memory includes, for example, the following functions or combinations thereof: random access, FIFO, stack, cache, page memory with MMU method.","In addition, in a preferred embodiment, the memory may be preloaded with data from the CT (e.g., constants, lookup tables, etc.).","Likewise, in an expanded embodiment, the CT may read back data from the memory via 0504 (e.g., for debugging or for changing tasks).","In another embodiment, the RAM-PAE may have a dedicated connection (0505) to a global bus.","The global bus connects a plurality of PAEs among one another and in a preferred embodiment also to modules for an external connection (e.g., interface modules).","The system described in German Patent Application No.","DE 197 04 742.4 may be used for such a bus system.","RAM-PAEs may be wired together in such a way that an n-fold larger memory is created from a plurality (n) of RAM-PAEs.","FIG.","6 shows an example of a wiring connection of ALU-PAEs (0601) and RAM-PAEs (0602) via a bus system 0104.FIG.","1 shows a preferred example of a wiring connection for a reconfigurable processor.","FIG.","7 shows a simple embodiment variant of an IO circuit corresponding to 0105.Addresses (ADR) and data (DTA) are transmitted together with synchronization lines (RDY/ACK) between the internal bus systems (0104) and an external bus system (0703).","The external bus system leads to IO-FIFOs and/or protocol converters, for example.","FIG.","7a shows a circuit for writing data.","The addresses and data arriving from 0104 are linked together (0701).","A FIFO stage for decoupling may be provided between 0104 and 0703 in the interface circuit (0701).","FIG.","7b shows a circuit for reading data, in which an acknowledgment circuit (0702, e.g., FIFO, counter) is provided for coordinating the outgoing addresses with the incoming data.","In 0701a and/or in 0701b, a FIFO stage for decoupling may be provided between 0104 and 0703.If a FIFO stage is provided in 0701b, it may also be used for acknowledgment circuit 0702.FIG.","8 shows a possible connection structure between interface modules and/or PAEs having a dedicated global bus (0801) and protocol converters (0802) to external (main) memories and/or other (peripheral) data streams.","Interface modules are connected (0803) to a PA, preferably via their network according to 0104.A bus system (0804a, 0804b) is provided between interface modules and/or PAEs having a dedicated global bus (0801) and protocol converters (0802).","In a preferred embodiment, 0804 is able to transmit pipelined data over a plurality of register stages.","0804a and 0804b are interconnected via switches (e.g., 0805) which are designed as transmission gates and/or tristate buffers and/or multiplexers, for example.","The multiplexers are triggered by rows and columns.","Triggering units (0806) control the data transfer of the interface modules and/or PAEs having a dedicated global bus (0801) to the protocol converters (0802), i.e., in the transfer direction 0804a to 0804b.","Triggering units (0807) control the data transfer of the protocol converters (0802) to the interface modules and/or the PAEs having a dedicated global bus (0801), i.e., in the transfer direction 0804b to 0804a.","The triggering units (0806) each decode address areas for selection of the protocol converters (0802); the triggering units (0807) each decode IOIDs for selection of the interface modules and/or PAEs having a dedicated global bus (0801).","Triggering units may operate according to different types of triggering, e.g., fixed connection without decoding; decoding of addresses and/or IOIDs; decoding of addresses and/or IOIDs and arbitration.","One or multiple data words/address words may be transmitted per arbitration.","Arbitration may be performed according to different rules.","The interface modules may preferably have a small FIFO for addresses and/or data in the output direction and/or input direction.","A particular arbitration rule preferably arbitrates an interface module having a FULL FIFO or an EMPTY FIFO or a FIFO to be emptied (FLUSH), for example.","Triggering units may be designed as described in German Patent Application No.","DE 199 26 538.0 (FIG.","32), for example.","These triggering units may be used for 0807 or 0806.When used as 0806, 0812 corresponds to 0804a, and 0813 corresponds to 0804b.","When used as 0807, 0812 corresponds to 0804b, and 0813 corresponds to 0804a.","Decoders (0810) decode the addresses/IOIDs of the incoming buses (0812) and trigger an arbiter (0811), which in turn switches the incoming buses to an output bus (0813) via a multiplexer.","The protocol converters are coupled to external bus systems (0808), a plurality of protocol converters optionally being connected to the same bus system (0809), so that they are able to utilize the same external resources.","The IO-FIFOs are preferably integrated into the protocol converters, a FIFO (BURST-FIFO) for controlling burst transfers for the external buses (0808) being connected downstream from them if necessary.","In a preferred embodiment, an additional FIFO stage (SYNC-FIFO) for synchronizing the outgoing addresses with the incoming data is connected downstream from the FIFOs.","Various programmable/configurable FIFO structures are depicted in 0820-0823, where A indicates the direction of travel of an address FIFO, D indicates the direction of travel of a data FIFO.","The direction of data transmission of the FIFOs depends on the direction of data transmission and the mode of operation.","If a VPU is operating as a bus master, then data and addresses are transmitted from internally to the external bus in the event of a write access (0820), and in the event of a read access (0821) addresses are transmitted from internally to externally and data from externally to internally.","If a VPU is operating as a bus slave, then data and addresses are transmitted from the external bus to internally in the event of a write access (0822) and in the event of a read access (0823) addresses are transmitted from externally to internally and data is transmitted from internally to externally.","In all data transfers, addresses and/or data and/or IOIDs and/or APIDs may be assigned and also stored in the FIFO stages.","In a particularly preferred embodiment, the transfer rate (operating frequency) of the bus systems 0104, 0804, and 0808/0809 may each be different due to the decoupling of the data transfers by the particular FIFO stages.","In particular the external bus systems (0808/0809) may operate at a higher transfer rate, for example, than the internal bus systems (0104) and/or (0804).","FIG.","9 shows a possible sequence of a data read transfer via the circuit according to FIG.","8.Addresses (preferably identifiers, e.g., with IOIDs and/or APIDs) are transmitted via internal bus system 0104 to interface modules and/or PAEs having a dedicated global bus, which preferably have an internal FIFO (0901).","The addresses are transmitted to an IO-FIFO (0903) via a bus system (e.g., 0804) which preferably operates as a pipeline (0902).","The addresses are transmitted to a BURST-FIFO (0905) via another bus (0904) which may be designed as a pipeline but which is preferably short and local.","The BURST-FIFO ensures correct handling of burst transfers via the external bus system, e.g., for controlling burst addresses and burst sequences and repeating burst cycles when errors occur.","IOIDs and/or APIDs of addresses (0906) which are transmitted via the external bus system may be transmitted together with the addresses and/or stored in an additional SYNC-FIFO (0907).","The SYNC-FIFO compensates for the latency between the outgoing address (0906) and the incoming data (0909).","Incoming data may be assigned IOIDs and/or APIDs (0908) of the addresses referencing them via the SYNC-FIFO (0910).","Data (and preferably IOIDs and/or APIDs) is buffered in an IO-FIFO (0911) and is subsequently transmitted via a bus system (e.g., 0804), which preferably functions as a pipeline (0912), to an interface module and/or PAE having a dedicated global bus (0913), preferably including an internal FIFO.","Data is transmitted from here to the internal bus system (0104).","Instead of to the IO-FIFO (0911), incoming data may optionally be directed first to a second BURST-FIFO (not shown), which behaves like BURST-FIFO 0905 if burst-error recovery is also necessary in read accesses.","Data is subsequently relayed to 0911.FIG.","10 corresponds in principle to FIG.","8, which is why the same reference numbers have been used.","In this embodiment, which is given as an example, fewer interface modules and/or PAEs having a dedicated global bus (0801) and fewer protocol converters (0802) to external (main) memories and/or other (peripheral) data streams are shown.","In addition, a collector (1001) is shown which is connected to bus systems 0804 in such a way that data is written from the interface modules and protocol converters into the collector and/or is read out from the collector.","The collector is switched to bus systems 0804a via triggering unit 1007 which corresponds to 0807, and the collector is switched to bus systems 0804b via triggering unit 1006, which corresponds to 0806.Multiple collectors may be implemented for which multiple triggering units 1006 and 1007 are used.","A collector may be segmented into multiple memory areas.","Each memory area may operate independently in different memory modes, e.g., as random access memory, FIFO, cache, MMU page, etc.","A translation table (TLB) (1002) may be assigned to a collector to permit an MMU-type mode of operation.","Page management may function, e.g., on the basis of segment addresses and/or other identifiers, e.g., APIDs and/or IOIDs.","A DMA or multiple DMAs are preferably assigned to a collector to perform data transfers with external (main) memories and/or other (peripheral) data streams, in particular to automatically permit the MMU function of page management (loading, writing).","DMAs may also access the TLB for address translation between external (main) memories and/or other (peripheral) data streams and collector.","In one possible mode of operation, DMAs may receive address specifications from the array (PA), e.g., via 0804.DMAs may be triggered by one or more of the following units: an MMU assigned to the collector, e.g., in the case of page faults; the array (PA); an external bus (e.g., 0809); an external processor; a higher-level loading unit (CT).","Collectors may have access to a dedicated bus interface (1004), preferably DMA-controlled and preferably master/slave capable, including a protocol converter, corresponding to or similar to protocol converters 0802 having access to external (main) memories and/or other (peripheral) data streams.","An external processor may have direct access to collectors (1007).","FIG.","11 corresponds in principle to FIG.","9, which is why the same reference numbers have been used.","A collector (1101) including assigned transfer control (e.g., DMA preferably with TLB) (1102) is integrated into the data stream.","The array (PA) now transmits data preferably using the collector (1103), which preferably exchanges data with external (main) memories and/or other (peripheral) data streams (1104), largely automatically and controlled via 1102.The collector preferably functions in a segmented MMU-type mode of operation, where different address areas and/or identifiers such as APIDs and/or IOIDs are assigned to different pages.","Preferably 1102 may be controlled by page faults.","FIG.","12 shows a flow chart of data transfers for different applications.","An array (PA) processes data according to the method described in German Patent Application No.","DE 196 54 846.2 by storing operands and results in memories 1202 and 1203.In addition, a data input channel (1204) and a data output channel (1205) are assigned to the PA, through which the operands and/or results are loaded and/or stored.","The channels may lead to external (main) memories and/or other (peripheral) data streams (1208).","The channels may include internal FIFO stages and/or PAE-RAMs/PAE-RAM pages and/or collectors/collector pages.","The addresses (CURR-ADR) may be computed currently by a configuration running in 1201 and/or may be computed in advance and/or computed by DMA operations of a (1003).","In particular, an address computation within 1201 (CURR-ADR) may be sent to a collector or its DMA to address and control the data transfers of the collector.","The data input channel may be preloaded by a configuration previously executed on 1201.The channels preferably function in a FIFO-like mode of operation to perform data transfers with 1208.In the example depicted here, a channel (1207), which has been filled by a previous configuration or application, is still being written to 1208 during data processing within 1201 described here.","This channel may also include internal FIFO stages and/or PAE-RAMs/PAE-RAM pages and/or collectors/collector pages.","The addresses may be computed currently by a configuration (OADR-CONF) running in parallel in 1201 and/or computed in advance and/or computed by DMA operations of a (1003).","In particular, an address computation within 1201 (OADR-CONF) may be sent to a collector or its DMA to address and control the data transfers of the collector.","In addition, data for a subsequent configuration or application is simultaneously loaded into another channel (1206).","This channel too may include internal FIFO stages and/or PAE-RAMs/PAE-RAM pages and/or collectors/collector pages.","The addresses may be computed currently by a configuration (IADR-CONF) running in parallel in 1201 and/or computed in advance and/or computed by DMA operations of a (1003).","In particular, an address computation within 1201 (IADR-CONF) may be sent to a collector or its DMA to address and control the data transfers of the collector.","Individual entries into the particular channels may have different identifiers, e.g., IOIDs and/or APIDs, enabling them to be assigned to a certain resource and/or memory location.","FIG.","13a shows a preferred implementation of a BURST-FIFO.","The function of an output FIFO which transmits its values to a burst-capable bus (BBUS) is to be described first.","A first pointer (1301) points to the data entry within a memory (1304) currently to be output to the BBUS.","With each data word output (1302), 1301 is moved by one position.","The value of pointer 1301 prior to the start of the current burst transfer has been stored in a register (1303).","If an error occurs during the burst transfer, 1301 is reloaded with the original value from 1303 and the burst transfer is restarted.","A second pointer (1305) points to the current data input position in the memory (1304) for data to be input (1306).","To prevent overwriting of any data still needed in the event of an error, pointer 1305 is compared (1307) with register 1303 to indicate that the BURST-FIFO is full.","The empty state of the BURST-FIFO may be ascertained by comparison (1308) of the output pointer (1301) with the input pointer (1305).","If the BURST-FIFO operates for input data from a burst transfer, the functions change as follows: 1301 becomes the input pointer for data 1306.If faulty data has been transmitted during the burst transfer, the position prior to the burst transfer is stored in 1303.If an error occurs during the burst transfer, 1301 is reloaded with the original value from 1303 and the burst transfer is restarted.","The pointer points to the readout position of the BURST-FIFO for reading out the data (1302).","To prevent premature readout of data of a burst transfer that has not been concluded correctly, 1305 is compared with the position stored in 1303 (1307) to indicate an empty BURST-FIFO.","A full BURST-FIFO is recognized by comparison (1308) of input pointer 1301 with the output pointer (1305).","FIG.","13b shows one possible implementation of a burst circuit which recognizes possible burst transfers and tests boundary limits.","The implementation has been kept simple and recognizes only linear address sequences.","Data transfers are basically started as burst transfers.","The burst transfer is aborted at the first nonlinear address.","Burst transfers of a certain length (e.g., 4) may also be detected and initialized by expanding a look-ahead logic, which checks multiple addresses in advance.","The address value (1313) of a first access is stored in a register (1310).","The address value of a subsequent data transfer is compared (1312) with the address value (1311) of 1310, which has been incremented by the address difference between the first data transfer and the second data transfer of the burst transfer (typically one word wide).","If the two values are the same, then the difference between the first address and the second address corresponds to the address difference of the burst transfer between two burst addresses.","Thus, this is a correct burst.","If the values are not the same, the burst transfer must be aborted.","The last address (1313) checked (the second address in the writing) is stored in 1310 and then compared with the next address (1313) accordingly.","To ascertain whether the burst limits (boundaries) have been maintained, the address bit(s) at which the boundary of the current address value (1313) is located is (are) compared with the address bits of the preceding address value (1310) (e.g., XOR 1314).","If the address bits are not the same, the boundary has been exceeded and the control of the burst must respond accordingly (e.g., termination of the burst transfer and restart).","FIG.","14 shows as an example various methods of connecting memories, in particular PAE-RAMs, to form a larger cohesive memory block.","FIGS.","14a-14d use the same reference numbers whenever possible.","Write data (1401) is preferably sent to the memories via pipeline stages (1402).","Read data (1403) is preferably removed from the memories also via pipeline stages (1404).","Pipeline stage 1404 includes a multiplexer, which forwards the particular active data path.","The active data path may be recognized, for example, by a RDY handshake applied.","A unit (RangeCheck, 1405) for monitoring the addresses (1406) for correct values within the address space may optionally be provided.","In FIG.","14a, the addresses are sent to the memories (1408a) via pipeline stages (1407a).","The memories compare the higher-value address part with a fixedly predetermined or configurable (e.g., by a higher-level configuration unit CT) reference address, which is unique for each memory.","If they are identical, that memory is selected.","The lower-value address part is used for selection of the memory location in the memory.","In FIG.","14b, the addresses are sent to the memories (1408b) via pipeline stages having an integrated decrementer (subtraction by 1) (1407b).","The memories compare the higher-value address part with the value zero.","If they are identical, that memory is selected.","The lower-value address part is used for selection of the memory location in the memory.","In FIG.","14c, the addresses are sent to the memories (1408c) via pipeline stages (1407c).","The memories compare the higher-level address part with a reference address, which is unique for each memory.","The reference address is generated by an adding or subtracting chain (1409), which preselects another unique reference address for each memory on the basis of a starting value (typically 0).","If they are identical, that memory is selected.","The lower-value address part is used for selection of the memory location in the memory.","In FIG.","14d, the addresses are sent to the memories (1408d) via pipeline stages (1407d).","The memories compare the higher-value address part with a reference address which is unique for each memory.","The reference address is generated by an addressing or subtracting chain (1410), which is integrated into the memories and preselects another unique reference address for each memory on the basis of a starting value (typically 0).","If they are identical, that memory is selected.","The lower-value address part is used for selection of the memory location in the memory.","For example, FREGs of the PAEs according to FIG.","5 may be used for 1402, 1404, and 1407.Depending on the direction of travel of the reference address, FREG or BREG may be used for 1409.The design shown here as an example has the advantage in particular that all the read/write accesses have the same latency because the addresses and data are sent to the BREG/FREG via register stages.","FIG.","15 shows the use of GlobalTrack bus systems (1501, 1502, 1503, 1504) for coupling configurations which were configured in any way as configuration macros (1506, 1507) within a system of PAEs (1505) (see also DE 198 07 872.2, DE 199 26 538.0, DE 100 28 397.7).","The configuration macros have (1508) their own internal bus connections, e.g., via internal buses (0104).","The configuration macros are interconnected via 1503 for data exchange.","1506 is connected to interface modules and/or local memories (RAM-PAEs) (1509, 1510) via 1501, 1502.1507 is connected to interface modules and/or local memories (RAM-PAEs) (1511) via 1504.Any other embodiments and combinations of the present inventions described here are possible and are self-evident in view of the foregoing, to those skilled in the art."]],"string":"[\n [\n \"Methods and devices for treating and/or processing data\",\n \"At the inputs and/or outputs, memories are assigned to a reconfigurable module to achieve decoupling of internal data processing and in particular decoupling of the reconfiguration cycles from the external data streams (to/from peripherals, memories, etc.\",\n \").\"\n ],\n [\n \"1-9.\",\n \"(canceled) 10.A method for decoupling data streams in a reconfigurable module, comprising: decoupling module-internal data transfers from external data transfers via at least one FIFO memory.\",\n \"11.A method for decoupling data streams in a reconfigurable module, comprising: providing multiple independent address generators.\",\n \"12.The method as recited in claim 11, further comprising: configuring the address generators independently of data processing.\",\n \"13.A method for programming a reconfigurable module, comprising: breaking down, by a compiler, address computations into multiple configurations.\",\n \"14.A device for data processing, comprising: a field of reconfigurable modules; and an interface module configured for an exchange of data to be processed between the field of reconfigurable modules and an external circuit configuration, wherein the interface module includes at least one FIFO memory in which data may be stored and from which data may be retrieved.\",\n \"15.The device as recited in claim 14, further comprising: a circuit configuration to determine data which will be needed in a future configuration and to preloading the determined data into the interface configuration.\",\n \"16.The device according to claim 15, wherein the circuit configuration includes a memory area addressable rapidly via the interface module.\",\n \"17.The device as recited in claim 16 wherein the rapidly addressable memory area includes at least one of a cache memory, an external RAM, a FIFO memory, and a register.\",\n \"18.The device as recited in claim 14, further comprising: means for backing up data of a configuration which has previously been executed and is at least partially no longer active at the time of data retrieval, the configuration data being stored in an interface memory, the configuration data already canceled by reconfiguration of participating field elements.\",\n \"19.The device as recited in claim 14, further comprising: means for backing up data of a configuration which has previously been executed and is at least partially no longer active at the time of data retrieval, for at least one of preventing deletion, and overwriting of data already sent once but not yet known to have been satisfactorily received.\",\n \"20.The device as recited in claim 14, further comprising: at least one of multiple field elements and field elements grouped into configurations of addressable memories to store data at least one of non-locally and temporarily.\",\n \"21.The device as recited in claim 20, wherein the addressable memory are RAM cells.\"\n ],\n [\n \"<SOH> BACKGROUND INFORMATION <EOH>The present invention relates to reconfigurable components in general, and in particular but not exclusively the decoupling of data processing within the reconfigurable component and/or within parts of the reconfigurable component and data streams, specifically both within the reconfigurable component and also to and from peripherals, mass memories, host processors, and the like (see, e.g., German Patent Application Nos.\",\n \"DE 101 10 530.4 and DE 102 02 044.2).\",\n \"Memories are assigned to a reconfigurable module (VPU) at the inputs and/or outputs to achieve decoupling of internal data processing, the reconfiguration cycles in particular, from the external data streams (to/from peripherals, memories, etc.).\",\n \"Reconfigurable architecture includes modules (VPUs) having a configurable function and/or interconnection, in particular integrated modules having a plurality of unidimensionally or multidimensionally positioned arithmetic and/or logic and/or analog and/or storage and/or internally/externally interconnecting modules, which are interconnected directly or via a bus system.\",\n \"These generic modules include in particular systolic arrays, neural networks, multiprocessor systems, processors having a plurality of arithmetic units and/or logic cells and/or communication/peripheral cells (IO), interconnecting and networking modules such as crossbar switches, as well as conventional modules including FPGA, DPGA, Chameleon, XPUTER, etc.\",\n \"Reference is also made in particular in this context to the following patents and patent applications of the same applicant: P 44 16 881.0-53, DE 197 81 412.3, DE 197 81 483.2, DE 196 54 846.2-53, DE 196 54 593.5-53, DE 197 04 044.6-53, DE 198 80 129.7, DE 198 61 088.2-53, DE 199 80 312.9, PCT/DE 00/01869, DE 100 36 627.9-33, DE 100 28 397.7, DE 101 10 530.4, DE 101 11 014.6, PCT/EP 00/10516, EP 01 102 674.7, DE 196 51 075.9, DE 196 54 846.2, DE 196 54 593.5, DE 197 04 728.9, DE 198 07 872.2, DE 101 39 170.6, DE 199 26 538.0, DE 101 42 904.5, DE 101 10 530.4, DE 102 02 044.2, DE 102 06 857.7, DE 101 35 210.7, EP 02 001 331.4, EP 01 129 923.7 as well as the particular parallel patent applications thereto.\",\n \"The entire disclosure of these documents are incorporated herein by reference.\",\n \"The above-mentioned architecture is used as an example to illustrate the present invention and is referred to hereinafter as VPU.\",\n \"The architecture includes an arbitrary number of arithmetic, logic (including memory) and/or memory cells and/or networking cells and/or communication/peripheral (IO) cells (PAEs—Processing Array Elements), which may be positioned to form a unidimensional or multidimensional matrix (PA); the matrix may have different cells of any desired configuration.\",\n \"Bus systems are also understood here as cells.\",\n \"A configuration unit (CT) which affects the interconnection and function of the PA is assigned to the entire matrix or parts thereof.\",\n \"Memory access methods for reconfigurable modules which operate according to a DMA principle are described in German Patent No.\",\n \"P 44 16 881.0, where one or more DMAs are formed by configuration.\",\n \"In German Patent Application No.\",\n \"196 54 595.1, DMAs are fixedly implemented in the interface modules and may be triggered by the PA or the CT. German Patent Application No.\",\n \"DE 196 54 846.2 describes how internal memories are written by external data streams and data is read out of the memory back into external units.\",\n \"German Patent Application No.\",\n \"DE 199 26 538.0 describes expanded memory concepts according to DE 196 54 846.2 for achieving more efficient and easier-to-program data transmission.\",\n \"U.S. Pat.\",\n \"No.\",\n \"6,347,346 describes a memory system which corresponds in all essential points to German Patent Application No.\",\n \"DE 196 54 846.2, having an explicit bus (global system port) to a global memory.\",\n \"U.S. Pat.\",\n \"No.\",\n \"6,341,318 describes a method for decoupling external data streams from internal data processing by using a double-buffer method, in which one buffer records/reads out the external data while another buffer records/reads out the internal data; as soon as the buffers are full/empty, depending on their function, the buffers are switched, i.e., the buffer formerly responsible for the internal data now sends its data to the periphery (or reads new data from the periphery) and the buffer formerly responsible for the external data now sends its data to the PA (reads new data from the PA).\",\n \"These double buffers are used in the application to buffer a cohesive data area.\",\n \"Such double-buffer configurations have enormous disadvantages in the data-stream area in particular, i.e., in data streaming, in which large volumes of data streaming successively into a processor field or the like must always be processed in the same way.\"\n ],\n [\n \"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.\",\n \"1 shows an example reconfigurable processor.\",\n \"FIG.\",\n \"2 a shows a direct FIFO to PA coupling.\",\n \"FIG.\",\n \"2 b shows IO connected via RAM-PAEs.\",\n \"FIG.\",\n \"2 c shows FIFOs connected upstream from the IOs.\",\n \"FIGS.\",\n \"3 a - 3 e show an example data processing method in a VPU.\",\n \"FIGS.\",\n \"4 a - 4 e show another example data processing method in a VPU.\",\n \"FIG.\",\n \"5 shows an example embodiment of a PAE.\",\n \"FIG.\",\n \"6 shows an example of a wiring connection of ALU-PAEs and RAM-PAEs via a bus system.\",\n \"FIG.\",\n \"7 a shows a circuit for writing data.\",\n \"FIG.\",\n \"7 b shows a circuit for reading data.\",\n \"FIG.\",\n \"8 shows an example connection between interface modules and/or PAEs to numerous and/or other data streams.\",\n \"FIG.\",\n \"9 shows an example sequence of a data read transfer via the circuit of FIG.\",\n \"8 .\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"BACKGROUND INFORMATION The present invention relates to reconfigurable components in general, and in particular but not exclusively the decoupling of data processing within the reconfigurable component and/or within parts of the reconfigurable component and data streams, specifically both within the reconfigurable component and also to and from peripherals, mass memories, host processors, and the like (see, e.g., German Patent Application Nos.\",\n \"DE 101 10 530.4 and DE 102 02 044.2).\",\n \"Memories are assigned to a reconfigurable module (VPU) at the inputs and/or outputs to achieve decoupling of internal data processing, the reconfiguration cycles in particular, from the external data streams (to/from peripherals, memories, etc.).\",\n \"Reconfigurable architecture includes modules (VPUs) having a configurable function and/or interconnection, in particular integrated modules having a plurality of unidimensionally or multidimensionally positioned arithmetic and/or logic and/or analog and/or storage and/or internally/externally interconnecting modules, which are interconnected directly or via a bus system.\",\n \"These generic modules include in particular systolic arrays, neural networks, multiprocessor systems, processors having a plurality of arithmetic units and/or logic cells and/or communication/peripheral cells (IO), interconnecting and networking modules such as crossbar switches, as well as conventional modules including FPGA, DPGA, Chameleon, XPUTER, etc.\",\n \"Reference is also made in particular in this context to the following patents and patent applications of the same applicant: P 44 16 881.0-53, DE 197 81 412.3, DE 197 81 483.2, DE 196 54 846.2-53, DE 196 54 593.5-53, DE 197 04 044.6-53, DE 198 80 129.7, DE 198 61 088.2-53, DE 199 80 312.9, PCT/DE 00/01869, DE 100 36 627.9-33, DE 100 28 397.7, DE 101 10 530.4, DE 101 11 014.6, PCT/EP 00/10516, EP 01 102 674.7, DE 196 51 075.9, DE 196 54 846.2, DE 196 54 593.5, DE 197 04 728.9, DE 198 07 872.2, DE 101 39 170.6, DE 199 26 538.0, DE 101 42 904.5, DE 101 10 530.4, DE 102 02 044.2, DE 102 06 857.7, DE 101 35 210.7, EP 02 001 331.4, EP 01 129 923.7 as well as the particular parallel patent applications thereto.\",\n \"The entire disclosure of these documents are incorporated herein by reference.\",\n \"The above-mentioned architecture is used as an example to illustrate the present invention and is referred to hereinafter as VPU.\",\n \"The architecture includes an arbitrary number of arithmetic, logic (including memory) and/or memory cells and/or networking cells and/or communication/peripheral (IO) cells (PAEs—Processing Array Elements), which may be positioned to form a unidimensional or multidimensional matrix (PA); the matrix may have different cells of any desired configuration.\",\n \"Bus systems are also understood here as cells.\",\n \"A configuration unit (CT) which affects the interconnection and function of the PA is assigned to the entire matrix or parts thereof.\",\n \"Memory access methods for reconfigurable modules which operate according to a DMA principle are described in German Patent No.\",\n \"P 44 16 881.0, where one or more DMAs are formed by configuration.\",\n \"In German Patent Application No.\",\n \"196 54 595.1, DMAs are fixedly implemented in the interface modules and may be triggered by the PA or the CT. German Patent Application No.\",\n \"DE 196 54 846.2 describes how internal memories are written by external data streams and data is read out of the memory back into external units.\",\n \"German Patent Application No.\",\n \"DE 199 26 538.0 describes expanded memory concepts according to DE 196 54 846.2 for achieving more efficient and easier-to-program data transmission.\",\n \"U.S. Pat.\",\n \"No.\",\n \"6,347,346 describes a memory system which corresponds in all essential points to German Patent Application No.\",\n \"DE 196 54 846.2, having an explicit bus (global system port) to a global memory.\",\n \"U.S. Pat.\",\n \"No.\",\n \"6,341,318 describes a method for decoupling external data streams from internal data processing by using a double-buffer method, in which one buffer records/reads out the external data while another buffer records/reads out the internal data; as soon as the buffers are full/empty, depending on their function, the buffers are switched, i.e., the buffer formerly responsible for the internal data now sends its data to the periphery (or reads new data from the periphery) and the buffer formerly responsible for the external data now sends its data to the PA (reads new data from the PA).\",\n \"These double buffers are used in the application to buffer a cohesive data area.\",\n \"Such double-buffer configurations have enormous disadvantages in the data-stream area in particular, i.e., in data streaming, in which large volumes of data streaming successively into a processor field or the like must always be processed in the same way.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 shows an example reconfigurable processor.\",\n \"FIG.\",\n \"2a shows a direct FIFO to PA coupling.\",\n \"FIG.\",\n \"2b shows IO connected via RAM-PAEs.\",\n \"FIG.\",\n \"2c shows FIFOs connected upstream from the IOs.\",\n \"FIGS.\",\n \"3a-3e show an example data processing method in a VPU.\",\n \"FIGS.\",\n \"4a-4e show another example data processing method in a VPU.\",\n \"FIG.\",\n \"5 shows an example embodiment of a PAE.\",\n \"FIG.\",\n \"6 shows an example of a wiring connection of ALU-PAEs and RAM-PAEs via a bus system.\",\n \"FIG.\",\n \"7a shows a circuit for writing data.\",\n \"FIG.\",\n \"7b shows a circuit for reading data.\",\n \"FIG.\",\n \"8 shows an example connection between interface modules and/or PAEs to numerous and/or other data streams.\",\n \"FIG.\",\n \"9 shows an example sequence of a data read transfer via the circuit of FIG.\",\n \"8.DETAILED DESCRIPTION An object of the present invention is to provide a novel approach for commercial use.\",\n \"A method according to an example embodiment of the present invention, in contrast to the previously known related art, allows a significantly simpler means of controlling the buffers, i.e., memories, connected in between; the related art is disadvantageous in the core area of typical applications of reconfigurable processors in particular.\",\n \"External and internal bus systems may be operated at different transfer rates and/or clock frequencies with no problem due to the memory devices connected in between because data is stored temporarily by the buffers.\",\n \"In comparison with inferior designs from the related art, this method requires fewer memory devices, typically only half as many buffers, i.e., data transfer interface memory devices, thus greatly reducing the hardware costs.\",\n \"The estimated reduction in hardware costs amounts to 25% to 50%.\",\n \"It is also simpler to generate addresses and to program the configuration because the buffers are transparent for the programmer.\",\n \"Hardware is simpler to write and to debug.\",\n \"A paging method which buffers various data areas in particular for different configurations may be integrated.\",\n \"It should first be pointed out that various memory systems are known as interfaces to the IO.\",\n \"Reference is made to German Patent No.\",\n \"and German Patent Application Nos.\",\n \"P 44 16 881.0, DE 196 54 595.1, and DE 199 26 538.0.In addition, a method is described in German Patent Applciation No.\",\n \"DE 196 54 846.2 in which data is first loaded from the IO, (1) data is stored within a VPU after being computed, (2) the array (PA) is reconfigured, (3) data is read out from the internal memory and written back to another internal memory, (4) this is continued until the fully computed result is sent to the IO.\",\n \"Reconfiguration means, for example, that a function executed by a part of the field of reconfigurable units or the entire field and/or the data network and/or data and/or constants which are necessary in data processing is/are determined anew.\",\n \"Depending on the application and/or embodiment, VPUs are reconfigured only completely or also partially, for example.\",\n \"Different reconfiguration methods are implementable, e.g., complete reconfiguration by switching memory areas (see, e.g., German Patent Application Nos.\",\n \"DE 196 51 075.9, DE 196 54 846.2) and/or wave reconfiguration (see, e.g., German Patent Application-Nos.\",\n \"DE 198 07 872.2, DE 199 26 538.0, DE 100 28 397.7, DE 102 06 857.7) and/or simple configuring of addressable configuration memories (see, e.g., German Patent Application Nos.\",\n \"DE 196 51 075.9, DE 196 54 846.2, DE 196 54 593.5).\",\n \"The entire disclosure of each of the particular patent specifications is expressly incorporated herewith.\",\n \"In one example embodiment, a VPU is entirely or partially configurable by wave reconfiguration or by directly setting addressable configuration memories.\",\n \"Thus, one of the main operating principles of VPU modules is to copy data back and forth between multiple memories, with additional and optionally the same operations (e.g., long FIR filter) and/or other operations (e.g., FFT followed by Viterbi) being performed with the same data during each copying operation.\",\n \"Depending on the particular application, data is read out from one or more memories and written into one or more memories.\",\n \"For storing data streams and/or states (triggers, see, e.g., German Patent Application Nos.\",\n \"DE 197 04 728.9, DE 199 26 538.0), internal/external memories (e.g., as FIFOs) are used and corresponding address generators are utilized.\",\n \"Any appropriate memory architecture may be fixedly implemented specifically in the algorithm and/or flexibly configured.\",\n \"For performance reasons, the internal memories of the VPU are preferably used, but basically external memories may also be used.\",\n \"Assuming this, the following comments shall now be made regarding the basic design: Interface modules which communicate data between the bus systems of the PA and external units are assigned to an array (PA) (see, e.g., German Patent No.\",\n \"P 44 16 881.0, and German Patent Application No.\",\n \"DE 196 54 595.1).\",\n \"Interface modules connect address buses and data buses in such a way as to form a fixed allocation between addresses and data.\",\n \"Interface modules may preferably generate addresses or parts of addresses independently.\",\n \"Interface modules are assigned to FIFOs which decouple internal data processing from external data transmission.\",\n \"A FIFO here is a data-streamable buffer, i.e., input/output data memory, which need not be switched for data processing, in particular during execution of one and the same configuration.\",\n \"If other data-streamable buffers are known in addition to FIFO memories, they will subsequently also be covered by the term where applicable.\",\n \"In particular, ring memories having one or more pointers, in particular at least one write memory and one read memory, should also be mentioned.\",\n \"Thus, for example, during multiple reconfiguration cycles for processing an application, the external data stream may be maintained as largely constant, regardless of internal processing cycles.\",\n \"FIFOs are able to store incoming/outgoing data and/or addresses.\",\n \"FIFOs may be integrated into an interface module or assigned to one or more of them.\",\n \"Depending on the design, FIFOs may also be integrated into the interface modules, and at the same time additional FIFOs may be implemented separately.\",\n \"It is also possible to use data-streamable buffers integrated into the module, e.g., by integration of FIFO groups into a chip which forms a reconfigurable processor array.\",\n \"In one example embodiment, multiplexers for free allocation of interface modules and FIFOs may also be present between the FIFOs (including those that are separate) and the interface modules.\",\n \"In one configuration, the connection of FIFOs to external modules or internal parts of the processor field performed by a multiplexer may be specified based on the processor field, e.g., by the PAE sending and/or receiving data, but it may also be determined, if desired, by a unit at a higher level of the hierarchy, such as a host processor in the case of division of data processing into a highly parallel part of the task and a poorly parallelizable part of the task and/or the multiplexer circuit may be determined by external specifications, which may be appropriate if, for example, it is indicated with the data which type of data is involved and how it is to be processed.\",\n \"With regard to the external connection, units for protocol conversion between the internal and external bus protocols (e.g., RAMBUS, AMBA, PCI, etc.)\",\n \"are also provided.\",\n \"A plurality of different protocol converters may also be used within one embodiment.\",\n \"The protocol converters may be designed separately or integrated into the FIFOs or interface modules.\",\n \"In one possible embodiment, multiplexers for free assignment of interface modules/FIFOs and protocol converters may be provided between the (separate) protocol converters and the interface modules/FIFOs.\",\n \"Downstream from the protocol converters there may be another multiplexer stage, so that a plurality of AMBA bus interfaces may be connected to the same AMBA bus, for example.\",\n \"This multiplexer stage may also be formed, for example, by the property of an external bus of being able to address a plurality of units.\",\n \"In one example embodiment, the circuit operates in master and slave operating modes.\",\n \"In the master mode, addresses and bus accesses are generated by the circuit and/or the assigned PA; in slave mode, external units access the circuit, i.e., the PA.\",\n \"In other embodiments, additional buffer memories or data collectors may be provided within the circuit, depending on the application, for exchanging data between interface modules.\",\n \"These buffer memories preferably operate in a random access mode and/or an MMU paging mode and/or a stack mode and may have their own address generators.\",\n \"The buffer memories are preferably designed as multi-port memories to permit simultaneous access of a plurality of interface modules.\",\n \"It is possible to access the buffer memories from a higher-level data processing unit, in particular from processors such as DSPs, CPUs, microcontrollers, etc., assigned to the reconfigurable module (VPU).\",\n \"Now the decoupling of external data streams in particular will be described.\",\n \"According to one aspect of the present invention, the external data streams are decoupled by FIFOs (input/output FIFO, combined as IO-FIFO) which are used between protocol converters and interface modules.\",\n \"The data processing method functions as follows: Through one or more input FIFOs, incoming data is decoupled from data processing in the array (PA).\",\n \"Data processing may be performed in the following steps: 1.The input FIFO(s) is (are) read out, processed by the array (PA) and/or written into one or more (other) memories (RAM banki) assigned locally to the array and/or preferably connected laterally to the array.\",\n \"The lateral connection has the advantage that the chip architecture and/or its design is/are simplified.\",\n \"2.The array (PA) is reconfigured.\",\n \"The memories (e.g., RAM bankl) are read out, data is processed and written into one or more memories (e.g., RAM bank2 and/or RAM bank1) or, as an alternative, data may already be written to the output FIFOs according to step 4.3.The array (PA) is reconfigured again and data is again written into a memory.\",\n \"4.This is continued until the result is sent to one or more output FIFOs for output.\",\n \"5.Then new data is again read out from the input FIFO(s) and processed accordingly, i.e., data processing is continued in step 1.With the preferred design of the input/output FIFOs (IO-FIFOs) as multi-ported FIFOs, data processing may be performed by protocol converters simultaneously with writing into and/or reading out from the particular FIFOs.\",\n \"The method described above yields a time decoupling which permits “quasi-steady-state” processing of constant data streams in such a way that there is only a latency but no interruption in the data stream when the first data packets have passed through.\",\n \"In an expanded embodiment, the IO-FIFOs may be designed so that the number of IO-FIFOs and their depth may be selected according to the application.\",\n \"In other words, IO-FIFOs may be distributed or combined (e.g., via a transmission gate, multiplexer/demultiplexer, etc.)\",\n \"so that there are more IO-FIFOs or they are deeper.\",\n \"For example, 8 FIFOs of 1,024 words each may be implemented and configured so that 8 FIFOs of 1,024 words or 2 FIFOs of 4,096 words are configured or, for example, 1 FIFO may be configured with 4,096 words and 4 with 1,024 words.\",\n \"Modifications of the data processing method described here are possible, depending on the design of the system and the requirements of the algorithms.\",\n \"In an expanded embodiment, the FIFOs function in such a way that in the case of output FIFOs the addresses belonging to the data inputs are also stored in the FIFOs and/or input FIFOs are designed so that there is one FIFO for the reading addresses to be sent out/already sent out and one FIFO for the incoming data words assigned to the addresses.\",\n \"Below is a discussion of how a FIFO-RAM bank coupling, which is possible according to the present invention, may be implemented in a particularly preferred variant of the present invention.\",\n \"Depending on the application, it is possible to conduct the data transfer with the IO-FIFOs via one or more additional memory stages (RAM bank) which are assigned locally to the array or are preferably coupled laterally to the array and only then relay data to the data processing PAEs (e.g., ALU-PAEs described in, e.g., German Patent Appliation No.\",\n \"DE 196 51 075.9).\",\n \"In a preferred embodiment, RAM-PAEs have a plurality of data interfaces and address interfaces, they are thus designed as multi-port arrays.\",\n \"Designability of a data interface and/or address interface as a global system port should also be mentioned as a possibility.\",\n \"Additional memory stage(s) (RAM banks) may be implemented, for example, by memory modules corresponding to the RAM-PAEs, as described in, for example, German Patent Application No.\",\n \"DE 196 54 846.2 and/or German Patent Application No.\",\n \"DE 199 26 538.0 and/or International Patent Application No.\",\n \"PCT/EP 00/10516.In other words, a RAM-PAE may constitute a passive memory which is limited (essentially) to the memory function (see, e.g., German Patent Application No.\",\n \"DE 196 54 846.2) or an active memory which automatically generates and controls functions such as address computation and/or bus accesses (see, e.g., German Patent Application No.\",\n \"DE 199 26 538.0).\",\n \"In particular, in one possible embodiment, active address generation functions and/or data transfer functions may also be implemented for a “global system port.” Depending on the design, active memories may actively manage one or more data interfaces and address interfaces (active interfaces).\",\n \"Active interfaces may be implemented, for example, by additional modules such as sequencers/state machines and/or ALUs and/or registers, etc., within a RAM-PAE and/or by suitable hardwiring of an active interface to other PAEs whose function and networking are configured in one or more RAM-PAEs in accordance with the functions to be implemented.\",\n \"Different RAM-PAEs may be assigned to different other PAEs.\",\n \"RAM-PAEs preferably have one or more of the following functions, i.e., modes of operation: random access, FIFO, stack, cache, MMU paging.\",\n \"In a preferred embodiment, RAM-PAEs are connected via a bus to a higher-level configuration unit (CT) and may be configured by it in their function and/or interconnection and/or memory depth and/or mode of operation.\",\n \"In addition, there is preferably also the possibility of preloading and reading out the memory contents by the CT, for example, to set constants and/or lookup tables (cos/sin).\",\n \"Due to the use of multi-ported memories for the RAM-PAEs, writing and/or reading out of data into/from the IO-FIFOs and data access by the array (PA) may take place simultaneously, so that the RAM-PAEs may in turn again have a buffer property, as described in German Patent Application No.\",\n \"DE 196 54 846.2, for example.\",\n \"RAM-PAEs may be combined (as discussed in International Patent Application No.\",\n \"PCT/EP 00/10516, for example) in such a way that larger memory blocks are formed and/or the RAM-PAEs operate so that the function of a larger memory is obtained (e.g., one 1,024-word RAM-PAE from two 512-word RAM-PAEs).\",\n \"In an example embodiment, the units may be combined so that the same address is sent to multiple memories.\",\n \"The address is subdivided so that one portion addresses the entries in the memories and another portion indicates the number of the memory selected (SEL).\",\n \"Each memory has a unique number and may be selected unambiguously by comparing it with SEL.\",\n \"In a preferred embodiment, the number for each memory is configurable.\",\n \"In another and/or additional example embodiment, an address is relayed from one memory to the next.\",\n \"This address is subdivided so that one portion addresses the entries in the memories and another portion indicates the number (SEL) of the memory selected.\",\n \"This is modified each time data is relayed; for example, a 1 may be subtracted from this each time data is relayed.\",\n \"The memory in which this address part has a certain value (e.g., zero) is activated.\",\n \"In an example embodiment, the units may be combined so that the same address is sent to a plurality of memories.\",\n \"The address is subdivided so that one part addresses the entries in the memories and another part indicates the number (SEL) of the memory selected.\",\n \"A bus runs between memories, namely from one memory to the next, which has a reference address such that the address has a certain value (e.g., zero) in the first memory and this value is modified each time data is relayed (e.g., incremented by 1).\",\n \"Therefore, each memory has a different unique reference address.\",\n \"The portion of the address having the number of the selected memory is compared with the reference address in each case.\",\n \"If they are identical, the particular memory is selected.\",\n \"Depending on the design, the reference bus may be constructed using the ordinary data bus system or a separated bus.\",\n \"In an example embodiment, there may be an area check of the address part SEL to rule out faulty addressing.\",\n \"It should now be pointed out that RAM-PAEs may be used as FIFOs.\",\n \"This may be preferred in particular when a comparatively large memory capacity is provided by RAM-PAEs.\",\n \"Thus, in particular when using multi-ported memories for the RAM-PAEs, this yields the design option of dispensing with explicit IO-FIFOs and/or configuring a corresponding number of RAM-PAEs as FIFOs in addition to the IO-FIFOs and sending data from the IO to the corresponding memory ports.\",\n \"This embodiment may be regarded as particularly cost efficient because no additional memories need be provided, but instead the memories of the VPU architecture, which are configurable in their function and/or interconnection (see, e.g., German Patent Application No.\",\n \"DE 196 54 846.2, DE 199 26 538.0 and International Patent Application No.\",\n \"PCT/EP 00/10516), are configured corresponding to the character of configurable processors.\",\n \"It is also possible to provide a multiplexer/demultiplexer upstream and/or downstream from the FIFO.\",\n \"Incoming and/or outgoing data streams may be formed from one or more data records.\",\n \"For example, the following function uses two incoming data streams (a and b) and one outgoing data stream (x): function example (a, b : integer) -> x : integer for i:= 1 to 100 for j:= 1 to 100 x[i]:= a[i] * b[j].\",\n \"This requirement may be met by using two approaches, for example: a) The number of IO channels implemented is exactly equal to the number of data streams required (see, e.g., German Patent No.\",\n \"P 44 16 881.0; German Patent Application No.\",\n \"DE 196 54 595.1); in the stated function, for example, three I/O channels would thus be necessary; or b) By using internal memories for decoupling data streams, more or less as a register set (see, e.g., German Patent Application Nos.\",\n \"DE 199 26 538.0, DE 196 54 846.2).\",\n \"The different data streams are exchanged between one or more memories and the IO (e.g., memory, peripheral, etc.)\",\n \"by a time multiplex method, for example.\",\n \"Data may then be exchanged internally in parallel with a plurality of memories, if necessary, if the IO data is sorted (split) accordingly during the transfer between these memories and the IO.\",\n \"Approach a) is supported according to the present invention by making available a sufficient number of IO channels and IO-FIFOs.\",\n \"However, this simple approach is unsatisfactory because an algorithm-dependent and very expensive number of IO channels, which cannot be determined precisely, must be made available.\",\n \"Therefore, approach b) or a suitable combination of a) and b) may be preferred, e.g., two IO channels, one input and one output, data streams being multiplexed on each channel if necessary.\",\n \"It should be pointed out that the interfaces should be capable of processing data streams, i.e., a sufficiently high clock frequency and/or sufficiently short latencies should be provided on the internal and/or external buses.\",\n \"This may be the reason why a combination of the two variants may be particularly preferred, because by providing a plurality of parallel IO channels, the required clocking of external and/or internal buses may be reduced accordingly.\",\n \"For approach b) or approaches based at least partially on approach b), it may be necessary to provide multiplexers and/or demultiplexers and to separate the data streams of one data channel (e.g., a) and b) should be separated from the input channel) or to combine a plurality of result channels on one output channel.\",\n \"One or more multiplexers/demultiplexers (MuxDemux stage) may be located at different positions, depending on the technical hardware implementation and/or the functions to be executed.\",\n \"For example, a) a MuxDemux stage may be connected between the input/output interface (e.g., described in German Patent Application No.\",\n \"DE 196 54 595.1) and the FIFO stage (IO-FIFO and/or RAM-PAE as FIFO), b) a MuxDemux stage may be connected downstream from the FIFO stage (IO-FIFO and/or RAM-PAE as FIFO), i.e., between the FIFO stage and the PA, c) a MuxDemux stage may be connected between the IO-FIFO and the RAM-PAEs.\",\n \"The MuxDemux stage may in turn either be fixedly implemented in the hardware and/or formed by a suitable configuration of any PAEs designed accordingly.\",\n \"The position of the multiplexers/demultiplexers of the MuxDemux stage is determined by the configuration by a CT and/or the array (PA) and/or the IO itself, which may also be dynamically influenced, e.g., on the basis of the degree of filling of the FIFO(s) and/or on the basis of pending data transfers (arbitration).\",\n \"In an example embodiment, the multiplexer/demultiplexer structure is formed by a configurable bus system (e.g., according to or resembling the bus system between the RAM/ALU/etc.—PAEs), whereby the bus system may in particular also be physically the same which is also used either by resource sharing or by a time multiplex method which may be implemented through a suitable reconfiguration.\",\n \"It may be particularly preferred if addresses are generated in a particular manner, as is evident from the following discussion.\",\n \"Addresses for internal or external memories may be computed by address generators.\",\n \"For example, groups of PAEs may be configured accordingly and/or explicit address generators, implemented separately and specially, if necessary (e.g., DMAs such as those described in German Patent No.\",\n \"DE 44 16 881) or within interface cells (such as those described in German Patent Application No.\",\n \"DE 196 54 595.1) may be used.\",\n \"In other words, either fixedly implemented address generators, which are integrated into a VPU or are implemented externally, may be used and/or the addresses may be calculated by a configuration of PAEs according to the requirements of an algorithm.\",\n \"Simple address generators are preferably fixedly implemented in the interface modules and/or active memories (e.g., RAM-PAES).\",\n \"For generation of complex address sequences (e.g., nonlinear, multidimensional, etc.\",\n \"), PAEs may be configured accordingly and connected to the interface cells.\",\n \"Such methods having the corresponding configurations are described in International Patent Application No.\",\n \"PCT/EP 00/10516.Configured address generators may belong to another configuration (ConfigID, see, e.g., German Patent Application Nos.\",\n \"DE 198 07 872.2, DE 199 26 538.0 and DE 100 28 397.7) other than data processing.\",\n \"This makes a decoupling of address generation from data processing possible, so that in a preferred method, for example, addresses may already be generated and the corresponding data already loaded before or during the time when the data processing configuration is being configured.\",\n \"It should be pointed out that such data preloading and/or address pregeneration is particularly preferred for increasing processor performance, in particular by reducing latency and/or the wait clock cycle.\",\n \"Accordingly, the result data and its addresses may still be processed during or after removal of the data processing/generating configuration.\",\n \"In particular, it is possible through the use of memories and/or buffers such as the FIFOs described here, for example, to further decouple data processing from memory access and/or IO access.\",\n \"In a preferred procedure, it may be particularly effective to combine fixedly implemented address generators (HARD-AG) (see, e.g., German Patent Application No.\",\n \"DE 196 54 595.1) and configurable address generators in the PA (SOFT-AG) in such a way that HARD-AGs are used for implementation of simple addressing schemes, while complex addressing sequences are computed by the SOFT-AG and then sent to the HARD-AG.\",\n \"In other words, individual address generators may overload and reset one another.\",\n \"Interface modules for reconfigurable components are described in German Patent Application No.\",\n \"DE 196 54 595.1.The interface modules disclosed therein and their operation could still be improved further to increase processor efficiency and/or performance.\",\n \"Therefore, within the scope of the present invention, a particular embodiment of interface modules is proposed below such as that disclosed in particular in German Patent Application No.\",\n \"DE 196 54 595.1.Each interface module may have its own unique identifier (IOID) which is transmitted from/to a protocol converter and is used for assigning data transfers to a certain interface module or for addressing a certain interface module.\",\n \"The IOID is preferably CT-configurable.\",\n \"For example, the IOID may be used to select a certain interface module for a data transfer in the case of accesses by an external master.\",\n \"In addition, the IOID may be used to assign the correct interface module to incoming read data.\",\n \"To do so, the IOID is, for example, transmitted with the address of a data-read access to the IO-FIFOs and either stored there and/or relayed further to the external bus.\",\n \"IO-FIFOs assign the IOIDs of the addresses sent out to the incoming read data and/or the IOIDs are also transmitted via the external bus and assigned by external devices or memories to the read data sent back.\",\n \"IOIDs may then address the multiplexers (e.g., upstream from the interface modules) so that they direct the incoming read data to the correct interface module.\",\n \"Interface modules and/or protocol converters conventionally operate as bus masters.\",\n \"In a special embodiment, it is now proposed that interface modules and/or protocol converters shall function alternatively and/or fixedly and/or temporarily as bus slaves, in particular in a selectable manner, e.g., in response to certain events, states of state machines in PAEs, requirements of a central configuration administration unit (CT), etc.\",\n \"In an additional embodiment, the interface modules are expanded so that generated addresses, in particular addresses generated in SOFT-AGs, are assigned a certain data packet.\",\n \"A preferred embodiment of an interface module is described below: A preferred coupling of an interface module is accomplished by connecting any PAEs (RAM, ALU, etc.)\",\n \"and/or the array (PA) via a bus (preferably configurable) to interface modules which are either connected to the protocol converters or have the protocol converters integrated into them.\",\n \"In a variant embodiment, IO-FIFOs are integrated into the interface modules.\",\n \"For write access (the VPU sends data to external IOs, e.g., memories/peripherals, etc.)\",\n \"it is advantageous to link the address output to the data output, i.e., a data transfer takes place with the IO precisely when a valid address word and a valid data word are applied at the interface module, the two words may be originating from different sources.\",\n \"Validity may be identified by a handshake protocol (RDY/ACK) according to German Patent Application Nos.\",\n \"DE 196 51 075.9 or DE 101 10 530.4, for example.\",\n \"Through suitable logic gating (e.g., AND) of RDY signals of address word and data word, the presence of two valid words is detectable, and IO access may be executed.\",\n \"On execution of the IO access, the data words and the address words may be acknowledged by generating a corresponding ACK for the two transfers.\",\n \"The IO access including the address and data, as well as the associated status signals, if necessary, may be decoupled in output FIFOs according to the present invention.\",\n \"Bus control signals are preferably generated in the protocol converters.\",\n \"For read access (the VPU receives data from external IOs, e.g., memories/peripherals, etc.\",\n \"), the addresses for the access are first generated by an address generator (HARD-AG and/or SOFT-AG) and the address transfer is executed.\",\n \"Read data may arrive in the same clock cycle or, at high frequencies, may arrive pipelined one or more clock cycles later.\",\n \"Both addresses and data may be decoupled through IO-FIFOs.\",\n \"The conventional RDY/ACK protocol may be used for acknowledgment of the data, and it may also be pipelined (see, e.g., German Patent Application Nos.\",\n \"DE 196 54 595.1, DE 197 04 742.4, DE 199 26 538.0, DE 100 28 397.7 and DE 101 10 530.4).\",\n \"The conventional RDY/ACK protocol may also be used for acknowledgment of the addresses.\",\n \"However, acknowledgment of the addresses by the receiver results in a very long latency, which may have a negative effect on the performance of VPUs.\",\n \"The latency may be bypassed in that the interface module acknowledges receipt of the address and synchronizes the incoming data assigned to the address with the address.\",\n \"Acknowledgment and synchronization may be performed by any suitable acknowledgment circuit.\",\n \"Two possible embodiments are explained in greater detail below, although in a non-limiting fashion: a) FIFO A FIFO stores the outgoing address cycles of the external bus transfers.\",\n \"With each incoming data word as a response to an external-bus access, the FIFO is instructed accordingly.\",\n \"Due to the FIFO character, the sequence of outgoing addresses corresponds to the sequence of outgoing data words.\",\n \"The depth of the FIFO (i.e., the number of possible entries) is preferably adapted to the latency of the external system, so that any outgoing address may be acknowledged without latency and optimum data throughput is achieved.\",\n \"Incoming data words are acknowledged according to the FIFO entry of the assigned address.\",\n \"If the FIFO is full, the external system is no longer able to accept any additional addresses and the current outgoing address is not acknowledged and is thus held until data words of a preceding bus transfer have been received and one FIFO entry has been removed.\",\n \"If the FIFO is empty, no valid bus transfer is executed and possibly incoming data words are not acknowledged.\",\n \"b) Credit Counter Each outgoing address of external bus transfers is acknowledged and added to a counter (credit counter).\",\n \"Incoming data words as a response to an external bus transfer are subtracted from the counter.\",\n \"If the counter reaches a defined maximum value, the external system can no longer accept any more addresses and the current outgoing address is not acknowledged and is thus held until data words of a preceding bus transfer have been received and the counter has been decremented.\",\n \"If the counter content is zero, no valid bus transfer is executed and incoming data words are not acknowledged.\",\n \"To optimally support burst transfers, the method using a) (FIFO) is particularly preferred, and in particular FIFOs may be used like the FIFOs described below for handling burst accesses and the assignment of IOIDs to the read data.\",\n \"The IO-FIFOs described here may be integrated into the interface modules.\",\n \"In particular, an IO-FIFO may also be used for embodiment variant a).\",\n \"The optional possibility of providing protocol converters is discussed above.\",\n \"With regard to particularly advantageous possible embodiments of protocol converters, the following comments should be made: A protocol converter is responsible for managing and controlling an external bus.\",\n \"The detailed structure and functioning of a protocol converter depend on the design of the external bus.\",\n \"For example, an AMBA bus requires a protocol converter different from a RAMBUS.\",\n \"Different protocol converters are connectable to the interface modules, and within one embodiment of a VPU, a plurality of, in particular, different protocol converters may be implemented.\",\n \"In one preferred embodiment, the protocol converters are integrated into the IO-FIFOs of the present invention.\",\n \"It is possible according to the present invention to provide burst bus access.\",\n \"Modern bus systems and SoC bus systems transmit large volumes of data via burst sequences.\",\n \"An address is first transmitted and data is then transmitted exclusively for a number of cycles (see AMBA Specification 2.0, ARM Limited).\",\n \"For correctly executing burst accesses, several tasks are to be carried out: 1) Recognizing Burst Cycles Linear bus accesses, which may be converted into bursts, must be recognized to trigger burst transfers on the external bus.\",\n \"For recognizing linear address sequences, a counter (TCOUNTER) may be used; it is first loaded with a first address of a first access and counts linearly up/down after each access.\",\n \"If the subsequent address corresponds to the counter content, there is a linear and burst-capable sequence.\",\n \"2) Aborting at Boundaries Some bus systems (e.g., AMBA) allow bursts (a) only up to a certain length and/or (b) only up to certain address limits (e.g., 1024 address blocks).\",\n \"For (a), a simple counter may be implemented according to the present invention, which counts from the first desired or necessary bus access the number of data transmissions and at a certain-value which corresponds to the maximum length of the burst transfer, signals the boundary limits using a comparator, for example.\",\n \"For L(b), the corresponding bit (e.g., the 10th bit for 1024 address limits) which represents the boundary limit may be compared between TCOUNTER and the current address (e.g., by an XOR function).\",\n \"If the bit in the TCOUNTER is not equal to the bit in the current address, there has been a transfer beyond a boundary limit which is signaled accordingly.\",\n \"3) Defining the Length If the external bus system does not require any information regarding the length of a burst cycle, it is possible and preferable according to the present invention to perform burst transfers of an indefinite length (cf.\",\n \"AMBA).\",\n \"If length information is expected and/or certain burst lengths are predetermined, the following procedure may be used according to the present invention.\",\n \"Data and addresses to be transmitted are written into a FIFO, preferably with the joint use of the IO-FIFO, and are known on the basis of the number of addresses in the (IO-)FIFO.\",\n \"For the addresses, an address FIFO is used, transmitting in master mode the addresses from the interface modules to the external bus and/or operating conversely in slave mode.\",\n \"Data is written into a data FIFO, which transmits data according to the transmission (read/write).\",\n \"In particular, a different FIFO may be used for write transfers and for read transfers.\",\n \"The bus transfers may then be subdivided into fixed burst lengths, so that they are known before the individual burst transfers and may be stated on initiation of the burst, burst transfers of the maximum burst length preferably being formed first and if the number of remaining (IO-)FIFO entries is smaller than the current burst length, a next smaller burst length is used in each case.\",\n \"For example, ten (IO-;)FIFO entries may be transmitted at a maximum burst length of 4 with 4, 4, 2 burst transfers.\",\n \"4) Error Recovery Many external bus systems (cf.\",\n \"AMBA) provide methods for error elimination in which failed bus transfers are repeated, for example.\",\n \"The information as to whether a bus transfer has failed is transmitted at the end of a bus transfer, more or less as an acknowledgment for the bus transfer.\",\n \"To repeat a bus transfer, it is now necessary for all the addresses to be available, and in the case of write access, the data to be written away must also be available.\",\n \"According to the present invention, the address FIFOs (preferably the address FIFOs of the IO-FIFOs) are modified so that the read pointer is stored before each burst transfer.\",\n \"Thus, a FIFO read pointer position memory means is provided, in particular an address FIFO read pointer position memory means.\",\n \"This may form an integral part of the address FIFO in which, for example, a flag is provided, indicating that information stored in the FIFO represents a read pointer position or it may be provided separately from the FIFO.\",\n \"As an alternative, a status indicating deletability could also be assigned to data stored in the FIFO, this status also being stored and reset to “deletable” if successful data transmission has been acknowledged.\",\n \"If an error has occurred, the read pointer is reset at the position stored previously and the burst transfer is repeated.\",\n \"If no error has occurred, the next burst transfer is executed and the read pointer is restored accordingly.\",\n \"To prevent the write pointer from arriving at a current burst transfer and thus overwriting values which might still be needed in a repeat of the burst transfer, the full status of the FIFOs is determined by comparing the stored read pointer with the write pointer.\",\n \"IO-FIFOs and/or FIFOs for managing burst transfers may preferably be expanded to incoming read data using the function of address assignment, which is known from the interface modules.\",\n \"Incoming read data may also be assigned the IOID which is preferably stored in the FIFOs together with the addresses.\",\n \"Through the assignment of the IOID to incoming read data, the assignment of the read data to the corresponding interface modules is possible by switching the multiplexers according to the IOIDs, for example.\",\n \"According to the present invention, it is possible to use certain bus systems and/or to design bus systems in different ways.\",\n \"This is described in further detail below.\",\n \"Depending on the design, different bus systems may be used between the individual units, in particular the interface modules, the IO-FIFOs, the protocol converters, and a different bus system may be implemented between each of two units.\",\n \"Different designs are implementable, the functions of a plurality of designs being combinable within one design.\",\n \"A few design options are described below.\",\n \"The simplest possible design is a direct connection of two units.\",\n \"In an expanded embodiment, multiplexers are provided between the units, which may have different designs.\",\n \"This example embodiment is preferred in particular when using a plurality of the particular units.\",\n \"A multiplex function may be obtained using a configurable bus, which is configurable by a higher-level configuration unit (CT), specifically for a period of time for the connection of certain units.\",\n \"In an example embodiment, the connections are defined by selectors which decode a portion of an address and/or an IOID, for example, by triggering the multiplexers for the interconnection of the units.\",\n \"In a particularly preferred embodiment, the selectors are designed in such a way that a plurality of units may select a different unit at the same time, each of the units being arbitrated for selection in chronological sequence.\",\n \"An example of a suitable bus system is described in, e.g., German Patent Application No.\",\n \"DE 199 26 538.0.Additional states may be used for arbitration.\",\n \"For example, data transfers between the interface modules and the IO-FIFOs may be optimized as follows: In each case one block of a defined size of data to be transmitted is combined within the FIFO stages.\",\n \"As soon as a block is full/empty, a bus access is signaled to the arbiter for transmitting the data.\",\n \"Data is transmitted in a type of burst transfer, i.e., the entire data block is transmitted by the arbiter during a bus allocation phase.\",\n \"In other words, a bus allocation may take place in a manner determined by FIFO states of the connected FIFOs, data blocks being used for the determination of state within a FIFO.\",\n \"If a FIFO is full, it may arbitrate the bus for emptying; if a FIFO is empty, it may arbitrate the bus for filling.\",\n \"Additional states may be provided, e.g., in flush, which is used for emptying only partially full FIFOs and/or for filling only partially empty FIFOs.\",\n \"For example, flush may be used in a change of configuration (reconfiguration).\",\n \"In a preferred embodiment, the bus systems are designed as pipelines in order to achieve high data transfer rates and clock rates by using suitable register stages and may also function as FIFOs themselves, for example.\",\n \"In a preferred embodiment, the multiplexer stage may also be designed as a pipeline.\",\n \"According to the present invention, it is possible to connect a plurality of modules to one IO and to provide communication among the modules.\",\n \"In this regard, the following should be pointed out: configuration modules which include a certain function and are reusable and/or relocatable within the PA are described in, for example, German Patent Application Nos.\",\n \"DE 198 07 872.2, DE 199 26 538.0, and DE 100 28 397.7.A plurality of these configuration modules may be configured simultaneously into the PA, dependently and/or independently of one another.\",\n \"The configuration modules must be hardwired to a limited IO, which is typically provided in particular only at certain locations and is therefore not relocatable, in such a way that the configuration modules are able to use the IOs simultaneously and data is assigned to the correct modules.\",\n \"In addition, configuration modules that belong together (dependent) must be hardwired together in such a way that free relocation of the configuration modules is possible among one another in the PA.\",\n \"Such a flexible design is in most cases not possible through the conventional networks (see, e.g., German Patent Nos.\",\n \"P 44 16 881.0, 02, 03, 08), because this network must usually be explicitly allocated and routed through a router.\",\n \"German Patent Application No.\",\n \"DE 197 04 742.4 describes a method of constructing flexible data channels within a PAE matrix according to the algorithms to be executed so that a direct connection through and in accordance with a data transmission is created and subsequently dismantled again.\",\n \"Data to be transmitted may be precisely assigned to one source and/or one destination.\",\n \"In addition and/or as an alternative to German Patent Application No.\",\n \"DE 197 04 742.4 and the procedures and configurations described therein, additional possibilities are now provided through the present invention, and methods (hereinafter referred to jointly as GlobalTrack) that permit flexible allocation and interconnection during run time may be used, e.g., serial buses, parallel buses and fiber optics, each with suitable protocols (e.g., Ethernet, Firewire, USB).\",\n \"Reference is made here explicitly to transmission by light using a light-conducting substrate, in particular with appropriate modulation for decoupling of the channels.\",\n \"Another particular feature of the present invention with respect to memory addressing, in particular paging and MMU options, is described below.\",\n \"Data channels of one or multiple GlobalTracks may be connected via mediating nodes to an ordinary network, e.g., according to German Patent Nos.\",\n \"P 44 16 881.0, 02, 03, 08.Depending on the implementation, the mediating nodes may be configured differently in the PA, e.g., assigned to each PAE, to a group and/or hierarchy of PAEs, and/or to every nth PAE.\",\n \"In a particularly preferred embodiment, all PAEs, interface modules, etc., have a dedicated connection to a GlobalTrack.\",\n \"A configuration module is designed in such a way that it has access to one or a plurality of these mediating nodes.\",\n \"A plurality of configuration modules among one another and/or configuration modules and IOs may now be connected via the GlobalTrack.\",\n \"With proper implementation (e.g., German Patent Application No.\",\n \"DE 197 04 742.4) a plurality of connections may now be established and used simultaneously.\",\n \"The connection between transmitters and receivers may be established in an addressed manner to permit individual data transfer.\",\n \"In other words, transmitters and receivers are identifiable via GlobalTrack.\",\n \"An unambiguous assignment of transmitted data is thus possible.\",\n \"Using an expanded IO, which also transmits the transmitter address and receiver address—as is described in German Patent Application No.\",\n \"DE 101 10 530.4, for example—and the multiplexing methods described in German Patent Application No.\",\n \"DE 196 54 595.1, data for different modules may be transmitted via the IO and may also be assigned unambiguously.\",\n \"In a preferred embodiment, data transfer is synchronized by handshake signals, for example.\",\n \"In addition, data transfer may also be pipelined, i.e., via a plurality of registers implemented in the GlobalTrack or assigned to it.\",\n \"In a very complex design for large-scale VPUs or for their interconnection, a GlobalTrack may be designed in a network topology using switches and routers; for example, Ethernet could be used.\",\n \"It should be pointed out that different media may be used for GlobalTrack topologies, e.g., the method described in German Patent Application No.\",\n \"DE 197 04 742.4 for VPU-internal connections and Ethernet for connections among VPUs.\",\n \"Memories (e.g., RAM-PAEs) may be equipped with an MMU-like paging method.\",\n \"For example, a large external memory could then be broken down into segments (pages), which in the case of data access within a segment would be loaded into one of the internal memories and, at a later point in time, after termination of data access, would be written back into the external memory.\",\n \"In a preferred embodiment, addresses sent to a (internal) memory are broken down into an address area, which is within the internal memory (MEMADR) (e.g., the lower 10 bits in a 1,024-entry memory) and a page address (the bits above the lower 10).\",\n \"The size of a page is thus determined by MEMADR.\",\n \"The page address is compared with a register (page register) assigned to the internal memory.\",\n \"The register stores the value of the page address last transferred from a higher-level external (main) memory into the internal memory.\",\n \"If the page address matches the page register, free access to the internal memory may take place.\",\n \"If the address does not match (page fault), the current page content is written, preferably linearly, into the external (main) memory at the location indicated by the page register.\",\n \"The memory area in the external (main) memory (page) which begins at the location of the current new page address is written into the internal memory.\",\n \"In a particularly preferred embodiment, it is possible to specify by configuration whether or not, in the event of a page fault, the new page is to be transferred from the external (main) memory into the internal memory.\",\n \"In a particularly preferred embodiment, it is possible to specify by configuration whether or not, in the event of a page fault, the old page is to be transferred from the internal memory into the external (main) memory.\",\n \"The comparison of the page address with the page register preferably takes place within the particular memory.\",\n \"Data transfer control in the event of page faults may be configured accordingly by any PAEs and/or may take place via DMAs (e.g., in the interface modules or external DMAs).\",\n \"In a particularly preferred embodiment, the internal memories are designed as active memories having integrated data transfer control (see, e.g., German Patent Application No.\",\n \"DE 199 26 538.0).\",\n \"In another possible embodiment, an internal memory may have a plurality (p) of pages, the size of a page then preferably being equal to the size of the memory divided by p. A translation table (translation look-aside buffer=TLB) which is preferably designed like a fully associative cache replaces the page register and translates page addresses to addresses in the internal memory; in other words, a virtual address may be translated into a physical address.\",\n \"If a page is not included in the translation table (TLB), a page fault occurs.\",\n \"If the translation table has no room for new additional pages, pages may be transferred from the internal memory into the external (main) memory and removed from the translation table so that free space is again available in the internal memory.\",\n \"It should be pointed out explicitly that a detailed discussion is not necessary because a plurality of conventional MMU methods may be used and may be used with only minor and obvious modifications.\",\n \"The possibility of providing a collector memory, as it is known, has been mentioned above.\",\n \"In this regard, the following details should also be mentioned.\",\n \"A collector memory (collector) capable of storing larger volumes of data may be connected between the interface modules and IO-FIFOs.\",\n \"The collector may be used for exchanging data between the interface modules, i.e., between memories assigned to the array (e.g., RAM-PAEs).\",\n \"The collector may be used as a buffer between data within a reconfigurable module and external data.\",\n \"A collector may function as a buffer for data between different reconfiguration steps; for example, it may store data of different configurations while different configurations are active and are being configured.\",\n \"At deactivation of configurations, the collector stores their data, and data of the newly configured and active configurations is transmitted to the PA, e.g., to memories assigned to the array (RAM-PAEs).\",\n \"A plurality of interface modules may have access to the collector and may manage data in separate and/or jointly accessible memory areas.\",\n \"In a preferred embodiment, the collector may have multiple terminals for interface modules, which may be accessed simultaneously (i.e., it is designed as a multi-port collector device).\",\n \"The collector has one or more terminals to an external memory and/or external peripherals.\",\n \"These terminals may be connected to the IO-FIFOs in particular.\",\n \"In an expanded embodiment, processors assigned to the VPU, such as DSPs, CPUs and microcontrollers, may access the collector.\",\n \"This is preferably accomplished via another multi-port interface.\",\n \"In a preferred embodiment, an address translation table is assigned to the collector.\",\n \"Each interface may have its own address translation table or all the interfaces may share one address translation table.\",\n \"The address translation table may be managed by the PA and/or a CT and/or an external unit.\",\n \"The address translation table is used to assign collector memory areas to any addresses and it operates like an MMU system.\",\n \"If an address area (page) is not present within the collector (pagemiss), this address area may be loaded into the collector from an external memory.\",\n \"In addition, address areas (pages) may be written from the collector into the external memory.\",\n \"For data transfer to or between the external memory, a DMA is preferably used.\",\n \"A memory area within the collector may be indicated to the DMA for reading or writing transmission; the corresponding addresses in the external memory may be indicated separately or preferably removed by the DMA from the address translation table.\",\n \"A collector and its address generators (e.g., DMAs) may preferably operate according to or like MMU systems, which are conventional for processors according to the related art.\",\n \"Addresses may be translated by using translation tables (TLB) for access to the collector.\",\n \"According to the present invention, all MMU embodiments and methods described for internal memories may also be used on a collector.\",\n \"The operational specifics will not be discussed further here because they correspond to or closely resemble the related art.\",\n \"In an expanded or preferred embodiment, a plurality of collectors may be implemented.\",\n \"According to the present invention, it is possible to optimize access to memory.\",\n \"The following should be pointed out in this regard: One basic property of the preferred reconfigurable VPU architecture PACT-XPP is the possibility of superimposing reconfiguration and data processing (see, e.g., German Patent No.\",\n \"P 44 16 881.0, and German Patent Application Nos.\",\n \"DE 196 51 075.9, DE 196 54 846.2, DE 196 54 593.5, DE 198 07 872.2, DE 199 26 538.0, DE 100 28 397.7, DE 102 06 857.7).\",\n \"In other words, for example: a) the next configuration may already be preloaded during data processing; and/or b) data processing in other already-configured elements may already begin while a number of configurable elements or certain configurations are not yet configured or are in the process of being configured; and/or c) the configuration of various activities is superimposed or decoupled in such a way that they run with a mutual time offset at optimum performance (see 8.1 address generation).\",\n \"Modern memory protocols (e.g., SDRAM, DDRAM, RAMBUS) usually have the following sequence or a sequence having a similar effect, but steps 2 and 3 may possibly also occur in the opposite order: 1.Initializing access with the address given; 2.A long latency; 3.Rapid transmission of data blocks, usually as a burst.\",\n \"This property may be utilized in a performance-efficient manner in VPU technology.\",\n \"For example, it is possible to separate the steps of computation of the address(es), initialization of memory access, data transfer and data processing in the array (PA) in such a way that different (chronological) configurations occur, so that largely optimum superpositioning of the memory cycles and data processing cycles may be achieved.\",\n \"Multiple steps may also be combined, depending on the application.\",\n \"For example, the following method corresponds to this principle: The application AP, which includes a plurality of configurations (ap=1, 2, .\",\n \".\",\n \".\",\n \", z), is to be executed.\",\n \"Furthermore, additional applications/configurations which are combined under WA are to be executed on the VPU: 1.Read addresses are first computed (in an ap configuration of AP) and the data transfers and IO-FIFOs are initialized; 2.Data transmitted for AP and now present in IO-FIFOs is processed (in an (ap+1) configuration) and, if necessary, stored in FIFOs, buffers or intermediate memories, etc.\",\n \"; 2a.\",\n \"Computation of results may require a plurality of configuration cycles (n) at the end of which the results are stored in an IO-FIFO, and 3.The addresses of the results are computed and the data transfer is initialized; this may take place in parallel or later in the same configuration or in an (ap+n+2) configuration; at the same time or with a time offset, data is then written from the IO-FIFOs into the memories.\",\n \"Between the steps, any configuration from WA may be executed, e.g., when a waiting time is necessary between steps, because data is not yet available.\",\n \"Likewise, in parallel with the processing of AP, configurations from WA may be executed during the steps, e.g., if AP does not use the resources required for WA.\",\n \"It will be self-evident to those skilled in the art that variously modified embodiments of this method are also possible.\",\n \"In one possible embodiment, the processing method may take place as shown below (Z marks a configuration cycle, i.e., a unit of time): Other configurations Z Configuration AP (WA) 1 Compute read addresses, Any other initialize access configurations 2 Input of data and/or data 3 + k Process data processing, read/ (if necessary in a plurality of write processes (k) configuration cycles) using IO-FIFOs 4 + k Compute write addresses, and/or RAM-PAEs in initialize access other resources or 5 + k Output of data time-multiplexed resources via configuration cycles This sequence may be utilized efficiently by the data processing method described in, for example, German Patent Application No.\",\n \"DE 102 02 044.2 in particular.\",\n \"The methods and devices described above are preferably operated using special compilers, which are expanded in particular in comparison with traditional compilers.\",\n \"The following should be pointed out in this regard: For generating configurations, compilers that run on any computer system are used.\",\n \"Typical compilers include, for example, C-compilers and/or even NML compilers for VPU technology, for example.\",\n \"Particularly suitable compiler methods are described in German Patent Application Nos.\",\n \"DE 101 39 170.6, and DE 101 29 237.6, and European Patent No.\",\n \"EP 02 001 331.4, for example.\",\n \"The compiler, at least partially, preferably takes into account the following particular factors: Separation of addressing into 1.external addressing, i.e., data transfers with external modules, 2.internal addressing, i.e., data transfers among PAEs, in particular between RAM-PAEs and ALU-PAEs, 3.in addition, time decoupling also deserves special attention.\",\n \"Bus transfers are broken down into internal and external transfers.\",\n \"bt1) External read accesses are separated and, in one possible embodiment, they are also translated into a separate configuration.\",\n \"Data is transmitted from an external memory to an internal memory.\",\n \"bt2) Internal accesses are coupled to data processing, i.e., internal memories are read and/or written for data processing.\",\n \"bt3) External write accesses are separated and, in one possible embodiment, they are also translated into a separate configuration.\",\n \"Data is transmitted from an internal memory into an external memory.\",\n \"bt1, bt2, and bt3 may be translated into different configurations which may, if necessary, be executed at a different point in time.\",\n \"This method will now be illustrated on the basis of the following example: function example (a, b:integer)→x:integer for i := 1 to 100 for j := 1 to 100 x[i] := a[i] * b[j].\",\n \"This function is transformed by the compiler into three parts, i.e., configurations (subconfig): example#dload: Loads data from externally (memories, peripherals, etc.)\",\n \"and writes it into internal memories.\",\n \"Internal memories are indicated by r# and the name of the original variable.\",\n \"example#process: Corresponds to the actual data processing.\",\n \"This reads data out of internal operands and writes the results back into internal memories.\",\n \"example#dstore: Writes the results from the internal memory into externally (memories, peripherals, etc.).\",\n \"function example# (a, b : integer) -> x : integer subconfig example#dload for i := 1 to 100 r#a[i] := a[i] for j := 1 to 100 r#b[j] := b[j] subconfig example#process for i := 1 to 100 for j := 1 to 100 r#x[i] := r#a[i] * r#b[j] subconfig example#dstore for i := 1 to 100 x[i] := r#x[i].\",\n \"An effect of the example method is that instead of i*j=100*100=10,000 external accesses, only i+j=100+100=200 external accesses are performed for reading the operands.\",\n \"These accesses are also completely linear, which greatly accelerates the transfer rate in modern bus systems (burst) and/or memories (SDRAM, DDRAM, RAMBUS, etc.).\",\n \"Internal memory accesses take place in parallel, because different memories have been assigned to the operands.\",\n \"For writing the results, i=100 external accesses are necessary and may again be performed linearly at maximum performance.\",\n \"If the number of data transfers is not known in advance (e.g., WHILE loop) or is very large, a method may be used which reloads the operands as necessary through subprogram call instructions and/or writes the results externally.\",\n \"In a preferred embodiment, the states of the FIFOs may (also) be queried: “empty” if the FIFO is empty and “full” if the FIFO is full.\",\n \"The program flow responds according to the states.\",\n \"It should be pointed out that certain variables (e.g., ai, bi, xi) are defined globally.\",\n \"For performance optimization, a scheduler may execute the configurations example#dloada, example#dloadb before calling up example#process according to the methods already described, so that data is already preloaded.\",\n \"Likewise, example#dstore(n) may still be called up after termination of example#process in order to empty r#x.\",\n \"subconfig example#dloada(n) while !full(r#a) AND ai <= n r#a[ai] := a[ai] ai++ subconfig example#dloadb(n) while !full(r#b) AND bi <= n r#b[bi] := b[bi] bi++ subconfig example#dstore (n) while !empty(r#x) AND xi <= n x[xi] := r#x[xi] xi++ subconfig example#process for i := 1 to n for j := 1 to m if empty(r#a) then example#dloada(n) if empty(r#b) then example#dloadb(m) if full(r#x) then example#dstore(n) r#x[i] := r#a[i] + r#b[j] bj := 1.The subprogram call instructions and managing of the global variables are comparatively complex for reconfigurable architectures.\",\n \"Therefore, in a preferred embodiment, the following optimization may be performed; in this optimized method, all configurations are run largely independently and are terminated after being completely processed (terminate).\",\n \"Since data b[j] is required repeatedly, example#dloadb must accordingly be run through repeatedly.\",\n \"To do so, for example, two alternatives will be described: Alternative 1: example#dloadb terminates after each run-through and is reconfigured for each new start by example#process.\",\n \"Alternative 2: example#dloadb runs infinitely and is terminated by example#process.\",\n \"While “idle,” a configuration is inactive (waiting).\",\n \"subconfig example#dloada(n) for i := 1 to n while full(r#a) idle r#a[i] :=a[i] terminate subconfig example#dloadb(n) while 1 // ALTERNATIVE 2 for i := 1 to n while full(r#b) idle r#b[i] := a[i] terminate subconfig example#dstore(n) for i := 1 to n while empty(r#b) idle x[i] := r#x[i] terminate subconfig example#process for i := 1 to n for j := 1 to m while empty(r#a) or empty(r#b) or full(r#x) idle r#x[i] := r#a[i] * r#b[j] config example#dloadb(n) // ALTERNATIVE 1 terminate example#dloadb(n) // ALTERNATIVE 2 terminate To avoid waiting cycles, configurations may also be terminated as soon as they are temporarily no longer able to continue fulfilling their function.\",\n \"The corresponding configuration is removed from the reconfigurable module but remains in the scheduler.\",\n \"Therefore, the “reenter” instruction is used for this below.\",\n \"The relevant variables are saved before termination and are restored when configuration is repeated: subconfig example#dloada(n) for ai := 1 to n if full(r#a) reenter r#a[ai] := a[ai] terminate subconfig example#dloadb(n) while 1 // ALTERNATIVE 2 for bi := 1 to n if full(r#b) reenter r#b[bi] := a[bi] terminate subconfig example#dstore(n) for xi := 1 to n if empty(r#b) reenter x[xi] := r#x[xi] terminate subconfig example#process for i := 1 to n for j := 1 to m if empty(r#a) or empty(r#b) or full(r#x) reenter r#x[i] := r#a[i] * r#b[j] config example#dloadb(n) // ALTERNATIVE 1 terminate example#dloadb(n) // ALTERNATIVE 2 terminate With regard to the preceding discussion and to the following, the possibility of using a ‘context switch’ according to the present invention should also be pointed out.\",\n \"In this regard, the following should be noted: Repeated start of configurations, e.g., “reenter,” requires that local data (e.g., ai, bi, xi) be backed up and restored.\",\n \"Known related-art methods provide explicit interfaces to memories or to a CT to transmit data.\",\n \"All of these methods may be inconsistent and/or may require additional hardware.\",\n \"The context switch according to the present invention is implemented in such a way that a first configuration is removed; data to be backed up remains in the corresponding memories (REGs) (memories, registers, counters, etc.).\",\n \"A second configuration is loaded; this connects the REGs in a suitable manner and in a defined sequence to one or multiple global memory (memories).\",\n \"The configuration may use address generators, for example, to access the global memory (memories).\",\n \"The configuration may use address generators, for example, to access REGs designed as memories.\",\n \"According to the configured connection between the REGs, the contents of the REGs are written into the global memory in a defined sequence, the particular addresses being predetermined by address generators.\",\n \"The address generator generates the addresses for the global memory (memories) in such a way that the memory areas (PUSHAREA) that have been written are unambiguously assigned to the first configuration removed.\",\n \"In other words, different address areas are preferably provided for different configurations.\",\n \"The configuration corresponds to a PUSH of ordinary processors.\",\n \"Other configurations subsequently use the resources.\",\n \"The first configuration is to be started again, but first a third configuration which connects the REGs of the first configuration in a defined sequence is started.\",\n \"The configuration may use address generators, for example, to access the global memory or memories.\",\n \"The configuration may use address generators, for example, to access REGs designed as memories.\",\n \"An address generator generates addresses, so that correct access to the PUSHAREA assigned to the first configuration takes place.\",\n \"The generated addresses and the configured sequence of the REGs are such that data of the REGs is written from the memories into the REGs in the original order.\",\n \"The configuration corresponds to a POP of ordinary processors.\",\n \"The first configuration is restarted.\",\n \"In summary, a context switch is implemented in such a way that data to be backed up is exchanged with a global memory by loading particular configurations which operate like processor architectures known from PUSH/POP.\",\n \"There is also the possibility of providing a special task switch and/or multiconfiguration handling.\",\n \"In a preferred mode of operation, different data blocks of different configurations may be partitioned.\",\n \"These partitions may be accessed in a time-optimized manner by preloading a portion of the operands of a subsequent configuration P from external (main) memories and/or other (peripheral) data streams into the internal memories, e.g., during execution of a configuration Q, and during the execution of P, the results of Q as a portion of the total result from the internal memories are written into external (main) memories and/or other (peripheral) data streams.\",\n \"The functioning here differs considerably from that described in, for example, U.S. Pat.\",\n \"No.\",\n \"6,341,318.A data stream or data block is preferably decoupled by a FIFO structure (e.g., IO-FIFO).\",\n \"Different data streams or data blocks of different configurations in particular are preferably decoupled by different memories and/or FIFO areas and/or assignment marks in the FIFOs.\",\n \"The optional MMU methods described above may be used for decoupling and buffering external data.\",\n \"In one type of application, a large external data block may be broken down into a plurality of segments, each may be processed within a VPU.\",\n \"In an additional preferred mode of operation, different data blocks of different configurations may be broken down into partitions according to the method described above, these partitions now being defined as pages for an MMU.\",\n \"In this way, time-optimized access is possible by preloading the operands of a subsequent configuration P as a page from external (main) memories and/or other (peripheral) data streams into the internal memories, e.g., during execution of a configuration Q in the PA, and during the execution of P, the results of Q as a page from the internal memories are written into external (main) memories and/or other (peripheral) data streams.\",\n \"For the methods described above, preferably internal memories capable of managing a plurality of partitions and/or pages are used.\",\n \"These methods may be used for RAM-PAEs and/or collector memories.\",\n \"Memories having a plurality of bus interfaces (multi-port) are preferably used to permit simultaneous access of MMUs and/or the PA and/or additional address generators/data transfer devices.\",\n \"In one embodiment, identifiers are also transmitted in the data transfers, permitting an assignment of data to a resource and/or an application.\",\n \"For example, the method described in German Patent Application No.\",\n \"DE 101 10 530.4 may be used.\",\n \"Different identifiers may also be used simnultaneously.\",\n \"In a particularly preferred embodiment, an application identifier (APID) is also transmitted in each data transfer along with the addresses and/or data.\",\n \"An application includes a plurality of configurations.\",\n \"On the basis of the APID, the transmitted data is assigned to an application and/or to the memories or other resources (e.g., PAEs, buses, etc.)\",\n \"intended for an application.\",\n \"To this end, the APIDs may be used in different ways.\",\n \"Interface modules, for example, may be selected by APIDs accordingly.\",\n \"Memories, for example, may be selected by APIDs accordingly.\",\n \"PAEs, for example, may be selected by APIDs accordingly.\",\n \"For example, memory segments in internal memories (e.g., RAM-PAEs, collector(s)) may be assigned by APIDs.\",\n \"To do so, the APIDS, like an address part, may be entered into a TLB assigned to an internal memory so that a certain memory area (page) is assigned and selected as a function of an APID.\",\n \"This method yields the possibility of efficiently managing and accessing data of different applications within a VPU.\",\n \"There is the option of explicitly deleting data of certain APIDs (APID-DEL) and/or writing into external (main) memories and/or other (peripheral) data streams (APID-FLUSH).\",\n \"This may take place whenever an application is terminated.\",\n \"APID-DEL and/or APID-FLUSH may be triggered by a configuration and/or by a higher-level loading unit (CT) and/or externally.\",\n \"The following processing example is presented to illustrate the method.\",\n \"An application Q (e.g., APID=Q) may include a configuration for reading operands (e.g., ConfigID=j), a configuration for processing operands (e.g., ConfigID=w), and a configuration for writing results (e.g., ConfigID=s).\",\n \"Configuration j is executed first to read the operands chronologically optimally decoupled.\",\n \"Configurations of other applications may be executed simultaneously.\",\n \"The operands are written from external (main) memories and/or (peripheral) data streams into certain internal memories and/or memory areas according to the APID identifier.\",\n \"Configuration w is executed to process the stored operands.\",\n \"To do so, the corresponding operands in the internal memories and/or memory areas are accessed by citation of APIDs.\",\n \"Results are written into internal memories and/or memory areas accordingly by citation of APIDs.\",\n \"Configurations of other applications may be executed simultaneously.\",\n \"In conclusion, configuration s writes the stored results from the internal memories and/or memory areas into external (main) memories and/or other (peripheral) data streams.\",\n \"Configurations of other applications may be executed simultaneously.\",\n \"To this extent, the basic sequence of the method corresponds to that described above for optimization of memory access.\",\n \"If data for a certain APID is not present in the memories or if there is no longer any free memory space for this data, a page fault may be triggered for transmission of the data.\",\n \"While a module was initially assumed in which a field of reconfigurable elements is provided having little additional wiring, such as memories, FIFOs, and the like, it is also possible to use the ideas according to the present invention for systems known as “systems on a chip” (SoC).\",\n \"For SoCs the terms “internal” and “external” are not completely applicable in the traditional terminology, e.g., when a VPU is linked to other modules (e.g., peripherals, other processors, and in particular memories) on a single chip.\",\n \"The following definition of terms may then apply; this should not be interpreted as restricting the scope of the invention but instead is given only as an example of how the ideas of the present invention may be applied with no problem to constructs which traditionally use a different terminology: internal: within a VPU architecture and/or areas belonging to the VPU architecture and IP, external: outside of a VPU architecture, i.e., all other modules, e.g., peripherals, other processors, and in particular memories on a SoC and/or outside the chip in which the VPU architecture is located.\",\n \"A preferred embodiment will now be described.\",\n \"In a particularly preferred embodiment, data processing PAEs are located and connected locally in the PA (e.g., ALUs, logic, etc.).\",\n \"RAM-PAEs may be incorporated locally into the PA, but in a particularly preferred embodiment they are remote from the PA or are placed at its edges (see, e.g., German Patent Application No.\",\n \"DE 100 50 442.6).\",\n \"This takes place so as not to interfere with the homogeneity of the PA in the case of large RAM-PAE memories, where the space required is much greater than with ALU-PAEs and because of a gate/transistor layout (e.g., GDS2) of memory cells, which usually varies greatly.\",\n \"If the RAM-PAEs have dedicated connections to an external bus system (e.g., global bus), they are preferably located at the edges of a PA for reasons of layout, floor plan, and manufacturing.\",\n \"The configurable bus system of the PA is typically used for the physical connection.\",\n \"In an expanded embodiment, PAEs and interface modules, as well as additional configurable modules, if necessary, have a dedicated connection to a dedicated global bus, e.g., a GlobalTrack.\",\n \"Interface modules and in particular protocol converters are preferably remote from the PA and are placed outside of its configuration.\",\n \"This takes place so as not to interfere with the homogeneity of the PA and because of a gate/transistor layout (e.g., GDS2) of the interface modules/protocol converters, which usually varies greatly.\",\n \"In addition, the connections to external units are preferably placed at the edges of a PA for reasons of layout, floor plan, and manufacturing.\",\n \"The interface modules are preferably connected to the PA by the configurable bus system of the PA, the interface modules being connected to its outer edges.\",\n \"The bus system allows data exchange to take place configurably between interface modules and any PAEs within the PA.\",\n \"In other words, within one or different configurations, some interface modules may be connected to RAM-PAEs, for example, while other interface modules may be connected to ALU-PAEs, for example.\",\n \"The IO-FIFOs are preferably integrated into the protocol converter.\",\n \"To permit a greater flexibility in the assignment of the internal data streams to the external data streams, the interface modules and protocol converters are designed separately and are connected via a configurable bus system.\",\n \"The present invention is explained in greater detail below only as an example and in a nonrestrictive manner with reference to the drawings.\",\n \"FIG.\",\n \"1 shows a particularly preferred design of a reconfigurable processor which includes a core (array PA) (0103) including, for example, a configuration of ALU-PAEs (0101) (for performing computations) and RAM-PAEs (0102) (for saving data) and thus corresponds to the basic principle described in, for example, German Patent Application No.\",\n \"DE 196 54 846.2.The RAM-PAEs are preferably not integrated locally into the core, but instead are remote from the ALU-PAEs at the edges of or outside the core.\",\n \"This takes place so as not to interfere with the homogeneity of the PA in the case of large RAM-PAE memories where the space requirement is far greater than that of ALU-PAEs and because of a gate/transistor layout (e.g., GDS2) of memory cells which usually varies greatly.\",\n \"If the RAM-PAEs have dedicated connections to an external bus system (e.g., dedicated global bus; GlobalTrack; etc.\",\n \"), then they are preferably placed at the edges of a PA for reasons of layout, floor plan, and manufacturing.\",\n \"The individual units are interlinked via bus systems (0104).\",\n \"Interface modules (interface modules and protocol converters, if necessary) (0105) are located at the edges of the core and are connected to external buses (IO), as similarly described in German Patent Application No.\",\n \"DE 196 54 595.1.The interface modules may have different designs, depending on the implementation, and may fulfill one or more of the following functions, for example: 1.Combining and synchronizing a plurality of bus systems to synchronize addresses and data for example, 2.Address generators and/or DMAs, 3.FIFO stages for decoupling data and/or addresses, 4.Interface controllers (e.g., for AMBA bus, RAMBUS, RapidIO, USB, DDRRAM, etc.).\",\n \"FIG.\",\n \"2 shows a different embodiment of the architecture according to the present invention, depicting a configuration 0201 of ALU-PAEs (PA) linked to a plurality of RAM-PAEs (0202).\",\n \"External buses (IOs) (0204) are connected via FIFOs (0203).\",\n \"FIG.\",\n \"2a shows a direct FIFO to PA coupling.\",\n \"FIG.\",\n \"2b shows the IO (0204) connected to 0201 via the RAM-PAEs (0202).\",\n \"The connection occurs typically via the configurable bus system 0104 or a dedicated bus system.\",\n \"Multiplexers/demultiplexers (0205) switch a plurality of buses (0104) to the IOs (0204).\",\n \"The multiplexers are triggered by a configuration logic and/or address selector logic and/or an arbiter (0206).\",\n \"The multiplexers may also be triggered through the PA.\",\n \"FIG.\",\n \"2c corresponds to FIG.\",\n \"2b, but FIFOs (0203) have been connected upstream from the IOs.\",\n \"The diagrams in FIG.\",\n \"3 correspond to those in FIG.\",\n \"2, which is why the same reference numbers are used.\",\n \"FIG.\",\n \"3 illustrates the preferred data processing method in a VPU.\",\n \"FIG.\",\n \"3a: data passes through the IO (0204) into an input FIFO (0303 corresponding to 0203) and is loaded from this into the PA (0201) and/or beforehand into memory 0202.FIGS.\",\n \"3b-e show the data execution in which data is transmitted between the memories.\",\n \"During this period of time, the FIFOs may still transmit input data (0301) and/or output data (0302).\",\n \"In FIG.\",\n \"3f, data is loaded from the PA and/or from the memories into the output FIFO (0304).\",\n \"It should be pointed out again that input of data from the input FIFO into the RAM-PAEs or 0201 and writing of data from 0201 or the RAM-PAEs may take place simultaneously.\",\n \"It should likewise be pointed out that the input/output FIFOs are able to receive and/or send external data continuously during steps a-f.\",\n \"FIG.\",\n \"4 shows the same method in a slightly modified version in which multiplexers/demultiplexers (0401) are connected between the FIFOs and 0201 for simple data distribution.\",\n \"The multiplexers are triggered by a configuration logic and/or address selector logic and/or an arbiter (0402).\",\n \"Multiple configurations take place for data processing (a-e).\",\n \"The data may be read into memories and/or directly (0403) into the PA from the FIFOs (input FIFOs).\",\n \"During the input operation, data may be written from the PA and/or memories into FIFOs (output FIFOS) (0404).\",\n \"For data output, data may be written from the memories and/or directly (0405) from the PA into the FIFOs.\",\n \"Meanwhile, new data may be written from the input FIFOs into memories and/or the PA (0406).\",\n \"New data (0407) may already be entered during a last configuration, for example.\",\n \"During the entire processing, data may be transmitted from externally into the input FIFOs (0408) and/or from the output FIFOs to externally (0409).\",\n \"FIG.\",\n \"5 shows a possible embodiment of a PAE.\",\n \"A first bus system (0104a) is connected to a data processing unit (0501), the results of which are transmitted to a second bus system (0104b).\",\n \"The vertical data transfer is carried over two register/multiplexer stages (FREG 0502, BREG 0503), each with a different transfer direction.\",\n \"Preferably simple ALUs, e.g., for addition, subtraction, and multiplex operations, may be integrated into the FREG/BREG.\",\n \"The unit is configured in its function and interconnection by a configuration unit (CT) via an additional interface (0504).\",\n \"In a preferred embodiment, there is the possibility of setting constants in registers and/or memories for data processing.\",\n \"In another embodiment, a configuration unit (CT) may read out data from the working registers and/or memories.\",\n \"In an expanded embodiment, a PAE may additionally have a connection to a dedicated global bus (0505) (e.g., a GlobalTrack) and may thus communicate directly with a global, and if necessary also an external memory and/or peripheral unit, for example.\",\n \"In addition, a global bus may be designed so that different PAEs may communicate directly with one another via this bus, and in a preferred embodiment they may also communicate with modules for an external connection (e.g., interface modules).\",\n \"A bus system such as that described in German Patent Application No.\",\n \"DE 197 04 742.4, for example, may be used for such purposes.\",\n \"The data processing unit (0501) may be designed for ALU-PAEs as an arithmetic logic unit (ALU), for example.\",\n \"Different ALU-PAEs may use different ALUs and bus connection systems.\",\n \"One ALU may have more than two bus connections to 0104a and/or 0104b, for example.\",\n \"The data processing unit (0501) may be designed as a memory for RAM-PAEs, for example.\",\n \"Different RAM-PAEs may use different memories and bus connection systems.\",\n \"For example, a memory may have a plurality, in particular, more than two bus connections to 0104a and/or 0104b to allow access of a plurality of senders/receivers to one memory, for example.\",\n \"Accesses may preferably also take place simultaneously (multi-port).\",\n \"The function of the memory includes, for example, the following functions or combinations thereof: random access, FIFO, stack, cache, page memory with MMU method.\",\n \"In addition, in a preferred embodiment, the memory may be preloaded with data from the CT (e.g., constants, lookup tables, etc.).\",\n \"Likewise, in an expanded embodiment, the CT may read back data from the memory via 0504 (e.g., for debugging or for changing tasks).\",\n \"In another embodiment, the RAM-PAE may have a dedicated connection (0505) to a global bus.\",\n \"The global bus connects a plurality of PAEs among one another and in a preferred embodiment also to modules for an external connection (e.g., interface modules).\",\n \"The system described in German Patent Application No.\",\n \"DE 197 04 742.4 may be used for such a bus system.\",\n \"RAM-PAEs may be wired together in such a way that an n-fold larger memory is created from a plurality (n) of RAM-PAEs.\",\n \"FIG.\",\n \"6 shows an example of a wiring connection of ALU-PAEs (0601) and RAM-PAEs (0602) via a bus system 0104.FIG.\",\n \"1 shows a preferred example of a wiring connection for a reconfigurable processor.\",\n \"FIG.\",\n \"7 shows a simple embodiment variant of an IO circuit corresponding to 0105.Addresses (ADR) and data (DTA) are transmitted together with synchronization lines (RDY/ACK) between the internal bus systems (0104) and an external bus system (0703).\",\n \"The external bus system leads to IO-FIFOs and/or protocol converters, for example.\",\n \"FIG.\",\n \"7a shows a circuit for writing data.\",\n \"The addresses and data arriving from 0104 are linked together (0701).\",\n \"A FIFO stage for decoupling may be provided between 0104 and 0703 in the interface circuit (0701).\",\n \"FIG.\",\n \"7b shows a circuit for reading data, in which an acknowledgment circuit (0702, e.g., FIFO, counter) is provided for coordinating the outgoing addresses with the incoming data.\",\n \"In 0701a and/or in 0701b, a FIFO stage for decoupling may be provided between 0104 and 0703.If a FIFO stage is provided in 0701b, it may also be used for acknowledgment circuit 0702.FIG.\",\n \"8 shows a possible connection structure between interface modules and/or PAEs having a dedicated global bus (0801) and protocol converters (0802) to external (main) memories and/or other (peripheral) data streams.\",\n \"Interface modules are connected (0803) to a PA, preferably via their network according to 0104.A bus system (0804a, 0804b) is provided between interface modules and/or PAEs having a dedicated global bus (0801) and protocol converters (0802).\",\n \"In a preferred embodiment, 0804 is able to transmit pipelined data over a plurality of register stages.\",\n \"0804a and 0804b are interconnected via switches (e.g., 0805) which are designed as transmission gates and/or tristate buffers and/or multiplexers, for example.\",\n \"The multiplexers are triggered by rows and columns.\",\n \"Triggering units (0806) control the data transfer of the interface modules and/or PAEs having a dedicated global bus (0801) to the protocol converters (0802), i.e., in the transfer direction 0804a to 0804b.\",\n \"Triggering units (0807) control the data transfer of the protocol converters (0802) to the interface modules and/or the PAEs having a dedicated global bus (0801), i.e., in the transfer direction 0804b to 0804a.\",\n \"The triggering units (0806) each decode address areas for selection of the protocol converters (0802); the triggering units (0807) each decode IOIDs for selection of the interface modules and/or PAEs having a dedicated global bus (0801).\",\n \"Triggering units may operate according to different types of triggering, e.g., fixed connection without decoding; decoding of addresses and/or IOIDs; decoding of addresses and/or IOIDs and arbitration.\",\n \"One or multiple data words/address words may be transmitted per arbitration.\",\n \"Arbitration may be performed according to different rules.\",\n \"The interface modules may preferably have a small FIFO for addresses and/or data in the output direction and/or input direction.\",\n \"A particular arbitration rule preferably arbitrates an interface module having a FULL FIFO or an EMPTY FIFO or a FIFO to be emptied (FLUSH), for example.\",\n \"Triggering units may be designed as described in German Patent Application No.\",\n \"DE 199 26 538.0 (FIG.\",\n \"32), for example.\",\n \"These triggering units may be used for 0807 or 0806.When used as 0806, 0812 corresponds to 0804a, and 0813 corresponds to 0804b.\",\n \"When used as 0807, 0812 corresponds to 0804b, and 0813 corresponds to 0804a.\",\n \"Decoders (0810) decode the addresses/IOIDs of the incoming buses (0812) and trigger an arbiter (0811), which in turn switches the incoming buses to an output bus (0813) via a multiplexer.\",\n \"The protocol converters are coupled to external bus systems (0808), a plurality of protocol converters optionally being connected to the same bus system (0809), so that they are able to utilize the same external resources.\",\n \"The IO-FIFOs are preferably integrated into the protocol converters, a FIFO (BURST-FIFO) for controlling burst transfers for the external buses (0808) being connected downstream from them if necessary.\",\n \"In a preferred embodiment, an additional FIFO stage (SYNC-FIFO) for synchronizing the outgoing addresses with the incoming data is connected downstream from the FIFOs.\",\n \"Various programmable/configurable FIFO structures are depicted in 0820-0823, where A indicates the direction of travel of an address FIFO, D indicates the direction of travel of a data FIFO.\",\n \"The direction of data transmission of the FIFOs depends on the direction of data transmission and the mode of operation.\",\n \"If a VPU is operating as a bus master, then data and addresses are transmitted from internally to the external bus in the event of a write access (0820), and in the event of a read access (0821) addresses are transmitted from internally to externally and data from externally to internally.\",\n \"If a VPU is operating as a bus slave, then data and addresses are transmitted from the external bus to internally in the event of a write access (0822) and in the event of a read access (0823) addresses are transmitted from externally to internally and data is transmitted from internally to externally.\",\n \"In all data transfers, addresses and/or data and/or IOIDs and/or APIDs may be assigned and also stored in the FIFO stages.\",\n \"In a particularly preferred embodiment, the transfer rate (operating frequency) of the bus systems 0104, 0804, and 0808/0809 may each be different due to the decoupling of the data transfers by the particular FIFO stages.\",\n \"In particular the external bus systems (0808/0809) may operate at a higher transfer rate, for example, than the internal bus systems (0104) and/or (0804).\",\n \"FIG.\",\n \"9 shows a possible sequence of a data read transfer via the circuit according to FIG.\",\n \"8.Addresses (preferably identifiers, e.g., with IOIDs and/or APIDs) are transmitted via internal bus system 0104 to interface modules and/or PAEs having a dedicated global bus, which preferably have an internal FIFO (0901).\",\n \"The addresses are transmitted to an IO-FIFO (0903) via a bus system (e.g., 0804) which preferably operates as a pipeline (0902).\",\n \"The addresses are transmitted to a BURST-FIFO (0905) via another bus (0904) which may be designed as a pipeline but which is preferably short and local.\",\n \"The BURST-FIFO ensures correct handling of burst transfers via the external bus system, e.g., for controlling burst addresses and burst sequences and repeating burst cycles when errors occur.\",\n \"IOIDs and/or APIDs of addresses (0906) which are transmitted via the external bus system may be transmitted together with the addresses and/or stored in an additional SYNC-FIFO (0907).\",\n \"The SYNC-FIFO compensates for the latency between the outgoing address (0906) and the incoming data (0909).\",\n \"Incoming data may be assigned IOIDs and/or APIDs (0908) of the addresses referencing them via the SYNC-FIFO (0910).\",\n \"Data (and preferably IOIDs and/or APIDs) is buffered in an IO-FIFO (0911) and is subsequently transmitted via a bus system (e.g., 0804), which preferably functions as a pipeline (0912), to an interface module and/or PAE having a dedicated global bus (0913), preferably including an internal FIFO.\",\n \"Data is transmitted from here to the internal bus system (0104).\",\n \"Instead of to the IO-FIFO (0911), incoming data may optionally be directed first to a second BURST-FIFO (not shown), which behaves like BURST-FIFO 0905 if burst-error recovery is also necessary in read accesses.\",\n \"Data is subsequently relayed to 0911.FIG.\",\n \"10 corresponds in principle to FIG.\",\n \"8, which is why the same reference numbers have been used.\",\n \"In this embodiment, which is given as an example, fewer interface modules and/or PAEs having a dedicated global bus (0801) and fewer protocol converters (0802) to external (main) memories and/or other (peripheral) data streams are shown.\",\n \"In addition, a collector (1001) is shown which is connected to bus systems 0804 in such a way that data is written from the interface modules and protocol converters into the collector and/or is read out from the collector.\",\n \"The collector is switched to bus systems 0804a via triggering unit 1007 which corresponds to 0807, and the collector is switched to bus systems 0804b via triggering unit 1006, which corresponds to 0806.Multiple collectors may be implemented for which multiple triggering units 1006 and 1007 are used.\",\n \"A collector may be segmented into multiple memory areas.\",\n \"Each memory area may operate independently in different memory modes, e.g., as random access memory, FIFO, cache, MMU page, etc.\",\n \"A translation table (TLB) (1002) may be assigned to a collector to permit an MMU-type mode of operation.\",\n \"Page management may function, e.g., on the basis of segment addresses and/or other identifiers, e.g., APIDs and/or IOIDs.\",\n \"A DMA or multiple DMAs are preferably assigned to a collector to perform data transfers with external (main) memories and/or other (peripheral) data streams, in particular to automatically permit the MMU function of page management (loading, writing).\",\n \"DMAs may also access the TLB for address translation between external (main) memories and/or other (peripheral) data streams and collector.\",\n \"In one possible mode of operation, DMAs may receive address specifications from the array (PA), e.g., via 0804.DMAs may be triggered by one or more of the following units: an MMU assigned to the collector, e.g., in the case of page faults; the array (PA); an external bus (e.g., 0809); an external processor; a higher-level loading unit (CT).\",\n \"Collectors may have access to a dedicated bus interface (1004), preferably DMA-controlled and preferably master/slave capable, including a protocol converter, corresponding to or similar to protocol converters 0802 having access to external (main) memories and/or other (peripheral) data streams.\",\n \"An external processor may have direct access to collectors (1007).\",\n \"FIG.\",\n \"11 corresponds in principle to FIG.\",\n \"9, which is why the same reference numbers have been used.\",\n \"A collector (1101) including assigned transfer control (e.g., DMA preferably with TLB) (1102) is integrated into the data stream.\",\n \"The array (PA) now transmits data preferably using the collector (1103), which preferably exchanges data with external (main) memories and/or other (peripheral) data streams (1104), largely automatically and controlled via 1102.The collector preferably functions in a segmented MMU-type mode of operation, where different address areas and/or identifiers such as APIDs and/or IOIDs are assigned to different pages.\",\n \"Preferably 1102 may be controlled by page faults.\",\n \"FIG.\",\n \"12 shows a flow chart of data transfers for different applications.\",\n \"An array (PA) processes data according to the method described in German Patent Application No.\",\n \"DE 196 54 846.2 by storing operands and results in memories 1202 and 1203.In addition, a data input channel (1204) and a data output channel (1205) are assigned to the PA, through which the operands and/or results are loaded and/or stored.\",\n \"The channels may lead to external (main) memories and/or other (peripheral) data streams (1208).\",\n \"The channels may include internal FIFO stages and/or PAE-RAMs/PAE-RAM pages and/or collectors/collector pages.\",\n \"The addresses (CURR-ADR) may be computed currently by a configuration running in 1201 and/or may be computed in advance and/or computed by DMA operations of a (1003).\",\n \"In particular, an address computation within 1201 (CURR-ADR) may be sent to a collector or its DMA to address and control the data transfers of the collector.\",\n \"The data input channel may be preloaded by a configuration previously executed on 1201.The channels preferably function in a FIFO-like mode of operation to perform data transfers with 1208.In the example depicted here, a channel (1207), which has been filled by a previous configuration or application, is still being written to 1208 during data processing within 1201 described here.\",\n \"This channel may also include internal FIFO stages and/or PAE-RAMs/PAE-RAM pages and/or collectors/collector pages.\",\n \"The addresses may be computed currently by a configuration (OADR-CONF) running in parallel in 1201 and/or computed in advance and/or computed by DMA operations of a (1003).\",\n \"In particular, an address computation within 1201 (OADR-CONF) may be sent to a collector or its DMA to address and control the data transfers of the collector.\",\n \"In addition, data for a subsequent configuration or application is simultaneously loaded into another channel (1206).\",\n \"This channel too may include internal FIFO stages and/or PAE-RAMs/PAE-RAM pages and/or collectors/collector pages.\",\n \"The addresses may be computed currently by a configuration (IADR-CONF) running in parallel in 1201 and/or computed in advance and/or computed by DMA operations of a (1003).\",\n \"In particular, an address computation within 1201 (IADR-CONF) may be sent to a collector or its DMA to address and control the data transfers of the collector.\",\n \"Individual entries into the particular channels may have different identifiers, e.g., IOIDs and/or APIDs, enabling them to be assigned to a certain resource and/or memory location.\",\n \"FIG.\",\n \"13a shows a preferred implementation of a BURST-FIFO.\",\n \"The function of an output FIFO which transmits its values to a burst-capable bus (BBUS) is to be described first.\",\n \"A first pointer (1301) points to the data entry within a memory (1304) currently to be output to the BBUS.\",\n \"With each data word output (1302), 1301 is moved by one position.\",\n \"The value of pointer 1301 prior to the start of the current burst transfer has been stored in a register (1303).\",\n \"If an error occurs during the burst transfer, 1301 is reloaded with the original value from 1303 and the burst transfer is restarted.\",\n \"A second pointer (1305) points to the current data input position in the memory (1304) for data to be input (1306).\",\n \"To prevent overwriting of any data still needed in the event of an error, pointer 1305 is compared (1307) with register 1303 to indicate that the BURST-FIFO is full.\",\n \"The empty state of the BURST-FIFO may be ascertained by comparison (1308) of the output pointer (1301) with the input pointer (1305).\",\n \"If the BURST-FIFO operates for input data from a burst transfer, the functions change as follows: 1301 becomes the input pointer for data 1306.If faulty data has been transmitted during the burst transfer, the position prior to the burst transfer is stored in 1303.If an error occurs during the burst transfer, 1301 is reloaded with the original value from 1303 and the burst transfer is restarted.\",\n \"The pointer points to the readout position of the BURST-FIFO for reading out the data (1302).\",\n \"To prevent premature readout of data of a burst transfer that has not been concluded correctly, 1305 is compared with the position stored in 1303 (1307) to indicate an empty BURST-FIFO.\",\n \"A full BURST-FIFO is recognized by comparison (1308) of input pointer 1301 with the output pointer (1305).\",\n \"FIG.\",\n \"13b shows one possible implementation of a burst circuit which recognizes possible burst transfers and tests boundary limits.\",\n \"The implementation has been kept simple and recognizes only linear address sequences.\",\n \"Data transfers are basically started as burst transfers.\",\n \"The burst transfer is aborted at the first nonlinear address.\",\n \"Burst transfers of a certain length (e.g., 4) may also be detected and initialized by expanding a look-ahead logic, which checks multiple addresses in advance.\",\n \"The address value (1313) of a first access is stored in a register (1310).\",\n \"The address value of a subsequent data transfer is compared (1312) with the address value (1311) of 1310, which has been incremented by the address difference between the first data transfer and the second data transfer of the burst transfer (typically one word wide).\",\n \"If the two values are the same, then the difference between the first address and the second address corresponds to the address difference of the burst transfer between two burst addresses.\",\n \"Thus, this is a correct burst.\",\n \"If the values are not the same, the burst transfer must be aborted.\",\n \"The last address (1313) checked (the second address in the writing) is stored in 1310 and then compared with the next address (1313) accordingly.\",\n \"To ascertain whether the burst limits (boundaries) have been maintained, the address bit(s) at which the boundary of the current address value (1313) is located is (are) compared with the address bits of the preceding address value (1310) (e.g., XOR 1314).\",\n \"If the address bits are not the same, the boundary has been exceeded and the control of the burst must respond accordingly (e.g., termination of the burst transfer and restart).\",\n \"FIG.\",\n \"14 shows as an example various methods of connecting memories, in particular PAE-RAMs, to form a larger cohesive memory block.\",\n \"FIGS.\",\n \"14a-14d use the same reference numbers whenever possible.\",\n \"Write data (1401) is preferably sent to the memories via pipeline stages (1402).\",\n \"Read data (1403) is preferably removed from the memories also via pipeline stages (1404).\",\n \"Pipeline stage 1404 includes a multiplexer, which forwards the particular active data path.\",\n \"The active data path may be recognized, for example, by a RDY handshake applied.\",\n \"A unit (RangeCheck, 1405) for monitoring the addresses (1406) for correct values within the address space may optionally be provided.\",\n \"In FIG.\",\n \"14a, the addresses are sent to the memories (1408a) via pipeline stages (1407a).\",\n \"The memories compare the higher-value address part with a fixedly predetermined or configurable (e.g., by a higher-level configuration unit CT) reference address, which is unique for each memory.\",\n \"If they are identical, that memory is selected.\",\n \"The lower-value address part is used for selection of the memory location in the memory.\",\n \"In FIG.\",\n \"14b, the addresses are sent to the memories (1408b) via pipeline stages having an integrated decrementer (subtraction by 1) (1407b).\",\n \"The memories compare the higher-value address part with the value zero.\",\n \"If they are identical, that memory is selected.\",\n \"The lower-value address part is used for selection of the memory location in the memory.\",\n \"In FIG.\",\n \"14c, the addresses are sent to the memories (1408c) via pipeline stages (1407c).\",\n \"The memories compare the higher-level address part with a reference address, which is unique for each memory.\",\n \"The reference address is generated by an adding or subtracting chain (1409), which preselects another unique reference address for each memory on the basis of a starting value (typically 0).\",\n \"If they are identical, that memory is selected.\",\n \"The lower-value address part is used for selection of the memory location in the memory.\",\n \"In FIG.\",\n \"14d, the addresses are sent to the memories (1408d) via pipeline stages (1407d).\",\n \"The memories compare the higher-value address part with a reference address which is unique for each memory.\",\n \"The reference address is generated by an addressing or subtracting chain (1410), which is integrated into the memories and preselects another unique reference address for each memory on the basis of a starting value (typically 0).\",\n \"If they are identical, that memory is selected.\",\n \"The lower-value address part is used for selection of the memory location in the memory.\",\n \"For example, FREGs of the PAEs according to FIG.\",\n \"5 may be used for 1402, 1404, and 1407.Depending on the direction of travel of the reference address, FREG or BREG may be used for 1409.The design shown here as an example has the advantage in particular that all the read/write accesses have the same latency because the addresses and data are sent to the BREG/FREG via register stages.\",\n \"FIG.\",\n \"15 shows the use of GlobalTrack bus systems (1501, 1502, 1503, 1504) for coupling configurations which were configured in any way as configuration macros (1506, 1507) within a system of PAEs (1505) (see also DE 198 07 872.2, DE 199 26 538.0, DE 100 28 397.7).\",\n \"The configuration macros have (1508) their own internal bus connections, e.g., via internal buses (0104).\",\n \"The configuration macros are interconnected via 1503 for data exchange.\",\n \"1506 is connected to interface modules and/or local memories (RAM-PAEs) (1509, 1510) via 1501, 1502.1507 is connected to interface modules and/or local memories (RAM-PAEs) (1511) via 1504.Any other embodiments and combinations of the present inventions described here are possible and are self-evident in view of the foregoing, to those skilled in the art.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471061"}}},{"rowIdx":570,"cells":{"text":{"kind":"list like","value":[["Integration of e-mail with instant messaging services","A system for instant communication in real time is described wherein it is determined if a user with a given e-mail address is available for on-line messaging.","The system takes in a group of e-mail addresses and automatically looks for screen names that are available for instant messaging.","Once a screen name is available for online communication, an ion appears nest to the screen name.","Instant messaging is achieved by pressing the icon.","The screen names, the icons and the e-mail addresses all appear in the same window, thereby, integrating traditional e-mail addresses with instant messaging services making real time communication an effective tool."],["1.A computer implementation method for instant communication, comprising the steps of: receiving in a group of e-mail addresses; making a request for a list of screen names from an instant messaging service associated with said e-mail addresses; automatically verifying screen names that are available for real-time communication; correlating said screen names to said e-mail addresses; and establishing said real-time communication by selecting an icon associated with said screen name.","2.The method of claim 1, further comprising the step of manually verifying screen names associated with said e-mail addresses.","3.The method of claim 2, further comprising the steps of manually verifying screen names associated with said e-mail addresses that are available for real-time communication.","4.The method of claim 3, further comprising the step of making a list of said screen names associated with a particular e-mail address.","5.A computer implemented method for updating a recipient of available screen names for instant communication, comprising the steps of: receiving in a group of e-mail addresses; making a request for a list of screen names from an instant messaging service associated with said e-mail addresses; automatically verifying screen names that are available for real-time communication; correlating said screen names to said e-mail addresses; making a list of e-mail addresses with their associated screen names; updating said list periodically for establishing said real-time communication; and establishing said communication instantly by pressing an icon associated with a user's screen name.","6.The method of claim 5, further comprising the step of manually verifying screen names associated with said e-mail addresses for communication at a later time.","7.A method to start instant communication, comprising the steps of: receiving in a group of e-mail addresses; manually verifying screen names associated with said e-mail addresses that are available for real-time communication; sending an invitation for instant communication; obtaining screen names that are available for real-time communication; correlating said screen names to said e-mail addresses; and establishing said real-time communication by selecting an icon with said screen name.","8.The method of claim 7, further comprising the step of sending an invitation for instant communication via electronic mail.","9.An apparatus for enabling instant communication between users of a computer, comprising: a means for displaying a graphic window; a means for displaying e-mail addresses between said users of said computer for the purpose of online communication; a means for displaying screen names associated with said e-mail addresses for the purpose of said instant communication; and said graphic window including said e-mail addresses and said screen names in the same window for the purpose of enabling said instant communication between said users of said computer instantly.","10.An apparatus for enabling instant communication as in claim 9, further comprising a graphic icon situated next to said screen name of said graphic window for the purpose of indicating an availability of said screen name for instant messaging.","11.An apparatus for enabling instant communication as in claim 10, wherein said instant communication between users of said computer is enabled by activating said icon."],["<SOH> TECHNICAL FIELD <EOH>The invention relates generally to software solving communication problems.","More particularly, the invention relates to an integration of traditional e-mail with instant messaging systems in real time."],["<SOH> SUMMARY OF THE INVENTION <EOH>This invention provides a mechanism for the automatic integration of traditional e-mail with instant messaging services such that instant communication is provided in real-time.","In one embodiment of the invention, the system has a single generic graphics window with both e-mail addresses and on-line chat addresses next to it so that anyone with access to the e-mail address could start the AOL's free instant messaging service instantly.","The active screen names that are available for instant messaging would have an icon next to the screen name, such that instant messaging is achieved by pressing the icon as it appears next to the screen name.","In the particular embodiment of the invention, the system takes in a group of e-mail addresses and makes a request for a list of screen names from the instant messaging service associated with that e-mail address.","The system then automatically verifies each screen name that is available for online communication.","Once a particular screen name is available for instant communication, the system correlates that screen name to that particular e-mail address.","An icon appears next to the screen name that is available for online instant messaging.","Instant messaging is accomplished by pressing the icon as it appears next to the active screen name.","In one embodiment, the system sets up a list of screen names for tracking their online presence into a buddy list.","In yet another embodiment, the system is associated with a user interface (UI) that prevents others from knowing the person's screen name.","In yet another embodiment, the system allows a manual search when the screen name is not currently used but still associated with the same e-mail addresses.","Next, the system enquires of the user associated with that particular screen name for online communication.","The recipient can choose to communicate with the user by clicking the icon associated with the screen name that is currently available for instant communication.","Thus, the system establishes instant messaging in real time."],["TECHNICAL FIELD The invention relates generally to software solving communication problems.","More particularly, the invention relates to an integration of traditional e-mail with instant messaging systems in real time.","DESCRIPTION OF THE PRIOR ART With the advent of computers, communication has never been easier.","When friends and family are online, communication is much easier if invoked in real-time as it happens in instant messaging.","Instant messaging using a computer invokes a real-time communication protocol.","In instant messaging using a real time communication protocol, real-time communication is possible if the screen name of users is known before one begins instant messaging.","The list of screen names of users is not completely useful for communication in real-time if the user with that particular screen name is not online.","This means that for real-time communication to be possible, not only should the screen name of users be known but also the users should be logged on before one begins instant messaging.","The end user usually has an address, referred to as ‘e-mail address’, which is specific to the user for communicating electronically on a computer or similar other devices.","In electronic mail, messages can be sent, received, or stored at convenient times.","In conventional e-mail systems, messages are delivered in “envelopes” which are then “opened” by the recipient to be read.","Conventional e-mail systems, operating across local area network boundaries, do not allow the senders of messages to determine whether their messages have been read.","As a result, the sender of a message often must telephone the recipient to verify that the needed information has not only arrived at the recipient's system, but has been reviewed by the recipient.","Many proprietary e-mail systems, however, allow a sender to check a separate database to see if a message has been received.","Some proprietary e-mail systems include an acknowledgement feature that allows the recipient's e-mail system to send a notification to the sender when the recipient has opened a message.","In prior art e-mail systems, the sender of an e-mail message has to wait a while for a response.","In addition, the sender of the message is not sure if they would get an e-mail response.","When users are logged into an e-mail, instant messaging in terms of AOL Instant Messenger is possible.","America Online, Inc. of AOL Time Warner Inc. (AOL) pioneered the technology of instant messaging using a real time communication protocol.","In AOL Instant Messenger service, for example, the user could use any number of screen names while being logged into an e-mail address and communicate with other users instantly.","However, for instant communication to be possible, one has to look up manually the screen name that is actually online currently for the associated e-mail address of the other users.","The problem with that is because there could be more than one screen name associated with a single e-mail address, it becomes a difficult task to accomplish instant online messaging service manually with many active screen names.","In prior art systems, such as AOL's Mail Contacts Online, one has to be a member to make use of Instant Messaging service.","Usually, such memberships entail a fee paid by the customer for using such services.","In Mail Contacts Online service, the graphics window shows (FIG.","1) a divided window with e-mail communication window separate from the instant messaging window.","The instant messaging window shows a buddy list with a list of screen names taken from the adjacent e-mail window and only those screen names that are available for instant messaging.","The difficulty with prior art systems is that they are not universally available unless the user requests membership to use the e-mail and/or Instant Messaging and pays a membership fee.","With the availability of e-mail access to non-AOL members, the Instant Messaging service of prior art systems is limited in terms of number of screen names that are available in the buddy list.","“Instant Messaging” is an AOL TM, so it is used herein to refer to “AOL Instant Messenger” for AOL members.","Given the number of Instant Messaging type services currently available, there is a need for integrating Instant Messaging services to e-mail services such that a user with access to any e-mail service, not limited to AOL member services, can use AOL's free Instant Messaging service and, thereby, achieve communication in real-time.","What is needed, therefore, is a generic service where anyone with access to an e-mail and instant messaging service can chat with anyone else instantly.","The generic services should allow a graphic window with e-mail and instant messaging services to go simultaneously and smoothly so that the instant messaging is realized instantly in real time.","SUMMARY OF THE INVENTION This invention provides a mechanism for the automatic integration of traditional e-mail with instant messaging services such that instant communication is provided in real-time.","In one embodiment of the invention, the system has a single generic graphics window with both e-mail addresses and on-line chat addresses next to it so that anyone with access to the e-mail address could start the AOL's free instant messaging service instantly.","The active screen names that are available for instant messaging would have an icon next to the screen name, such that instant messaging is achieved by pressing the icon as it appears next to the screen name.","In the particular embodiment of the invention, the system takes in a group of e-mail addresses and makes a request for a list of screen names from the instant messaging service associated with that e-mail address.","The system then automatically verifies each screen name that is available for online communication.","Once a particular screen name is available for instant communication, the system correlates that screen name to that particular e-mail address.","An icon appears next to the screen name that is available for online instant messaging.","Instant messaging is accomplished by pressing the icon as it appears next to the active screen name.","In one embodiment, the system sets up a list of screen names for tracking their online presence into a buddy list.","In yet another embodiment, the system is associated with a user interface (UI) that prevents others from knowing the person's screen name.","In yet another embodiment, the system allows a manual search when the screen name is not currently used but still associated with the same e-mail addresses.","Next, the system enquires of the user associated with that particular screen name for online communication.","The recipient can choose to communicate with the user by clicking the icon associated with the screen name that is currently available for instant communication.","Thus, the system establishes instant messaging in real time.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 is a graphic representation of a prior art approach to indicating separate windows for e-mail addresses and instant messaging respectively; FIG.","1A is a representative block diagram of the elements of a preferred embodiment of the invention for integrating traditional e-mail with instant messaging services for the purpose of providing communication in real-time; FIG.","2 is a flow chart showing a mechanism for enabling an automatic real-time communication via e-mail according to this invention; FIG.","3 is a flow chart of another embodiment of the invention showing a mechanism for enabling availability of a manual option for real time communication; FIG.","4 is a flow chart showing a mechanism for inviting the user for instant communication to establish real-time communication according to the invention; and FIG.","5 is a flow chart showing a mechanism for enabling a list of screen names, ex.","a buddy list for instant messaging any time according to an alternative embodiment of the invention.","DETAILED DESCRIPTION OF THE INVENTION In commercially available electronic mail systems today, senders are able to send messages over a variety of networks such as local area networks, wide area networks, Internet, wireless networks, and the like.","This does not, however, mean senders of the message would get an e-mail response instantly.","When users are logged into an electronic mail or e-mail, instant messaging is possible if the recipient's screen name and e-mail address are known.","Because users could use any number of screen names while being logged into an e-mail, this communication is, however, not instant communication in real-time.","More often than not, it takes manually for a user to retrieve the screen name that is associated with that e-mail address and that is currently being used for on-line communication.","This invention (FIG.","1A) provides a system for automatic integration 1000 of traditional e-mail with instant messaging service, more particularly, AOL's free Instant Messaging service for the purpose of providing communication in real-time.","The invention comprises a system that takes in a group of e-mail addresses and makes a request for a list of screen names associated with that e-mail address from the instant messaging service.","The system is associated with a user interface (UI) that protects the user's screen name if the user is not interested in on-line communication (represented in FIG.","1A as OFF) and prefers to not make his screen name available to recipients of his messages.","When the user screen name is not available to the system, the system makes a manual search 200 for the screen names that are associated with that e-mail address.","The problem with a manual search 200 using only knowledge of available e-mail addresses is that when a user manually does a search for screen names, there is no contextual information to indicate if the person is actually online or which screen name is being used.","Because this becomes tedious, an invitation 300 is sent to the user for participation in on-line communication and a request for screen name applicable and used with that e-mail address.","Often the user is interested in on-line communication and would not mind if the screen names were available for on-line messaging.","This is shown symbolically as an ‘ON’ state on user interface (UI) in FIG.","1A and the invention allows for automatic search 100.The system then automatically 100 verifies each screen name that is available for online communication.","Once a particular screen name is available, the system then correlates that screen name to that particular e-mail address.","In one embodiment, the system periodically tracks a list of screen names for tracking their online presence and puts them into a buddy list 400.An online real-time communication is established once the icon 500 is activated next to the screen name for instant messaging.","The recipient can choose to communicate to the user by clicking on the icon associated with the screen name where the user is currently available for instant communication.","Thus, the invention establishes instant messaging in real time.","FIG.","2 is a flow chart showing a mechanism for enabling instant communication using an automatic 100 (FIG.","1A) real-time communication via e-mail.","When the user interface is enabled 10 (or is ON as in FIG.","1A), the method involves taking in a group of e-mail addresses 20 and making a request for a list of screen names from an instant messaging service associated with that e-mail addresses 30.An automatic verification of screen names available for real-time communication 50 is performed for each individual screen name.","If there is a screen name available for instant messaging, the system correlates the screen names available for that particular e-mail address 60 and establishes real-time communication 70 by pressing an icon 500 (FIG.","1A) with that particular screen name.","If there is no screen name available for instant messaging, the system cannot correlate the screen name with that particular e-mail address and simply continues the search for the next available screen name 40 for real-time messaging.","When real-time communication is established using the described automatic search 50, the system is said to have accomplished it's task and comes to a stop 80.FIG.","3 is a flow chart showing a mechanism for enabling real-time communication using a manual 200 (FIG.","1A) search for the screen name.","When the user interface is not enabled 110 (or is OFF as in FIG.","1A), the method involves taking in a group of e-mail addresses 120 and making a list of screen names by manually looking for screen names 130 associated with that e-mail address and establishing instant messaging 140 if screen name is available.","When real-time communication is established using the described manual search 130, the system then associates an icon (refer to 500 in FIG.","1A) 150 with the screen name that is available for instant messaging.","The system is said to have accomplished its task and comes to a stop 150.FIG.","4 is a flow chart showing another embodiment of the invention, which comprises, inviting the user for instant communication and requesting the user for screen name for real-time communication when the user interface (UI) is not enabled 210 (OFF in FIG.","1A).","This embodiment is useful in prompting the user for real-time communication when the user does not wish to disclose his screen name to all individuals on the internet.","The system takes in select group of e-mail addresses 220, manually tests for screen names available for instant messaging 230.If screen name is available 240 for instant messaging, the icon 500 (FIG.","1A) is pressed and instant messaging is established.","If the screen name is not available for instant messaging, then the system invites the user for instant communication 250 by sending an electronic mail or e-mail.","If the invitation is accepted 270, then the system obtains the user's screen name 280, and establishes a real-time communication 290 when the icon 500 (FIG.","1A) is pressed.","The system function is completed as real-time communication is established and comes to a stop 300 or if the invitation is not accepted 260.FIG.","5 is a flow chart showing another embodiment of the invention, which comprises enabling a list of screen names into a list, referred to as buddy list for instant messaging any time.","In this embodiment, the system sets up a list of screen names for tracking their online presence into a buddy list.","The system does an automatic look up of screen names 330 when the user interface is enabled (ON in FIG.","1A) given a list of e-mail addresses 320.It then verifies if the available screen name is associated with that particular e-mail address and available for real-time communication.","If it is not available 350, it looks back at it's list of screen names and goes to the next one until one is found that is available for instant messaging.","If the system does find the screen name for that e-mail address available for instant messaging, it makes a buddy list of e-mail address with their associated screen names 360.The buddy list is periodically tested for online presence 370 and updated for establishing real-time communication 390 or returned 380 for the next available screen name for that e-mail address.","When real-time communication is established, the icon 500 is pressed for communication with the individual whose screen name is displayed and the process ends 395 with satisfactory completion.","Although the invention is described herein with reference to the preferred embodiment, one skilled in the art will readily appreciate that other applications may be substituted for those set forth herein without departing from the spirit and scope of the present invention.","Accordingly, the invention should only be limited by the Claims included below."]],"string":"[\n [\n \"Integration of e-mail with instant messaging services\",\n \"A system for instant communication in real time is described wherein it is determined if a user with a given e-mail address is available for on-line messaging.\",\n \"The system takes in a group of e-mail addresses and automatically looks for screen names that are available for instant messaging.\",\n \"Once a screen name is available for online communication, an ion appears nest to the screen name.\",\n \"Instant messaging is achieved by pressing the icon.\",\n \"The screen names, the icons and the e-mail addresses all appear in the same window, thereby, integrating traditional e-mail addresses with instant messaging services making real time communication an effective tool.\"\n ],\n [\n \"1.A computer implementation method for instant communication, comprising the steps of: receiving in a group of e-mail addresses; making a request for a list of screen names from an instant messaging service associated with said e-mail addresses; automatically verifying screen names that are available for real-time communication; correlating said screen names to said e-mail addresses; and establishing said real-time communication by selecting an icon associated with said screen name.\",\n \"2.The method of claim 1, further comprising the step of manually verifying screen names associated with said e-mail addresses.\",\n \"3.The method of claim 2, further comprising the steps of manually verifying screen names associated with said e-mail addresses that are available for real-time communication.\",\n \"4.The method of claim 3, further comprising the step of making a list of said screen names associated with a particular e-mail address.\",\n \"5.A computer implemented method for updating a recipient of available screen names for instant communication, comprising the steps of: receiving in a group of e-mail addresses; making a request for a list of screen names from an instant messaging service associated with said e-mail addresses; automatically verifying screen names that are available for real-time communication; correlating said screen names to said e-mail addresses; making a list of e-mail addresses with their associated screen names; updating said list periodically for establishing said real-time communication; and establishing said communication instantly by pressing an icon associated with a user's screen name.\",\n \"6.The method of claim 5, further comprising the step of manually verifying screen names associated with said e-mail addresses for communication at a later time.\",\n \"7.A method to start instant communication, comprising the steps of: receiving in a group of e-mail addresses; manually verifying screen names associated with said e-mail addresses that are available for real-time communication; sending an invitation for instant communication; obtaining screen names that are available for real-time communication; correlating said screen names to said e-mail addresses; and establishing said real-time communication by selecting an icon with said screen name.\",\n \"8.The method of claim 7, further comprising the step of sending an invitation for instant communication via electronic mail.\",\n \"9.An apparatus for enabling instant communication between users of a computer, comprising: a means for displaying a graphic window; a means for displaying e-mail addresses between said users of said computer for the purpose of online communication; a means for displaying screen names associated with said e-mail addresses for the purpose of said instant communication; and said graphic window including said e-mail addresses and said screen names in the same window for the purpose of enabling said instant communication between said users of said computer instantly.\",\n \"10.An apparatus for enabling instant communication as in claim 9, further comprising a graphic icon situated next to said screen name of said graphic window for the purpose of indicating an availability of said screen name for instant messaging.\",\n \"11.An apparatus for enabling instant communication as in claim 10, wherein said instant communication between users of said computer is enabled by activating said icon.\"\n ],\n [\n \"<SOH> TECHNICAL FIELD <EOH>The invention relates generally to software solving communication problems.\",\n \"More particularly, the invention relates to an integration of traditional e-mail with instant messaging systems in real time.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>This invention provides a mechanism for the automatic integration of traditional e-mail with instant messaging services such that instant communication is provided in real-time.\",\n \"In one embodiment of the invention, the system has a single generic graphics window with both e-mail addresses and on-line chat addresses next to it so that anyone with access to the e-mail address could start the AOL's free instant messaging service instantly.\",\n \"The active screen names that are available for instant messaging would have an icon next to the screen name, such that instant messaging is achieved by pressing the icon as it appears next to the screen name.\",\n \"In the particular embodiment of the invention, the system takes in a group of e-mail addresses and makes a request for a list of screen names from the instant messaging service associated with that e-mail address.\",\n \"The system then automatically verifies each screen name that is available for online communication.\",\n \"Once a particular screen name is available for instant communication, the system correlates that screen name to that particular e-mail address.\",\n \"An icon appears next to the screen name that is available for online instant messaging.\",\n \"Instant messaging is accomplished by pressing the icon as it appears next to the active screen name.\",\n \"In one embodiment, the system sets up a list of screen names for tracking their online presence into a buddy list.\",\n \"In yet another embodiment, the system is associated with a user interface (UI) that prevents others from knowing the person's screen name.\",\n \"In yet another embodiment, the system allows a manual search when the screen name is not currently used but still associated with the same e-mail addresses.\",\n \"Next, the system enquires of the user associated with that particular screen name for online communication.\",\n \"The recipient can choose to communicate with the user by clicking the icon associated with the screen name that is currently available for instant communication.\",\n \"Thus, the system establishes instant messaging in real time.\"\n ],\n [\n \"TECHNICAL FIELD The invention relates generally to software solving communication problems.\",\n \"More particularly, the invention relates to an integration of traditional e-mail with instant messaging systems in real time.\",\n \"DESCRIPTION OF THE PRIOR ART With the advent of computers, communication has never been easier.\",\n \"When friends and family are online, communication is much easier if invoked in real-time as it happens in instant messaging.\",\n \"Instant messaging using a computer invokes a real-time communication protocol.\",\n \"In instant messaging using a real time communication protocol, real-time communication is possible if the screen name of users is known before one begins instant messaging.\",\n \"The list of screen names of users is not completely useful for communication in real-time if the user with that particular screen name is not online.\",\n \"This means that for real-time communication to be possible, not only should the screen name of users be known but also the users should be logged on before one begins instant messaging.\",\n \"The end user usually has an address, referred to as ‘e-mail address’, which is specific to the user for communicating electronically on a computer or similar other devices.\",\n \"In electronic mail, messages can be sent, received, or stored at convenient times.\",\n \"In conventional e-mail systems, messages are delivered in “envelopes” which are then “opened” by the recipient to be read.\",\n \"Conventional e-mail systems, operating across local area network boundaries, do not allow the senders of messages to determine whether their messages have been read.\",\n \"As a result, the sender of a message often must telephone the recipient to verify that the needed information has not only arrived at the recipient's system, but has been reviewed by the recipient.\",\n \"Many proprietary e-mail systems, however, allow a sender to check a separate database to see if a message has been received.\",\n \"Some proprietary e-mail systems include an acknowledgement feature that allows the recipient's e-mail system to send a notification to the sender when the recipient has opened a message.\",\n \"In prior art e-mail systems, the sender of an e-mail message has to wait a while for a response.\",\n \"In addition, the sender of the message is not sure if they would get an e-mail response.\",\n \"When users are logged into an e-mail, instant messaging in terms of AOL Instant Messenger is possible.\",\n \"America Online, Inc. of AOL Time Warner Inc. (AOL) pioneered the technology of instant messaging using a real time communication protocol.\",\n \"In AOL Instant Messenger service, for example, the user could use any number of screen names while being logged into an e-mail address and communicate with other users instantly.\",\n \"However, for instant communication to be possible, one has to look up manually the screen name that is actually online currently for the associated e-mail address of the other users.\",\n \"The problem with that is because there could be more than one screen name associated with a single e-mail address, it becomes a difficult task to accomplish instant online messaging service manually with many active screen names.\",\n \"In prior art systems, such as AOL's Mail Contacts Online, one has to be a member to make use of Instant Messaging service.\",\n \"Usually, such memberships entail a fee paid by the customer for using such services.\",\n \"In Mail Contacts Online service, the graphics window shows (FIG.\",\n \"1) a divided window with e-mail communication window separate from the instant messaging window.\",\n \"The instant messaging window shows a buddy list with a list of screen names taken from the adjacent e-mail window and only those screen names that are available for instant messaging.\",\n \"The difficulty with prior art systems is that they are not universally available unless the user requests membership to use the e-mail and/or Instant Messaging and pays a membership fee.\",\n \"With the availability of e-mail access to non-AOL members, the Instant Messaging service of prior art systems is limited in terms of number of screen names that are available in the buddy list.\",\n \"“Instant Messaging” is an AOL TM, so it is used herein to refer to “AOL Instant Messenger” for AOL members.\",\n \"Given the number of Instant Messaging type services currently available, there is a need for integrating Instant Messaging services to e-mail services such that a user with access to any e-mail service, not limited to AOL member services, can use AOL's free Instant Messaging service and, thereby, achieve communication in real-time.\",\n \"What is needed, therefore, is a generic service where anyone with access to an e-mail and instant messaging service can chat with anyone else instantly.\",\n \"The generic services should allow a graphic window with e-mail and instant messaging services to go simultaneously and smoothly so that the instant messaging is realized instantly in real time.\",\n \"SUMMARY OF THE INVENTION This invention provides a mechanism for the automatic integration of traditional e-mail with instant messaging services such that instant communication is provided in real-time.\",\n \"In one embodiment of the invention, the system has a single generic graphics window with both e-mail addresses and on-line chat addresses next to it so that anyone with access to the e-mail address could start the AOL's free instant messaging service instantly.\",\n \"The active screen names that are available for instant messaging would have an icon next to the screen name, such that instant messaging is achieved by pressing the icon as it appears next to the screen name.\",\n \"In the particular embodiment of the invention, the system takes in a group of e-mail addresses and makes a request for a list of screen names from the instant messaging service associated with that e-mail address.\",\n \"The system then automatically verifies each screen name that is available for online communication.\",\n \"Once a particular screen name is available for instant communication, the system correlates that screen name to that particular e-mail address.\",\n \"An icon appears next to the screen name that is available for online instant messaging.\",\n \"Instant messaging is accomplished by pressing the icon as it appears next to the active screen name.\",\n \"In one embodiment, the system sets up a list of screen names for tracking their online presence into a buddy list.\",\n \"In yet another embodiment, the system is associated with a user interface (UI) that prevents others from knowing the person's screen name.\",\n \"In yet another embodiment, the system allows a manual search when the screen name is not currently used but still associated with the same e-mail addresses.\",\n \"Next, the system enquires of the user associated with that particular screen name for online communication.\",\n \"The recipient can choose to communicate with the user by clicking the icon associated with the screen name that is currently available for instant communication.\",\n \"Thus, the system establishes instant messaging in real time.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is a graphic representation of a prior art approach to indicating separate windows for e-mail addresses and instant messaging respectively; FIG.\",\n \"1A is a representative block diagram of the elements of a preferred embodiment of the invention for integrating traditional e-mail with instant messaging services for the purpose of providing communication in real-time; FIG.\",\n \"2 is a flow chart showing a mechanism for enabling an automatic real-time communication via e-mail according to this invention; FIG.\",\n \"3 is a flow chart of another embodiment of the invention showing a mechanism for enabling availability of a manual option for real time communication; FIG.\",\n \"4 is a flow chart showing a mechanism for inviting the user for instant communication to establish real-time communication according to the invention; and FIG.\",\n \"5 is a flow chart showing a mechanism for enabling a list of screen names, ex.\",\n \"a buddy list for instant messaging any time according to an alternative embodiment of the invention.\",\n \"DETAILED DESCRIPTION OF THE INVENTION In commercially available electronic mail systems today, senders are able to send messages over a variety of networks such as local area networks, wide area networks, Internet, wireless networks, and the like.\",\n \"This does not, however, mean senders of the message would get an e-mail response instantly.\",\n \"When users are logged into an electronic mail or e-mail, instant messaging is possible if the recipient's screen name and e-mail address are known.\",\n \"Because users could use any number of screen names while being logged into an e-mail, this communication is, however, not instant communication in real-time.\",\n \"More often than not, it takes manually for a user to retrieve the screen name that is associated with that e-mail address and that is currently being used for on-line communication.\",\n \"This invention (FIG.\",\n \"1A) provides a system for automatic integration 1000 of traditional e-mail with instant messaging service, more particularly, AOL's free Instant Messaging service for the purpose of providing communication in real-time.\",\n \"The invention comprises a system that takes in a group of e-mail addresses and makes a request for a list of screen names associated with that e-mail address from the instant messaging service.\",\n \"The system is associated with a user interface (UI) that protects the user's screen name if the user is not interested in on-line communication (represented in FIG.\",\n \"1A as OFF) and prefers to not make his screen name available to recipients of his messages.\",\n \"When the user screen name is not available to the system, the system makes a manual search 200 for the screen names that are associated with that e-mail address.\",\n \"The problem with a manual search 200 using only knowledge of available e-mail addresses is that when a user manually does a search for screen names, there is no contextual information to indicate if the person is actually online or which screen name is being used.\",\n \"Because this becomes tedious, an invitation 300 is sent to the user for participation in on-line communication and a request for screen name applicable and used with that e-mail address.\",\n \"Often the user is interested in on-line communication and would not mind if the screen names were available for on-line messaging.\",\n \"This is shown symbolically as an ‘ON’ state on user interface (UI) in FIG.\",\n \"1A and the invention allows for automatic search 100.The system then automatically 100 verifies each screen name that is available for online communication.\",\n \"Once a particular screen name is available, the system then correlates that screen name to that particular e-mail address.\",\n \"In one embodiment, the system periodically tracks a list of screen names for tracking their online presence and puts them into a buddy list 400.An online real-time communication is established once the icon 500 is activated next to the screen name for instant messaging.\",\n \"The recipient can choose to communicate to the user by clicking on the icon associated with the screen name where the user is currently available for instant communication.\",\n \"Thus, the invention establishes instant messaging in real time.\",\n \"FIG.\",\n \"2 is a flow chart showing a mechanism for enabling instant communication using an automatic 100 (FIG.\",\n \"1A) real-time communication via e-mail.\",\n \"When the user interface is enabled 10 (or is ON as in FIG.\",\n \"1A), the method involves taking in a group of e-mail addresses 20 and making a request for a list of screen names from an instant messaging service associated with that e-mail addresses 30.An automatic verification of screen names available for real-time communication 50 is performed for each individual screen name.\",\n \"If there is a screen name available for instant messaging, the system correlates the screen names available for that particular e-mail address 60 and establishes real-time communication 70 by pressing an icon 500 (FIG.\",\n \"1A) with that particular screen name.\",\n \"If there is no screen name available for instant messaging, the system cannot correlate the screen name with that particular e-mail address and simply continues the search for the next available screen name 40 for real-time messaging.\",\n \"When real-time communication is established using the described automatic search 50, the system is said to have accomplished it's task and comes to a stop 80.FIG.\",\n \"3 is a flow chart showing a mechanism for enabling real-time communication using a manual 200 (FIG.\",\n \"1A) search for the screen name.\",\n \"When the user interface is not enabled 110 (or is OFF as in FIG.\",\n \"1A), the method involves taking in a group of e-mail addresses 120 and making a list of screen names by manually looking for screen names 130 associated with that e-mail address and establishing instant messaging 140 if screen name is available.\",\n \"When real-time communication is established using the described manual search 130, the system then associates an icon (refer to 500 in FIG.\",\n \"1A) 150 with the screen name that is available for instant messaging.\",\n \"The system is said to have accomplished its task and comes to a stop 150.FIG.\",\n \"4 is a flow chart showing another embodiment of the invention, which comprises, inviting the user for instant communication and requesting the user for screen name for real-time communication when the user interface (UI) is not enabled 210 (OFF in FIG.\",\n \"1A).\",\n \"This embodiment is useful in prompting the user for real-time communication when the user does not wish to disclose his screen name to all individuals on the internet.\",\n \"The system takes in select group of e-mail addresses 220, manually tests for screen names available for instant messaging 230.If screen name is available 240 for instant messaging, the icon 500 (FIG.\",\n \"1A) is pressed and instant messaging is established.\",\n \"If the screen name is not available for instant messaging, then the system invites the user for instant communication 250 by sending an electronic mail or e-mail.\",\n \"If the invitation is accepted 270, then the system obtains the user's screen name 280, and establishes a real-time communication 290 when the icon 500 (FIG.\",\n \"1A) is pressed.\",\n \"The system function is completed as real-time communication is established and comes to a stop 300 or if the invitation is not accepted 260.FIG.\",\n \"5 is a flow chart showing another embodiment of the invention, which comprises enabling a list of screen names into a list, referred to as buddy list for instant messaging any time.\",\n \"In this embodiment, the system sets up a list of screen names for tracking their online presence into a buddy list.\",\n \"The system does an automatic look up of screen names 330 when the user interface is enabled (ON in FIG.\",\n \"1A) given a list of e-mail addresses 320.It then verifies if the available screen name is associated with that particular e-mail address and available for real-time communication.\",\n \"If it is not available 350, it looks back at it's list of screen names and goes to the next one until one is found that is available for instant messaging.\",\n \"If the system does find the screen name for that e-mail address available for instant messaging, it makes a buddy list of e-mail address with their associated screen names 360.The buddy list is periodically tested for online presence 370 and updated for establishing real-time communication 390 or returned 380 for the next available screen name for that e-mail address.\",\n \"When real-time communication is established, the icon 500 is pressed for communication with the individual whose screen name is displayed and the process ends 395 with satisfactory completion.\",\n \"Although the invention is described herein with reference to the preferred embodiment, one skilled in the art will readily appreciate that other applications may be substituted for those set forth herein without departing from the spirit and scope of the present invention.\",\n \"Accordingly, the invention should only be limited by the Claims included below.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471068"}}},{"rowIdx":571,"cells":{"text":{"kind":"list like","value":[["Large touch-sensitive area with time-controlled and location-controlled emitter and receiver modules","The invention enables the realization of an optical and electronic structure of a touch-sensitive area on any flat display surface such as a monitor.","The optical structure does not require any additional optical components such as lenses.","Laser diodes are likewise not required for the operation of a structure of this type.","Large distances and, nevertheless, high sensitivity and high resolution of the touch-sensitive area are accomplished by a novel time-control and location-control of both the emitter diodes as well as the receiver diodes or receiver photo-transistors.","If still or moving images are displayed on the surface, the invention can be used as a touch screen and can interactively control the display.","A central module simulates a computer mouse and a keyboard."],["1.A touch-sensitive region on a display device for detecting a user input, comprising: emitter diodes for outputting light; receiver diodes/phototransistors for detecting the light, the emitter diodes and receiver diodes/phototransistors being arranged in vertical and horizontal emitter and receiver units set opposite to each other, one of the emitter diodes and one of the receiver diodes/phototransistors being optically set in a direct paired relationship to each other; and a central module; wherein the detection of a user input is realized through continuously repeating time-sequenced and position-sequenced on-and-off switching of individual emitter diodes and receiver diodes/phototransistors; wherein one of the emitter diodes and one of the receiver diodes/phototransistors are controlled in pairs; and wherein for evaluating and increasing the resolution, the signals of the emitter diodes are referenced, adjacent to the emitter diodes, and all electrical signals of the receiver diodes/phototransistors are assembled into the central module, and also the emitter diodes are controlled from the Central module, such that the emitter diodes output a plurality of pulses, whose integrative, quantitative evaluation is a measure of the position of the interruption of a light barrier during an evaluation period and provides information on the position of the interruption relative to the emitter diodes.","2.A touch-sensitive region according to claim 1, characterized in that the emitter diodes are operated during their short on-time with higher power than their nominal continuous power.","3.A touch-sensitive region according to claim 1, characterized in that the wavelength of the applied electromagnetic radiation is in the infrared range of the spectrum and the emitter diodes and receiver diodes/phototransistors are emitting and receiving semiconductor elements.","4.A touch-sensitive region according to claim 1, characterized in that the emitter diodes are operated with a burst signal during their on time, which means that the electrical alternating signal controlling them has a higher frequency f than the alternating frequency of the emitter diodes controlled one after the other; wherein for faster saturation, measures, such as increasing the start-up voltage amplitude/voltage rise time and necessary discharge of charge carriers from the emitter diodes after the saturation pulse, are used.","5.A touch-sensitive region according to claim 1, characterized in that the amplifier circuit of the receiver diodes or phototransistors has a large amplification at the burst frequency f and otherwise enables a high attenuation through an inductive operating resistor followed by an integrator.","6.A touch-sensitive region according to claim 1, characterized in that not only the emitter diodes arranged optically in pairs with a receiver diode or phototransistor are active during the on time of the receiver diode or phototransistor, but adjacent emitter diodes are also active.","7.A touch-sensitive region according to claim 1, characterized in that an optical aperture is arranged in front of the emitter diodes and receiver diodes or phototransistors.","8.A touch-sensitive region according to claim 1, characterized in that the optical paired relationship of emitter diodes and receiver diodes or phototransistors involves oppositely positioned emitter diodes and receiver diodes or phototransistors in opposing emitter and receiver units.","9.A touch-sensitive region according to claim 1, characterized in that the optically paired relationship of the emitter diodes and receiver diodes or phototransistors involves emitter diodes and receiver diodes or phototransistors positioned one next to the other in a mixed emitter and receiver unit and an oppositely positioned mirror surface.","10.A touch-sensitive region according to claim 1, characterized in that the optical relationship of the emitter diodes and receiver diodes or phototransistors involves emitter diodes and receiver diodes or phototransistors positioned one next to the other in a mixed emitter and receiver unit and an oppositely positioned absorbing surface.","11.A touch-sensitive region according to claim 1, characterized in that the total number of emitter diodes in the emitter units is exactly equal to or not equal to the number of receiver diodes or phototransistors in the receiver units.","12.A touch-sensitive region according to claim 1, characterized in that a microprocessor of the central module calculates the position of the user input from digital receiver module signals by means of a statistical analysis method.","13.A touch-sensitive region according to claim 1, characterized in that the central module can simulate any arbitrary interface relative to an attached computer.","14.A touch-sensitive region according to claim 2, characterized in that the wavelength of the applied electromagnetic radiation is in the infrared range of the spectrum and the emitter diodes and receiver diodes/phototransistors are emitting and receiving semiconductor elements.","15.A touch-sensitive region according to claim 2, characterized in that the emitter diodes are operated with a burst signal during their on time, which means that the electrical alternating signal controlling them has a higher frequency f than the alternating frequency of the emitter diodes controlled one after the other; wherein for faster saturation, measures, such as increasing the start-up voltage amplitude/voltage rise time and necessary discharge of charge carriers from the emitter diodes after the saturation pulse, are used.","16.A touch-sensitive region according to claim 3, characterized in that the emitter diodes are operated with a burst signal during their on time, which means that the electrical alternating signal controlling them has a higher frequency f than the alternating frequency of the emitter diodes controlled one after the other; wherein for faster saturation, measures, such as increasing the start-up voltage amplitude/voltage rise time and necessary discharge of charge carriers from the emitter diodes after the saturation pulse, are used.","17.A touch-sensitive region according to claim 2, characterized in that the amplifier circuit of the receiver diodes or phototransistors has a large amplification at the burst frequency f and otherwise enables a high attenuation through an inductive operating resistor followed by an integrator.","18.A touch-sensitive region according to claim 3, characterized in that the amplifier circuit of the receiver diodes or phototransistors has a large amplification at the burst frequency f and otherwise enables a high attenuation through an inductive operating resistor followed by an integrator.","19.A touch-sensitive region according to claim 4, characterized in that the amplifier circuit of the receiver diodes or phototransistors has a large amplification at the burst frequency f and otherwise enables a high attenuation through an inductive operating resistor followed by an integrator.","20.A touch-sensitive region according to claim 2, characterized in that not only the emitter diodes arranged optically in pairs with a receiver diode or phototransistor are active during the on time of the receiver diode or phototransistor, but adjacent emitter diodes are also active."],["The invention relates to the optical and electronic structure of a very large (possibly over 0.04 m2, typically: >1 m2) touch-sensitive area (also called a touchscreen) on any transparent surface (monitor), which is used to build multi-active display windows, multi-active boxes, information terminals, or security systems, e.g., in the form of personnel filters.","This invention describes the application of a multi-active display window, whose entire area, up to 8 m×8 m, can be configured interactively for the first time.","Here, the resolution of the scan equals ±1.5 mm.","For the application as a multi-active display window (FIG.","1), a focusing screen, projection film, or another projection surface (2), which replaces part of or the entire decoration (8), is set up behind the display window panel (1).","On this surface, a large-area image is projected (rear projection) from behind by means of a known video and data light projector (7).","These images are typically generated by a control computer.","At the border of the display window panel, a vandalism-proof coating is applied from the outside.","This contains a special infrared (IR) emitter and receiver module arrays (3, 4, 5, 6), which cover the display window (the touch-sensitive area) with an invisible light-barrier curtain.","From the front, the user can point to a certain area of the image (user input).","This area is identified by the structure according to the invention described in this patent specification and forwarded to the control computer.","This computer can control the further flow of images and graphics corresponding to the user input.","Thus, an interactive, i.e., user-controlled, presentation is possible.","Here, this user input need not absolutely lie on the area given by the rear projection; instead, a separately designated area (e.g., a keyboard or trackball) can also be on the touch-sensitive area.","Furthermore, the described detection structure can also be applied to other optical systems and image generators, which generate images on surfaces (plasma screens, large monitors, LCD monitors, rear-projection boxes, etc.).","The presentation processed in the control computer can consist of, e.g., an Internet presentation.","The multi-active display window can then be functionally compared with a computer mouse and its navigation properties.","Thus, for the first time economical (without personnel and around the clock) information and advertisements can be provided for any supplier at locations in cities that are heavily frequented by pedestrians.","For the current state of the art, touch-sensitive surfaces up to approximately 40″ (touchscreens) are realized preferably in the type in which a film or a number of thin conductive threads on the image surface detects the user input (touch) because an electrical property of the film or the thread (e.g., resistance, capacitance, field strength) changes when touched, and this change is converted into the exact two-dimensional position of the user input by evaluating the separately detected horizontal and vertical electrical signals.","The disadvantages of these methods are the following: 1) The cost of the structure greatly increases with the size of the touch-sensitive area.","Surfaces over approximately 0.5 m2 thus cannot be realized economically.","2) The accuracy of the touch detection decreases with larger surfaces.","3) The described methods are unsuitable for use in public areas, because they are not vandalism-proof.","This means that they can be easily destroyed and can no longer fulfill their intended function due to outside actions of a willfully destructive nature.","4) Most solutions of the state of the art require additional layers over the projected image and thus attenuate the brightness of the image.","Other possibilities include the acoustic (ultrasound) detection of the interrupt, wherein fewer emitters/receivers are necessary than for two-dimensional networks.","However, this technology is reliable only up to touchscreen sizes of approximately 1.5×1.5 m2 and shows disadvantages for the reliable detection of user input in the edge regions.","There are also active detection systems.","Active means that the user uses a special, active transmitting pointing device in order to perform the user input (flip-chart).","This system is not suitable if anonymous persons are to benefit from the interactivity, e.g., of the multi-active display window.","In addition, for small touchscreens (e.g., in automatic bank machines), detectors based on infrared emitters and receivers are used.","Here, the touch-sensitive surface is covered with invisible IR radiation and the interruption of a light barrier is detected.","The emitters and receivers are turned on permanently.","Usually, additional lenses are used for increasing the light-beam focusing for such structures.","Therefore, only small surfaces (<0.1 m2) can be detected, because for greater distances from the emitter units to the receiver units, strong cross-coupling prevents the detection of the user input and also the light intensity of the diodes is no longer sufficient to achieve reliable detection.","It is known, e.g., for infrared light-emitting diodes (IR-LED) from remote controls, that LEDs can be operated in a pulsed mode up to pulse duty factors of 1:100.This means that the LED is turned off one-hundred times longer than the time during which it is turned on (and thus emits light).","Here, pulse sequences (bursts) with rectangular signals are forwarded to the emitter, which produce a more or less sinusoidal light stream based on their self-capacitance and the pn-junction properties.","The electrical current during the short on period can then equal 100-times the nominal continuous current without significantly affecting the life of the LED.","Thus, considerably higher peak powers of the light radiation can be achieved in a short time period than would be possible for continuous operation.","It is further known that IR-LEDs and IR receivers (IR-sensitive photodiodes, phototransistors, or photoresistors) can be used in light barriers.","Here, the interruption of the optical connection between emitter and receiver (when the emitter and receiver “are looking at” each other) or the formation of the optical connection (when the emitter and receiver see each other only if a third object reflects the emitted light) is detected and, e.g., it is determined whether a person has passed through a certain space or whether a person is located in front of a certain point in space.","An embodiment of the invention is explained in more detail with reference to the enclosed drawings.","Shown are: FIG.","1, a schematic structure of a multi-active display window, FIG.","2, a block diagram of a large-area, interactive projection, FIG.","3, the structure of the time-controlled and position-controlled light barriers.","The disadvantages mentioned above of previous touchscreen solutions for large surfaces do not occur for a configuration of the touchscreen according to the invention.","In connection with a rear projection by means of contemporary high-power LCD or DMD video and data projectors, large display windows can be transformed into multi-active projection surfaces (FIG.","1), which fulfill the following properties: 1) Economical provision of the display window with a touch-sensitive surface.","2) Vandalism-proof structure.","3) Reliable detection of a user input for a finger width greater than 10 mm (small children) with a position resolution of ±1.5 mm without any light loss of the projection.","4) Completely automatic day and night operation possible due to high-power, remote-control capable projectors.","According to the invention, these requirements are fulfilled by detecting the user input through electromagnetic (typically in the infrared range of the spectrum) emitters and receivers, which are arranged in the horizontal and vertical direction and which are controlled in a suitable way in a time-sequenced and position-dependent pattern.","These transmitters and receivers are each arranged separately in modules (transmitters and receiver modules FIG.","3, 300, 310), which can be arranged in series in principle in an arbitrary amount (FIG.","2, 100, 110, 120, 130).","These modules are attached on the outside around the touch-sensitive area (190) to be defined, wherein the modules can be mounted at a large distance to the borders of the area (up to 8 m from each other), which creates the ability of “hiding” these components in terms of construction.","Because the modules are installed in stable aluminum, steel, or diecast housings, this arrangement is vandalism-proof.","The emitter and receiver modules 300, 310 are arranged opposite each other and “look” out over nearly the entire touch-sensitive area 190 (the distance from the display window surface here equals approximately 0.5 to 3 cm).","Each module (300, 310) has its own microcontroller, which is arranged in the module itself.","The entire arrangement represents a multiprocessor system.","Also, the central module 140 has a microcontroller with greater capability relative to the module controllers (greater RAM capacity, higher clock frequency, larger flash-RAM).","The emitter modules 300 receive their control signals from the central module 140.The measurement signals of the receiver modules 310 are analyzed statistically and then transmitted to the central module 140.The central module 140 assembles the entire interactive surface and recognizes a relevant user input.","This input is output, e.g., in a special protocol (e.g., standard serial interface for a mouse) to the PC.","Other output types, such as x-coordinates, y-coordinates (for special consoles), or PS2-mouse emulation can be set by the attached PC.","The central module allows the implementation of arbitrary mouse protocols or serial transmission protocols based on the microcontroller circuit.","The data is transmitted to the control computer 220 controlling the presentation.","A preferred configuration of the invention is described in the following (FIG.","2, FIG.","3): The touchscreen consists of IR-emitter and IR-receiver modules (300, 310), which are arranged together into corresponding horizontal and vertical emitter and receiver module arrays (100, 110, 120, 130).","Here, the number of active emitter modules in the horizontal (or vertical) direction n (or m) must agree with the number of active receiver modules in the horizontal (vertical) direction.","Here, m can not equal n. Thus, any rectangular area 190 can be defined.","In the preferred example, n, m should not be greater than 16.For example, for a structural module length of 16 cm (16 emitters 300 or receivers 310 are assembled on a module and the distance of the emitter diodes or receiver transistors to each module is 10 mm), user inputs on a maximum surface area of 2.56×2.56 m2 (6.55 m2) can be detected in this embodiment.","In principle, larger areas with a configuration according to the invention are possible.","The emitter and receiver module arrays (100, 110, 120, 130) must not be mounted directly on the touch-sensitive area 190, but instead can feature a distance, which in principle is arbitrarily large, from the area (FIG.","2).","The corresponding two emitter and receiver module arrays (which in turn consist of n or m modules) are connected to a central module 140.In the preferred configuration of the invention, the central module 140 automatically notices how many modules make up the units in the x-direction (horizontal) and y-direction (vertical).","In this preferred configuration, next to the active region 190, which is predetermined by the size of the projected image, yet another touch-sensitive area 170 can be defined, which can be used as a keyboard and/or trackball for data input to the control computer (PC) 220.Therefore, in the total possible touch-sensitive area, allowed zones (180 and 170) and not-allowed zones (175) are created.","The connection from the central module 140 to the control computer (PC) 220 is realized in this configuration by two interfaces, one functions as a mouse 150, one as a keyboard 160, and by a keyboard, mouse, and monitor changeover switch 210.An additional mouse 250 and an additional keyboard 240 can be installed for fitting the entire system directly at the site of the control computer (PC) 220, which can be in a different room.","In the following, the type of control of the individual emitter diodes and receiver phototransistors (320, 330) is described.","Instead of the phototransistors, naturally other optical receivers, such as, e.g., photodiodes or photo-elements can also be used.","The control is distinguished by the following fundamental properties: 1) Only one opposing pair 340 of emitter/receiver diodes of a emitter/receiver unit are ever turned on.","This means that an emitter diode emits (in general) IR light, while in this time period tD only the opposing receiver diode is turned on (only its signal amplifier amplifies the incident signal).","After a certain time Δdiode this pair is turned off and the next is pair turned on.","After one cycle (scan) over all the diode pairs is completed, the cycle begins again with the first pair.","It is useful to scan the horizontal and vertical diodes simultaneously (so that two emitter diodes and two receiver diodes are always turned on, one from the horizontal and one from the vertical emitter/receiver unit).","The sequence of diodes to be turned on is completely arbitrary, but for the sake of control simplicity they should be turned on in series.","A typical value for Δtdiode is currently around 200 μs.","A faster or slower control for other applications (e.g., security technology, monitoring areas) is possible.","Thus, using a maximum of 16 modules each with 16 diodes produces 256 diodes per emitter/receiver unit and a time of approximately 50 ms for one scan (this corresponds to a total scanning frequency of approximately 20 Hz).","2) The paired operation of the emitter and receiver diodes spares the use of expensive optics for focusing the emitted light.","These would otherwise be necessary to prevent cross-coupling between the receivers over such large distances.","For the same reason, simple IR-diodes with small beam angles are sufficient.","No laser diodes are used (which would be more expensive many times over).","3) During the short on time Δtdiode of an emitter diode 320, this diode is operated at a multiple of the nominal continuous power (current-regulated or voltage-regulated).","The light emission during time Δtdiode is thus a multiple over its nominal continuous light power.","Because each IR-LED is turned off during the remaining time of a scan, this means no restriction on the life of the diodes.","For this reason, the distance of the emitter and receiver units to the touch-sensitive surface can be large (up to 8 m total distance has already been demonstrated).","Because the emitter and receiver arrays are to enable the scanning of a large sensitive area, a normal pulsed solution, like those in remote controls with normal rectangular voltage level control or even sinusoidal control, is no longer possible.","Here, the diode must be considered as a component with depletion layer capacitance, charge carrier lifetime, and charge carrier mobility.","These factors produce the necessity of generating massive saturation at turn on through the turn-on voltage amplitude and through a build-up time that lies in the region of a few ns.","To achieve such control behavior, different methods, such as the use of high-power, fast switching transistors or corresponding logic with re-differentiation and amplification can be used.","Through this saturation, no pulse sequence in the kHz range is possible for the normal LED, because there are too many charge carriers in the pn junction.","A pulse sequence with this control produces a single flash of light, which fades in the range of μs after turning off the pulse sequence.","To prevent this property, the emitter diode must be discharged of charge carriers again directly after saturation.","For this purpose, there are various possibilities, e.g., the use of an additional discharging transistor or simply a parallel load resistor.","The result is the generation of short, very intense flashes of light, which are emitted as a sequence (burst) and which are used later in the receiver as the basis for a high signal-to-noise ratio.","The microcontrollers in the emitter modules permit both controls to be set individually in order to minimize possible reflections on the display window panel or projection plane.","In the preferred configuration of the invention, according to the size of the interactive surface, 10-15 pulses are emitted per burst.","4) During their on time, the IR-LEDs 320 are operated not at a constant power, but instead in the so-called burst-mode.","This means they are operated with a frequency f, which clearly lies over the frequency of the relaying of the diodes f>1/Δtdiode.","Typically, this frequency lies in the kHz range.","5) The amplification circuit of detectors 330 in the receiver unit features a high attenuation (low amplification) for frequencies not equal to the burst frequency f, but a low attenuation (high amplification) at the burst frequency f. Therefore, ambient lighting affects (daylight, 50 Hz ripple frequency from power-line lamps, etc.)","are effectively suppressed.","The input circuit of the receiver, however, has no normal filter (LC, RC) as a working resistor, but instead uses only an inductor as a working resistor.","Therefore, two significant advantages are produced: first, disruption by constant light (daylight, street lamps) or light with amplitude modulation in small frequency ranges (50 Hz, 100 Hz) is no longer possible.","In this case, the coil acts as a short circuit.","Second, the operating point of the phototransistors even for strong incident sunlight is not shifted so far that the usable signal can no longer be detected.","For burst analysis, no filter or PLL system is used.","After the build-up time, an integrator is turned on, which permits continuous average value formation.","The integration time has a ratio of 1:3 to the total time of one burst.","By means of a comparator, a nearly 100% detection likelihood of the interrupts of the light barrier is obtained for large distances between emitter and receiver.","The receiver components can be influenced in their sensitivity with the aid of the microcontroller by the beginning of the integration and by the period of the integration relative to the burst time.","This allows a flexible and automatic adaptation to the total distance of the emitter and receiver arrays.","6) For further increase of the reliable detection also of narrower interrupts and the resolution capability (positional resolution of the detection of the interrupt), not only can the diode lying exactly opposite the currently operating receiver (pair 340) be turned on, but also its direct neighbor.","The emitter on time then decreases to ⅓ of the receiver on time.","Other time sequences/positional combinations are also possible.","To touch the interactive surface, usually only one finger (index finger) is used.","The index finger of small children has an average diameter of 10 mm, that of adults approximately 18 mm.","The distance of the light barriers equals 10 mm in the x-direction and y-direction.","In the normal case, an adult always interrupts more than one light barrier and can thus be detected at 100%.","In the worst case, a small child can hold a finger between two light barriers, so that residual light radiation like from one-sided screen shading is always still seen by the receiver.","To achieve a higher detection likelihood for smaller objects, which can interrupt the beam path, the following controls of the receiver and emitter are possible.","Starting with emitter LEDs with 5 mm φ, 10 mm distance to neighbor LED, and a beam angle of ±8°, the following results were calculated for the statistical evaluation to resolve an interrupt with 100% reliability at any position within the active region (resolution of the position <±1.5 mm).","a) Turned on during the integration time are: receiver n and emitter n continuously: the finger thickness must be at least 15 mm in the entire interactive region to be detected at 100% with a resolution of ±1.5 mm with additional evaluation in the central module 140 (additional evaluation is described farther below).","b) The emitters n−1, n, and n+1 are turned on one after the other during the integration time of the receiver n. The receiver signals are evaluated as a function of which emitter is currently active.","c) The emitters n−3, n−1, n, n+1, n+3 are turned on one after the other during the integration time of the receiver n. The receiver signals are evaluated as a function of which emitter is currently active.","Other arbitrary combinations of emitter control relative to the receiver are conceivable.","The burst on-time period is here lengthened correspondingly.","Because the total distance between the emitter and receiver arrays is usually greater than the projected image and thus the desired interactive region, in most cases the control according to method b) makes the most sense.","However, by means of microcontroller technology, if desired, version a) or c) can also be set.","Other settings of the total control are possible.","All controls except 1) allow the reliable detection of a finger thickness>10 mm.","7) If the emitter and receiver units (100, 110, 120, 130) are located at a large distance to each other and directly in front of a very reflective surface (display window), then in addition to the electronic regulation, the emitter and receiver modules (300, 310) can also be installed in a housing, such that a screen is created only in the surface of the projection, which prevents the emitted light of the emitter diodes from reaching the reflective surface, and thus prevents beam interruption, and thus detection.","This screen can also be mounted as a separate part.","8) In addition to the combination of relative emitter changeover with reference to the integration of a receiver, which significantly increases the detection likelihood of smaller objects, a basic statistical analysis concerning interruptions of the light barriers in position and time is performed in the central module 140.This produces a higher resolution of the scanning up to approximately 1 mm.","An average value formation of the interruptions over 3 adjacent light barriers and in addition a time analysis over three scans in sequence permit the reliable detection of the user and the input location.","In addition, short interruptions, e.g., insects, are excluded.","Longer interruptions (opaque object on a light barrier area) can also be filtered out after the recognition time (20-40 s).","In addition, an event is interpreted as true only if x and y light barriers are interrupted for a minimum time.","9) The central module 140 emulates a standard serial mouse relative to the control computer (PC) 220.Thus, this system can be used immediately for all operating systems (e.g., Windows 95/98/2000 or Linux) and computer types (e.g., PCs, workstations).","If the user leaves the interactive region at a position x1, y1, then the pressing of the left mouse button at this position is reported to the control computer by the central module.","Because the central module is built with a microcontroller, the emulation of the direct output of x-coordinates and y-coordinates for video games or a PS2 mouse can also be executed in firmware.","Other interface protocols can be loaded by the control computer at any time into the central module.","In addition to the mouse emulation, the central module 140 can emulate a standard IBM keyboard (150, 160) if desired.","A typical user input and its detection then look like this: The user brings his finger close to the touch-sensitive surface (display window) to touch an image region displayed by the projector.","After a certain cycling of the scanning of all emitter and receiver diodes, several receiver diodes in the horizontal and vertical direction determine a light interruption.","This information is converted in the central module 140 into a position of the user interruption and can be transmitted to the control computer, e.g., as the position of the mouse pointer.","For the next scan cycle, the focus of the interrupt is possibly located at a different position (because one scan only lasts {fraction (1/20)} of a second, the position of the interrupt changes only slowly).","If after a certain scan an interruption is no longer determined, then the user has removed his finger from the sensitive zone.","This action can be transmitted to the control computer from the central module as a mouse click.","Then the control computer can display new image contents corresponding to the mouse click on the touch-sensitive surface by the projector.","According to the invention, there are also configurations with an unequal number of emitter and receiver diodes in the emitter and receiver units.","For large distances from the emitter unit to the touch-sensitive surface, emitter diodes can thus be spared.","The resolution is then determined by the distance of the receiver diodes.","Another configuration according to the invention consists in that the emitter and receiver diodes are not arranged in different modules and emitter and receiver units, but instead alternating or in parallel on one side.","On the opposite side, there is then either a reflective or absorbing unit.","In the first case, the detection is completely analogous to the description above.","In the second case, the light back-scattered by the user input (typically a finger) is used for detection (no light barrier interrupt is detected, instead the light barrier closing is detected).","The absorption device in the opposite region prevents an unintended closing of the light barriers.","The control of adjacent emitter and receiver diodes otherwise follows the path described in points 1-8.A preferred configuration of the installation of the touch-sensitive surface is that shown in FIG.","1.Here, a data and video projector illuminates from behind a rear-projection surface (normal focusing screen, holographic rear-projection screen, e.g., HoloPro© from Pronova for better suppression of ambient light) located behind a display window.","The touch-sensitive surface is built on the display window.","The holders of the emitter and receiver units can be “hidden” in the frame of the display window screen.","For a typical projection surface of 1.6 m2, a transmission of the focusing screen of 50%, a light-amplification factor of the focusing screen in the forward direction of 2, an ambient brightness on the display window of 2000 lx (shaded outside area), and a degree of reflection for this light of approximately 35%, a projector with an optical power of approximately 2300 ANSII lumens is required to achieve twice as much light as the ambient light on the focusing screen.","For the use of a HoloPro™ screen, this ratio is even more favorable.","A touch-sensitive display window can be used for advertisement anywhere in an outside area, whether in pedestrian zones, at exhibitions, in auto dealers, etc.","Other applications of the touch-sensitive surface are large, enclosed rear-projection boxes (multi-active boxes) or smaller information terminals in railroad stations or airports.","List of Reference Symbols FIG.","1 Key: 1 Glass panel of the display window 2 Projection surface 3 Strip with receiver modules 4 Strip with receiver modules 5 Strip with emitter modules 6 Strip with emitter modules 7 Video-data projector 8 Decoration FIG.","2 Key: 100 Strip with emitter modules 110 Strip with receiver modules 120 Strip with emitter modules 130 Strip with receiver modules 140 Central module 150 Mouse output 160 Keyboard output 170 Film with keyboard image 175 Not used area 180 Projection surface (image surface) 190 Touch-sensitive area 200 Video-data projector 210 Mouse-keyboard-monitor changeover switch 220 Computer 230 Monitor 240 Keyboard 250 Mouse 260 Printer 270 Card reader 280 LAN connection FIG.","3 Key: 300 Emitter module 310 Receiver module 320 Emitter diodes 330 Receiver phototransistors or diodes 340 Emitter-receiver pair 350 IR light beam"]],"string":"[\n [\n \"Large touch-sensitive area with time-controlled and location-controlled emitter and receiver modules\",\n \"The invention enables the realization of an optical and electronic structure of a touch-sensitive area on any flat display surface such as a monitor.\",\n \"The optical structure does not require any additional optical components such as lenses.\",\n \"Laser diodes are likewise not required for the operation of a structure of this type.\",\n \"Large distances and, nevertheless, high sensitivity and high resolution of the touch-sensitive area are accomplished by a novel time-control and location-control of both the emitter diodes as well as the receiver diodes or receiver photo-transistors.\",\n \"If still or moving images are displayed on the surface, the invention can be used as a touch screen and can interactively control the display.\",\n \"A central module simulates a computer mouse and a keyboard.\"\n ],\n [\n \"1.A touch-sensitive region on a display device for detecting a user input, comprising: emitter diodes for outputting light; receiver diodes/phototransistors for detecting the light, the emitter diodes and receiver diodes/phototransistors being arranged in vertical and horizontal emitter and receiver units set opposite to each other, one of the emitter diodes and one of the receiver diodes/phototransistors being optically set in a direct paired relationship to each other; and a central module; wherein the detection of a user input is realized through continuously repeating time-sequenced and position-sequenced on-and-off switching of individual emitter diodes and receiver diodes/phototransistors; wherein one of the emitter diodes and one of the receiver diodes/phototransistors are controlled in pairs; and wherein for evaluating and increasing the resolution, the signals of the emitter diodes are referenced, adjacent to the emitter diodes, and all electrical signals of the receiver diodes/phototransistors are assembled into the central module, and also the emitter diodes are controlled from the Central module, such that the emitter diodes output a plurality of pulses, whose integrative, quantitative evaluation is a measure of the position of the interruption of a light barrier during an evaluation period and provides information on the position of the interruption relative to the emitter diodes.\",\n \"2.A touch-sensitive region according to claim 1, characterized in that the emitter diodes are operated during their short on-time with higher power than their nominal continuous power.\",\n \"3.A touch-sensitive region according to claim 1, characterized in that the wavelength of the applied electromagnetic radiation is in the infrared range of the spectrum and the emitter diodes and receiver diodes/phototransistors are emitting and receiving semiconductor elements.\",\n \"4.A touch-sensitive region according to claim 1, characterized in that the emitter diodes are operated with a burst signal during their on time, which means that the electrical alternating signal controlling them has a higher frequency f than the alternating frequency of the emitter diodes controlled one after the other; wherein for faster saturation, measures, such as increasing the start-up voltage amplitude/voltage rise time and necessary discharge of charge carriers from the emitter diodes after the saturation pulse, are used.\",\n \"5.A touch-sensitive region according to claim 1, characterized in that the amplifier circuit of the receiver diodes or phototransistors has a large amplification at the burst frequency f and otherwise enables a high attenuation through an inductive operating resistor followed by an integrator.\",\n \"6.A touch-sensitive region according to claim 1, characterized in that not only the emitter diodes arranged optically in pairs with a receiver diode or phototransistor are active during the on time of the receiver diode or phototransistor, but adjacent emitter diodes are also active.\",\n \"7.A touch-sensitive region according to claim 1, characterized in that an optical aperture is arranged in front of the emitter diodes and receiver diodes or phototransistors.\",\n \"8.A touch-sensitive region according to claim 1, characterized in that the optical paired relationship of emitter diodes and receiver diodes or phototransistors involves oppositely positioned emitter diodes and receiver diodes or phototransistors in opposing emitter and receiver units.\",\n \"9.A touch-sensitive region according to claim 1, characterized in that the optically paired relationship of the emitter diodes and receiver diodes or phototransistors involves emitter diodes and receiver diodes or phototransistors positioned one next to the other in a mixed emitter and receiver unit and an oppositely positioned mirror surface.\",\n \"10.A touch-sensitive region according to claim 1, characterized in that the optical relationship of the emitter diodes and receiver diodes or phototransistors involves emitter diodes and receiver diodes or phototransistors positioned one next to the other in a mixed emitter and receiver unit and an oppositely positioned absorbing surface.\",\n \"11.A touch-sensitive region according to claim 1, characterized in that the total number of emitter diodes in the emitter units is exactly equal to or not equal to the number of receiver diodes or phototransistors in the receiver units.\",\n \"12.A touch-sensitive region according to claim 1, characterized in that a microprocessor of the central module calculates the position of the user input from digital receiver module signals by means of a statistical analysis method.\",\n \"13.A touch-sensitive region according to claim 1, characterized in that the central module can simulate any arbitrary interface relative to an attached computer.\",\n \"14.A touch-sensitive region according to claim 2, characterized in that the wavelength of the applied electromagnetic radiation is in the infrared range of the spectrum and the emitter diodes and receiver diodes/phototransistors are emitting and receiving semiconductor elements.\",\n \"15.A touch-sensitive region according to claim 2, characterized in that the emitter diodes are operated with a burst signal during their on time, which means that the electrical alternating signal controlling them has a higher frequency f than the alternating frequency of the emitter diodes controlled one after the other; wherein for faster saturation, measures, such as increasing the start-up voltage amplitude/voltage rise time and necessary discharge of charge carriers from the emitter diodes after the saturation pulse, are used.\",\n \"16.A touch-sensitive region according to claim 3, characterized in that the emitter diodes are operated with a burst signal during their on time, which means that the electrical alternating signal controlling them has a higher frequency f than the alternating frequency of the emitter diodes controlled one after the other; wherein for faster saturation, measures, such as increasing the start-up voltage amplitude/voltage rise time and necessary discharge of charge carriers from the emitter diodes after the saturation pulse, are used.\",\n \"17.A touch-sensitive region according to claim 2, characterized in that the amplifier circuit of the receiver diodes or phototransistors has a large amplification at the burst frequency f and otherwise enables a high attenuation through an inductive operating resistor followed by an integrator.\",\n \"18.A touch-sensitive region according to claim 3, characterized in that the amplifier circuit of the receiver diodes or phototransistors has a large amplification at the burst frequency f and otherwise enables a high attenuation through an inductive operating resistor followed by an integrator.\",\n \"19.A touch-sensitive region according to claim 4, characterized in that the amplifier circuit of the receiver diodes or phototransistors has a large amplification at the burst frequency f and otherwise enables a high attenuation through an inductive operating resistor followed by an integrator.\",\n \"20.A touch-sensitive region according to claim 2, characterized in that not only the emitter diodes arranged optically in pairs with a receiver diode or phototransistor are active during the on time of the receiver diode or phototransistor, but adjacent emitter diodes are also active.\"\n ],\n [\n \"The invention relates to the optical and electronic structure of a very large (possibly over 0.04 m2, typically: >1 m2) touch-sensitive area (also called a touchscreen) on any transparent surface (monitor), which is used to build multi-active display windows, multi-active boxes, information terminals, or security systems, e.g., in the form of personnel filters.\",\n \"This invention describes the application of a multi-active display window, whose entire area, up to 8 m×8 m, can be configured interactively for the first time.\",\n \"Here, the resolution of the scan equals ±1.5 mm.\",\n \"For the application as a multi-active display window (FIG.\",\n \"1), a focusing screen, projection film, or another projection surface (2), which replaces part of or the entire decoration (8), is set up behind the display window panel (1).\",\n \"On this surface, a large-area image is projected (rear projection) from behind by means of a known video and data light projector (7).\",\n \"These images are typically generated by a control computer.\",\n \"At the border of the display window panel, a vandalism-proof coating is applied from the outside.\",\n \"This contains a special infrared (IR) emitter and receiver module arrays (3, 4, 5, 6), which cover the display window (the touch-sensitive area) with an invisible light-barrier curtain.\",\n \"From the front, the user can point to a certain area of the image (user input).\",\n \"This area is identified by the structure according to the invention described in this patent specification and forwarded to the control computer.\",\n \"This computer can control the further flow of images and graphics corresponding to the user input.\",\n \"Thus, an interactive, i.e., user-controlled, presentation is possible.\",\n \"Here, this user input need not absolutely lie on the area given by the rear projection; instead, a separately designated area (e.g., a keyboard or trackball) can also be on the touch-sensitive area.\",\n \"Furthermore, the described detection structure can also be applied to other optical systems and image generators, which generate images on surfaces (plasma screens, large monitors, LCD monitors, rear-projection boxes, etc.).\",\n \"The presentation processed in the control computer can consist of, e.g., an Internet presentation.\",\n \"The multi-active display window can then be functionally compared with a computer mouse and its navigation properties.\",\n \"Thus, for the first time economical (without personnel and around the clock) information and advertisements can be provided for any supplier at locations in cities that are heavily frequented by pedestrians.\",\n \"For the current state of the art, touch-sensitive surfaces up to approximately 40″ (touchscreens) are realized preferably in the type in which a film or a number of thin conductive threads on the image surface detects the user input (touch) because an electrical property of the film or the thread (e.g., resistance, capacitance, field strength) changes when touched, and this change is converted into the exact two-dimensional position of the user input by evaluating the separately detected horizontal and vertical electrical signals.\",\n \"The disadvantages of these methods are the following: 1) The cost of the structure greatly increases with the size of the touch-sensitive area.\",\n \"Surfaces over approximately 0.5 m2 thus cannot be realized economically.\",\n \"2) The accuracy of the touch detection decreases with larger surfaces.\",\n \"3) The described methods are unsuitable for use in public areas, because they are not vandalism-proof.\",\n \"This means that they can be easily destroyed and can no longer fulfill their intended function due to outside actions of a willfully destructive nature.\",\n \"4) Most solutions of the state of the art require additional layers over the projected image and thus attenuate the brightness of the image.\",\n \"Other possibilities include the acoustic (ultrasound) detection of the interrupt, wherein fewer emitters/receivers are necessary than for two-dimensional networks.\",\n \"However, this technology is reliable only up to touchscreen sizes of approximately 1.5×1.5 m2 and shows disadvantages for the reliable detection of user input in the edge regions.\",\n \"There are also active detection systems.\",\n \"Active means that the user uses a special, active transmitting pointing device in order to perform the user input (flip-chart).\",\n \"This system is not suitable if anonymous persons are to benefit from the interactivity, e.g., of the multi-active display window.\",\n \"In addition, for small touchscreens (e.g., in automatic bank machines), detectors based on infrared emitters and receivers are used.\",\n \"Here, the touch-sensitive surface is covered with invisible IR radiation and the interruption of a light barrier is detected.\",\n \"The emitters and receivers are turned on permanently.\",\n \"Usually, additional lenses are used for increasing the light-beam focusing for such structures.\",\n \"Therefore, only small surfaces (<0.1 m2) can be detected, because for greater distances from the emitter units to the receiver units, strong cross-coupling prevents the detection of the user input and also the light intensity of the diodes is no longer sufficient to achieve reliable detection.\",\n \"It is known, e.g., for infrared light-emitting diodes (IR-LED) from remote controls, that LEDs can be operated in a pulsed mode up to pulse duty factors of 1:100.This means that the LED is turned off one-hundred times longer than the time during which it is turned on (and thus emits light).\",\n \"Here, pulse sequences (bursts) with rectangular signals are forwarded to the emitter, which produce a more or less sinusoidal light stream based on their self-capacitance and the pn-junction properties.\",\n \"The electrical current during the short on period can then equal 100-times the nominal continuous current without significantly affecting the life of the LED.\",\n \"Thus, considerably higher peak powers of the light radiation can be achieved in a short time period than would be possible for continuous operation.\",\n \"It is further known that IR-LEDs and IR receivers (IR-sensitive photodiodes, phototransistors, or photoresistors) can be used in light barriers.\",\n \"Here, the interruption of the optical connection between emitter and receiver (when the emitter and receiver “are looking at” each other) or the formation of the optical connection (when the emitter and receiver see each other only if a third object reflects the emitted light) is detected and, e.g., it is determined whether a person has passed through a certain space or whether a person is located in front of a certain point in space.\",\n \"An embodiment of the invention is explained in more detail with reference to the enclosed drawings.\",\n \"Shown are: FIG.\",\n \"1, a schematic structure of a multi-active display window, FIG.\",\n \"2, a block diagram of a large-area, interactive projection, FIG.\",\n \"3, the structure of the time-controlled and position-controlled light barriers.\",\n \"The disadvantages mentioned above of previous touchscreen solutions for large surfaces do not occur for a configuration of the touchscreen according to the invention.\",\n \"In connection with a rear projection by means of contemporary high-power LCD or DMD video and data projectors, large display windows can be transformed into multi-active projection surfaces (FIG.\",\n \"1), which fulfill the following properties: 1) Economical provision of the display window with a touch-sensitive surface.\",\n \"2) Vandalism-proof structure.\",\n \"3) Reliable detection of a user input for a finger width greater than 10 mm (small children) with a position resolution of ±1.5 mm without any light loss of the projection.\",\n \"4) Completely automatic day and night operation possible due to high-power, remote-control capable projectors.\",\n \"According to the invention, these requirements are fulfilled by detecting the user input through electromagnetic (typically in the infrared range of the spectrum) emitters and receivers, which are arranged in the horizontal and vertical direction and which are controlled in a suitable way in a time-sequenced and position-dependent pattern.\",\n \"These transmitters and receivers are each arranged separately in modules (transmitters and receiver modules FIG.\",\n \"3, 300, 310), which can be arranged in series in principle in an arbitrary amount (FIG.\",\n \"2, 100, 110, 120, 130).\",\n \"These modules are attached on the outside around the touch-sensitive area (190) to be defined, wherein the modules can be mounted at a large distance to the borders of the area (up to 8 m from each other), which creates the ability of “hiding” these components in terms of construction.\",\n \"Because the modules are installed in stable aluminum, steel, or diecast housings, this arrangement is vandalism-proof.\",\n \"The emitter and receiver modules 300, 310 are arranged opposite each other and “look” out over nearly the entire touch-sensitive area 190 (the distance from the display window surface here equals approximately 0.5 to 3 cm).\",\n \"Each module (300, 310) has its own microcontroller, which is arranged in the module itself.\",\n \"The entire arrangement represents a multiprocessor system.\",\n \"Also, the central module 140 has a microcontroller with greater capability relative to the module controllers (greater RAM capacity, higher clock frequency, larger flash-RAM).\",\n \"The emitter modules 300 receive their control signals from the central module 140.The measurement signals of the receiver modules 310 are analyzed statistically and then transmitted to the central module 140.The central module 140 assembles the entire interactive surface and recognizes a relevant user input.\",\n \"This input is output, e.g., in a special protocol (e.g., standard serial interface for a mouse) to the PC.\",\n \"Other output types, such as x-coordinates, y-coordinates (for special consoles), or PS2-mouse emulation can be set by the attached PC.\",\n \"The central module allows the implementation of arbitrary mouse protocols or serial transmission protocols based on the microcontroller circuit.\",\n \"The data is transmitted to the control computer 220 controlling the presentation.\",\n \"A preferred configuration of the invention is described in the following (FIG.\",\n \"2, FIG.\",\n \"3): The touchscreen consists of IR-emitter and IR-receiver modules (300, 310), which are arranged together into corresponding horizontal and vertical emitter and receiver module arrays (100, 110, 120, 130).\",\n \"Here, the number of active emitter modules in the horizontal (or vertical) direction n (or m) must agree with the number of active receiver modules in the horizontal (vertical) direction.\",\n \"Here, m can not equal n. Thus, any rectangular area 190 can be defined.\",\n \"In the preferred example, n, m should not be greater than 16.For example, for a structural module length of 16 cm (16 emitters 300 or receivers 310 are assembled on a module and the distance of the emitter diodes or receiver transistors to each module is 10 mm), user inputs on a maximum surface area of 2.56×2.56 m2 (6.55 m2) can be detected in this embodiment.\",\n \"In principle, larger areas with a configuration according to the invention are possible.\",\n \"The emitter and receiver module arrays (100, 110, 120, 130) must not be mounted directly on the touch-sensitive area 190, but instead can feature a distance, which in principle is arbitrarily large, from the area (FIG.\",\n \"2).\",\n \"The corresponding two emitter and receiver module arrays (which in turn consist of n or m modules) are connected to a central module 140.In the preferred configuration of the invention, the central module 140 automatically notices how many modules make up the units in the x-direction (horizontal) and y-direction (vertical).\",\n \"In this preferred configuration, next to the active region 190, which is predetermined by the size of the projected image, yet another touch-sensitive area 170 can be defined, which can be used as a keyboard and/or trackball for data input to the control computer (PC) 220.Therefore, in the total possible touch-sensitive area, allowed zones (180 and 170) and not-allowed zones (175) are created.\",\n \"The connection from the central module 140 to the control computer (PC) 220 is realized in this configuration by two interfaces, one functions as a mouse 150, one as a keyboard 160, and by a keyboard, mouse, and monitor changeover switch 210.An additional mouse 250 and an additional keyboard 240 can be installed for fitting the entire system directly at the site of the control computer (PC) 220, which can be in a different room.\",\n \"In the following, the type of control of the individual emitter diodes and receiver phototransistors (320, 330) is described.\",\n \"Instead of the phototransistors, naturally other optical receivers, such as, e.g., photodiodes or photo-elements can also be used.\",\n \"The control is distinguished by the following fundamental properties: 1) Only one opposing pair 340 of emitter/receiver diodes of a emitter/receiver unit are ever turned on.\",\n \"This means that an emitter diode emits (in general) IR light, while in this time period tD only the opposing receiver diode is turned on (only its signal amplifier amplifies the incident signal).\",\n \"After a certain time Δdiode this pair is turned off and the next is pair turned on.\",\n \"After one cycle (scan) over all the diode pairs is completed, the cycle begins again with the first pair.\",\n \"It is useful to scan the horizontal and vertical diodes simultaneously (so that two emitter diodes and two receiver diodes are always turned on, one from the horizontal and one from the vertical emitter/receiver unit).\",\n \"The sequence of diodes to be turned on is completely arbitrary, but for the sake of control simplicity they should be turned on in series.\",\n \"A typical value for Δtdiode is currently around 200 μs.\",\n \"A faster or slower control for other applications (e.g., security technology, monitoring areas) is possible.\",\n \"Thus, using a maximum of 16 modules each with 16 diodes produces 256 diodes per emitter/receiver unit and a time of approximately 50 ms for one scan (this corresponds to a total scanning frequency of approximately 20 Hz).\",\n \"2) The paired operation of the emitter and receiver diodes spares the use of expensive optics for focusing the emitted light.\",\n \"These would otherwise be necessary to prevent cross-coupling between the receivers over such large distances.\",\n \"For the same reason, simple IR-diodes with small beam angles are sufficient.\",\n \"No laser diodes are used (which would be more expensive many times over).\",\n \"3) During the short on time Δtdiode of an emitter diode 320, this diode is operated at a multiple of the nominal continuous power (current-regulated or voltage-regulated).\",\n \"The light emission during time Δtdiode is thus a multiple over its nominal continuous light power.\",\n \"Because each IR-LED is turned off during the remaining time of a scan, this means no restriction on the life of the diodes.\",\n \"For this reason, the distance of the emitter and receiver units to the touch-sensitive surface can be large (up to 8 m total distance has already been demonstrated).\",\n \"Because the emitter and receiver arrays are to enable the scanning of a large sensitive area, a normal pulsed solution, like those in remote controls with normal rectangular voltage level control or even sinusoidal control, is no longer possible.\",\n \"Here, the diode must be considered as a component with depletion layer capacitance, charge carrier lifetime, and charge carrier mobility.\",\n \"These factors produce the necessity of generating massive saturation at turn on through the turn-on voltage amplitude and through a build-up time that lies in the region of a few ns.\",\n \"To achieve such control behavior, different methods, such as the use of high-power, fast switching transistors or corresponding logic with re-differentiation and amplification can be used.\",\n \"Through this saturation, no pulse sequence in the kHz range is possible for the normal LED, because there are too many charge carriers in the pn junction.\",\n \"A pulse sequence with this control produces a single flash of light, which fades in the range of μs after turning off the pulse sequence.\",\n \"To prevent this property, the emitter diode must be discharged of charge carriers again directly after saturation.\",\n \"For this purpose, there are various possibilities, e.g., the use of an additional discharging transistor or simply a parallel load resistor.\",\n \"The result is the generation of short, very intense flashes of light, which are emitted as a sequence (burst) and which are used later in the receiver as the basis for a high signal-to-noise ratio.\",\n \"The microcontrollers in the emitter modules permit both controls to be set individually in order to minimize possible reflections on the display window panel or projection plane.\",\n \"In the preferred configuration of the invention, according to the size of the interactive surface, 10-15 pulses are emitted per burst.\",\n \"4) During their on time, the IR-LEDs 320 are operated not at a constant power, but instead in the so-called burst-mode.\",\n \"This means they are operated with a frequency f, which clearly lies over the frequency of the relaying of the diodes f>1/Δtdiode.\",\n \"Typically, this frequency lies in the kHz range.\",\n \"5) The amplification circuit of detectors 330 in the receiver unit features a high attenuation (low amplification) for frequencies not equal to the burst frequency f, but a low attenuation (high amplification) at the burst frequency f. Therefore, ambient lighting affects (daylight, 50 Hz ripple frequency from power-line lamps, etc.)\",\n \"are effectively suppressed.\",\n \"The input circuit of the receiver, however, has no normal filter (LC, RC) as a working resistor, but instead uses only an inductor as a working resistor.\",\n \"Therefore, two significant advantages are produced: first, disruption by constant light (daylight, street lamps) or light with amplitude modulation in small frequency ranges (50 Hz, 100 Hz) is no longer possible.\",\n \"In this case, the coil acts as a short circuit.\",\n \"Second, the operating point of the phototransistors even for strong incident sunlight is not shifted so far that the usable signal can no longer be detected.\",\n \"For burst analysis, no filter or PLL system is used.\",\n \"After the build-up time, an integrator is turned on, which permits continuous average value formation.\",\n \"The integration time has a ratio of 1:3 to the total time of one burst.\",\n \"By means of a comparator, a nearly 100% detection likelihood of the interrupts of the light barrier is obtained for large distances between emitter and receiver.\",\n \"The receiver components can be influenced in their sensitivity with the aid of the microcontroller by the beginning of the integration and by the period of the integration relative to the burst time.\",\n \"This allows a flexible and automatic adaptation to the total distance of the emitter and receiver arrays.\",\n \"6) For further increase of the reliable detection also of narrower interrupts and the resolution capability (positional resolution of the detection of the interrupt), not only can the diode lying exactly opposite the currently operating receiver (pair 340) be turned on, but also its direct neighbor.\",\n \"The emitter on time then decreases to ⅓ of the receiver on time.\",\n \"Other time sequences/positional combinations are also possible.\",\n \"To touch the interactive surface, usually only one finger (index finger) is used.\",\n \"The index finger of small children has an average diameter of 10 mm, that of adults approximately 18 mm.\",\n \"The distance of the light barriers equals 10 mm in the x-direction and y-direction.\",\n \"In the normal case, an adult always interrupts more than one light barrier and can thus be detected at 100%.\",\n \"In the worst case, a small child can hold a finger between two light barriers, so that residual light radiation like from one-sided screen shading is always still seen by the receiver.\",\n \"To achieve a higher detection likelihood for smaller objects, which can interrupt the beam path, the following controls of the receiver and emitter are possible.\",\n \"Starting with emitter LEDs with 5 mm φ, 10 mm distance to neighbor LED, and a beam angle of ±8°, the following results were calculated for the statistical evaluation to resolve an interrupt with 100% reliability at any position within the active region (resolution of the position <±1.5 mm).\",\n \"a) Turned on during the integration time are: receiver n and emitter n continuously: the finger thickness must be at least 15 mm in the entire interactive region to be detected at 100% with a resolution of ±1.5 mm with additional evaluation in the central module 140 (additional evaluation is described farther below).\",\n \"b) The emitters n−1, n, and n+1 are turned on one after the other during the integration time of the receiver n. The receiver signals are evaluated as a function of which emitter is currently active.\",\n \"c) The emitters n−3, n−1, n, n+1, n+3 are turned on one after the other during the integration time of the receiver n. The receiver signals are evaluated as a function of which emitter is currently active.\",\n \"Other arbitrary combinations of emitter control relative to the receiver are conceivable.\",\n \"The burst on-time period is here lengthened correspondingly.\",\n \"Because the total distance between the emitter and receiver arrays is usually greater than the projected image and thus the desired interactive region, in most cases the control according to method b) makes the most sense.\",\n \"However, by means of microcontroller technology, if desired, version a) or c) can also be set.\",\n \"Other settings of the total control are possible.\",\n \"All controls except 1) allow the reliable detection of a finger thickness>10 mm.\",\n \"7) If the emitter and receiver units (100, 110, 120, 130) are located at a large distance to each other and directly in front of a very reflective surface (display window), then in addition to the electronic regulation, the emitter and receiver modules (300, 310) can also be installed in a housing, such that a screen is created only in the surface of the projection, which prevents the emitted light of the emitter diodes from reaching the reflective surface, and thus prevents beam interruption, and thus detection.\",\n \"This screen can also be mounted as a separate part.\",\n \"8) In addition to the combination of relative emitter changeover with reference to the integration of a receiver, which significantly increases the detection likelihood of smaller objects, a basic statistical analysis concerning interruptions of the light barriers in position and time is performed in the central module 140.This produces a higher resolution of the scanning up to approximately 1 mm.\",\n \"An average value formation of the interruptions over 3 adjacent light barriers and in addition a time analysis over three scans in sequence permit the reliable detection of the user and the input location.\",\n \"In addition, short interruptions, e.g., insects, are excluded.\",\n \"Longer interruptions (opaque object on a light barrier area) can also be filtered out after the recognition time (20-40 s).\",\n \"In addition, an event is interpreted as true only if x and y light barriers are interrupted for a minimum time.\",\n \"9) The central module 140 emulates a standard serial mouse relative to the control computer (PC) 220.Thus, this system can be used immediately for all operating systems (e.g., Windows 95/98/2000 or Linux) and computer types (e.g., PCs, workstations).\",\n \"If the user leaves the interactive region at a position x1, y1, then the pressing of the left mouse button at this position is reported to the control computer by the central module.\",\n \"Because the central module is built with a microcontroller, the emulation of the direct output of x-coordinates and y-coordinates for video games or a PS2 mouse can also be executed in firmware.\",\n \"Other interface protocols can be loaded by the control computer at any time into the central module.\",\n \"In addition to the mouse emulation, the central module 140 can emulate a standard IBM keyboard (150, 160) if desired.\",\n \"A typical user input and its detection then look like this: The user brings his finger close to the touch-sensitive surface (display window) to touch an image region displayed by the projector.\",\n \"After a certain cycling of the scanning of all emitter and receiver diodes, several receiver diodes in the horizontal and vertical direction determine a light interruption.\",\n \"This information is converted in the central module 140 into a position of the user interruption and can be transmitted to the control computer, e.g., as the position of the mouse pointer.\",\n \"For the next scan cycle, the focus of the interrupt is possibly located at a different position (because one scan only lasts {fraction (1/20)} of a second, the position of the interrupt changes only slowly).\",\n \"If after a certain scan an interruption is no longer determined, then the user has removed his finger from the sensitive zone.\",\n \"This action can be transmitted to the control computer from the central module as a mouse click.\",\n \"Then the control computer can display new image contents corresponding to the mouse click on the touch-sensitive surface by the projector.\",\n \"According to the invention, there are also configurations with an unequal number of emitter and receiver diodes in the emitter and receiver units.\",\n \"For large distances from the emitter unit to the touch-sensitive surface, emitter diodes can thus be spared.\",\n \"The resolution is then determined by the distance of the receiver diodes.\",\n \"Another configuration according to the invention consists in that the emitter and receiver diodes are not arranged in different modules and emitter and receiver units, but instead alternating or in parallel on one side.\",\n \"On the opposite side, there is then either a reflective or absorbing unit.\",\n \"In the first case, the detection is completely analogous to the description above.\",\n \"In the second case, the light back-scattered by the user input (typically a finger) is used for detection (no light barrier interrupt is detected, instead the light barrier closing is detected).\",\n \"The absorption device in the opposite region prevents an unintended closing of the light barriers.\",\n \"The control of adjacent emitter and receiver diodes otherwise follows the path described in points 1-8.A preferred configuration of the installation of the touch-sensitive surface is that shown in FIG.\",\n \"1.Here, a data and video projector illuminates from behind a rear-projection surface (normal focusing screen, holographic rear-projection screen, e.g., HoloPro© from Pronova for better suppression of ambient light) located behind a display window.\",\n \"The touch-sensitive surface is built on the display window.\",\n \"The holders of the emitter and receiver units can be “hidden” in the frame of the display window screen.\",\n \"For a typical projection surface of 1.6 m2, a transmission of the focusing screen of 50%, a light-amplification factor of the focusing screen in the forward direction of 2, an ambient brightness on the display window of 2000 lx (shaded outside area), and a degree of reflection for this light of approximately 35%, a projector with an optical power of approximately 2300 ANSII lumens is required to achieve twice as much light as the ambient light on the focusing screen.\",\n \"For the use of a HoloPro™ screen, this ratio is even more favorable.\",\n \"A touch-sensitive display window can be used for advertisement anywhere in an outside area, whether in pedestrian zones, at exhibitions, in auto dealers, etc.\",\n \"Other applications of the touch-sensitive surface are large, enclosed rear-projection boxes (multi-active boxes) or smaller information terminals in railroad stations or airports.\",\n \"List of Reference Symbols FIG.\",\n \"1 Key: 1 Glass panel of the display window 2 Projection surface 3 Strip with receiver modules 4 Strip with receiver modules 5 Strip with emitter modules 6 Strip with emitter modules 7 Video-data projector 8 Decoration FIG.\",\n \"2 Key: 100 Strip with emitter modules 110 Strip with receiver modules 120 Strip with emitter modules 130 Strip with receiver modules 140 Central module 150 Mouse output 160 Keyboard output 170 Film with keyboard image 175 Not used area 180 Projection surface (image surface) 190 Touch-sensitive area 200 Video-data projector 210 Mouse-keyboard-monitor changeover switch 220 Computer 230 Monitor 240 Keyboard 250 Mouse 260 Printer 270 Card reader 280 LAN connection FIG.\",\n \"3 Key: 300 Emitter module 310 Receiver module 320 Emitter diodes 330 Receiver phototransistors or diodes 340 Emitter-receiver pair 350 IR light beam\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471069"}}},{"rowIdx":572,"cells":{"text":{"kind":"list like","value":[["Method for the amplification and detection of hbv dna using a transcription based amplification","The present invention provides a method for the transcription based amplification of a target HBV nucleic acid sequence starting from HBV DNA optionally present in a sample, comprising the steps of,—incubating the sample, suspected to contain HBV, in an amplification buffer with one or more restriction enzymes capable of cleaving the HBV DNA at a selected restriction site, said restriction enzyme creating a defined 3′ end of the said HBV DNA strand(s), a promotor-primer, said promotor-primer having a 5′ region comprising the sequence of a promotor recognized by a DNA-dependent RNA polymerase and a 3′ region complementary to the define 3′ end of the DNA strand, a second or reverse primer, having the opposite polarity of the promotor-primer and comprising the 5′ end of the said target sequence, and in case of HBV ssDNA as the target sequence, a restriction primer,—maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for a digestion by the restriction enzyme to take place,—subjecting the sample thus obtained to a heat treatment at a temperature and time sufficient to inactivate the restriction enzyme and/or to render at least partially a double strand single stranded,—adding the following reagents to the sample: an enzyme having RNA dependent DNA polymerase activity, an enzyme having DNA dependent DNA polymerase activity, an enzyme having Rnase H activity, an enzyme having RNA polymerase activity, and—maintaining the thus created reaction mixture under the appropriate conditions to a sufficient amount of time for the amplification to take place."],["1.A method for the transcription based amplification of a target HBV nucleic acid sequence starting from HBV DNA optionally present in a sample, said method comprising: -a) incubating the sample, suspected to contain HBV, in an amplification buffer with i) one or more restriction enzymes capable of cleaving the HBV DNA at a selected restriction site, said restriction enzyme creating a defined 3′ end of the said HBV DNA strand(s), ii) a promoter-primer, said promoter-primer having a 5′ region comprising the sequence of a promoter recognized by a DNA-dependent RNA polymerase and a 3′ region complementary to the defined 3′ end of the DNA strand, iii) a second or reverse primer, having the opposite polarity of the promoter-primer and comprising the 5′ end of the said target sequence, and iv) in case of HBV ssDNA as the target sequence, a restriction primer; -b) maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for a digestion by the restriction enzyme to take place; -c) subjecting the sample thus obtained to a heat treatment at a temperature and time sufficient to inactivate the restriction enzyme and/or to render at least partially a double strand single stranded; -d) adding the following reagents to the sample: i) an enzyme having RNA dependent DNA polymerase activity ii) an enzyme having DNA dependent DNA polymerase activity iii) an enzyme having Rnase H activity iv) an enzyme having RNA polymerase activity; and e) maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for the amplification to take place.","2.The method according to claim 1, wherein the DNA is double stranded HBV DNA.","3.The method according to claim 1, wherein the DNA is single stranded and the promoter primer and the restriction primer are combined in using a combined promoter and restriction primer comprising a sequence complementary to the region including the restriction site of the target ssDNA and the sequence of a promoter recognized by a DNA-dependent RNA polymerase.","4.The method according to claim 1 in which nucleoside triphosphates are added to the initial incubation mixture prior to the heat treatment.","5.The method according to claim 1 in which a reverse transcriptase is used combining the activities of the enzyme having RNA dependent DNA polymerase activity and the enzyme having DNA dependent DNA activity.","6.The method according to claim 1, in which a reverse transcriptase is used having inherent RNase H activity replacing 3 enzymes, namely the enzyme having RNA dependent DNA polymerase activity, the enzyme having DNA dependent DNA activity as well as the enzyme having Rnase H activity.","7.The method according to claim 1, in which the incubation temperature is from about 35° C. to about 45° C. 8.The method according to claim 1 in which to the heating step is carried out at a temperature between about 92° C. and about 98° C. 9.The method according to claim 1, wherein a restriction enzyme is used that cuts the HBV DNA at a site that is conserved among the different genotypes of HBV.","10.The method according to claim 1, wherein the restriction site is located in the part of the HBV genome that encodes the surface antigen.","11.The method according to claim 10, wherein the restriction site is an XbaI site located at nucleotides 247-252 according to the EcoRI site, the BssSI site located at nucleotides 253-258 according to the EcoRI site or an AvrII site located at nucleotides 178-183 according to the EcoRI site, and the restriction enzyme used is the XbaI, BssSI or AvrII restriction enzyme.","12.The method according to claim 1, wherein the restriction primer is an oligonucleotide containing from about 10 to about 35 nucleotides which hybridize with the HBV target and comprise at least 10 nucleotides flanking the XbaI site located at nucleotides 247-252 according to the EcoRI site, the BssSI site located at nucleotides 253-258 according to the EcoRI site or the AvrII site located at nucleotides 178-183 according to the EcoRI site.","13.The method according to claim 12, wherein the restriction primer has the oligonucleotide sequence of SEQ ID No.","8 or SEQ ID No 9.14.The method according to claim 1, wherein the promoter or forward primer is an oligonucleotide containing from about 10 to about 35 nucleotides which hybridize with the HBV target and comprising at least 10 nucleotides, counted from the cleavage site, of the nucleotide sequence of SEQ ID No 10 in combination with the restriction enzyme XbaI, or of SEQ ID No 11 in combination with BssSI or of SEQ ID No 12 in combination with AvrII, linked to a promoter sequence.","15.The method according to claim 14, wherein the promoter oligonucleotide has the nucleotide sequence of SEQ ID No 1, SEQ ID No 2, or SEQ ID No.","3.16.The method according to claim 1, wherein the amplified HBV nucleic acid is additionally detected using a hybridization probe comprising an oligonucleotide sequence containing from about 10 to about 35 nucleotides which hybridize with the amplified HBV target and comprise at least 10 nucleotides of SEQ ID No.","13 or SEQ ID No 14.17.The method according to claim 16, wherein the probe has the oligonucleotide sequence of SEQ ID No 6 or SEQ ID No 7.18.The method according to claim 1 wherein the second or reverse primer is an oligonucleotide containing from about 10 to about 35 nucleotides and comprising at least 10 nucleotides of the nucleotide sequence of SEQ ID No.","4 or SEQ ID No.","5.19.An oligonucleotide with an nucleotide sequence selected from the group consisting of SEQ ID No 1, SEQ ID No.","2, SEQ ID No.","3, SEQ ID No.","4, SEQ ID No.","5, SEQ ID No.","6, SEQ ID No.","7, SEQ ID No.","8, SEQ ID No.","9, SEQ ID No.","10, SEQ ID No.","11, SEQ ID No.","12, SEQ ID No.","13, and SEQ ID No.","14.20.A set of oligonucleotide primers suitable for use in the amplification of HBV nucleic acid according to the method of claim 1 comprising a promoter primer, wherein the promoter primer is an oligonucleotide containing from about 10 to about 35 nucleotides which hybridize with the HBV target and comprising at least 10 nucleotides, counted from the cleavage site, of the nucleotide sequence of SEQ ID No.","10 in combination with the restriction enzyme XbaI, or of SEQ ID No.","11 in combination with BssSI or of SEQ ID No.","12 in combination with AvrII, linked to a promoter sequence and a reverse primer, wherein the reverse primer is an oligonucleotide containing from about 10 to about 35 nucleotides and comprising at least 10 nucleotides of the nucleotide sequence of SEQ ID No.","4 or SEQ ID No.","5.21.A test kit suitable for carrying out the transcription based amplification and detection of HBV DNA according to claim 1 comprising a restriction enzyme capable of cleaving the HBV DNA at a selected restriction site, said restriction enzyme creating a defined 3′ end of the said HBV DNA strand(s), a forward primer that corresponds with the cleavage site of the restriction enzyme chosen, said forward primer having a 5′ region comprising the sequence of a promoter recognized by a DNA-dependent RNA polymerase and a 3′ region complementary to the defined 3′ end of the DNA strand, and is provided with a promoter sequence, other reagents for carrying out a transcription based amplification reaction, and means for detecting the amplified HBV DNA.","22.The method according to claim 1, in which the incubation temperature is from about 37° C. to about 41° C. 23.The method according to claim 1, in which the heating step is carried out at a temperature of about 95° C. 24.The method according to claim 1, wherein the restriction primer is an oligonucleotide containing from about 15 to about 30 nucleotides which hybridize with the HBV target and comprise at least 15 nucleotides flanking the XbaI site located at nucleotides 247-252 according to the EcoRI site, the BssSI site located at nucleotides 253-258 according to the EcoRI site or the AvrII site located at nucleotides 178-183 according to the EcoRI site.","25.The method according to claim 1, wherein the promoter or forward primer is an oligonucleotide containing from about 15 to about 30 nucleotides which hybridize with the HBV target and comprising at least 15 nucleotides, counted from the cleavage site, of the nucleotide sequence of SEQ ID No.","10 in combination with the restriction enzyme XbaI, or of SEQ ID No.","11 in combination with BssSI or of SEQ ID No.","12 in combination with AvrII, linked to a promoter sequence.","26.The method according to claim 1, wherein the amplified HBV nucleic acid is additionally detected using a hybridization probe comprising an oligonucleotide sequence containing from about 15 to about 30 nucleotides which hybridize with the amplified HBV target and comprise at least 15 nucleotides of SEQ ID No.","13 or SEQ ID No.","14.27.The method according to claim 1, wherein the second or reverse primer is an oligonucleotide containing from about 15 to about 30 nucleotides and comprising at least 15 nucleotides of the nucleotide sequence of SEQ ID No.","4 or SEQ ID No.","5.28.A set of oligonucleotide primers comprising a promoter primer, wherein the promoter primer is an oligonucleotide containing from about 15 to about 30 nucleotides which hybridize with the HBV target and comprising at least 15 nucleotides, counted from the cleavage site, of the nucleotide sequence of SEQ ID No.","10 in combination with the restriction enzyme XbaI, or of SEQ ID No.","11 in combination with BssSI or of SEQ ID No.","12 in combination with AvrII, linked to a promoter sequence and a reverse primer, wherein the reverse primer is an oligonucleotide containing from about 15 to about 30 nucleotides and comprising at least 15 nucleotides of the nucleotide sequence of SEQ ID No.","4 or SEQ ID No.","5."],["<SOH> BRIEF DESCRIPTION OF THE FIGURES <EOH>FIG.","1 : Schematic presentation of DNA NASBA including restriction enzyme digestion.","The restriction enzyme (arrow) is only active during the initiation phase of NASBA.","After digestion, the forward primer is hybridized to the template.","AMV RT will extend the 3′ end of the target strand (black) of the DNA, using the forward primer, including the T7 promoter sequence (dark grey) as template.","The T7 DdRp will recognize the double stranded T7 promoter sequence and RNA amplicon (light grey) production will begin.","The RNA amplicon sequence is complementary to the target DNA strand.","During the cyclic phase, the RNA amplicon will be amplified and detected by molecular beacon technology.","RNase H and the reverse primer are only required during the cyclic phase.","FIG.","2 : NASBA of HBV DNA with and without digestion with XbaI in combination with forward primer S-p3.8.After digestion with XbaI, S-p3.8 can be used as template for the extension of target DNA.","Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.","A sample without template (NT) is used as negative control.","FIG.","3 : NASBA of HBV DNA with and without digestion with BssSI in combination with forward primer S-p3.10.After digestion with BssSI, S-p3.10 can be used as template for the extension of target DNA.","Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.","A sample without template (NT) is used as negative control.","FIG.","4 .","NASBA of HBV DNA with and without digestion with XbaI in combination with forward primer S-p3.10.S-p3.10 can not be used as template for the extension of target DNA, after digestion with XbaI.","Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.","A sample without template (NT) is used as negative control.","FIG.","5 .","NASBA of HBV DNA with and without digestion with BssSI in combination with forward primer S-p3.8.S-p3.8 can be used as template for the extension of target DNA, after digestion with BssSI.","Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.","A sample without template (NT) is used as negative control.","FIG.","6 .","NASBA of HBV DNA with and without digestion with AvrII in combination with forward primer S-p3.5.S-p3.5 can be used as template for the extension of target DNA, after digestion with AvrII.","Primer S-p4.4 is used as reverse primer and molecular beacon S-WT4 as probe.","A sample without template (NT) is used as negative control.","The invention is further exemplified by the following Examples.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["The present invention is directed to a transcription based amplification method for the amplification of HBV DNA.","Nucleic acid amplification methods are used in the field of molecular biology and recombinant DNA technology.","These methods are used to increase the number of copies of a particular nucleic acid sequence, present in small amounts and often in an environment in which a wide variety of other nucleic acid sequences, both RNA and DNA, are also present.","In particular, nucleic acid amplification methods are used to facilitate the detection or quantification of nucleic acid and are important for diagnosing for example infectious diseases, inherited diseases and various types of cancer.","Nucleic acid amplification methods have also found their applications in other fields where samples are investigated in which nucleic acid may be present in minute amounts, such as forensic sciences, archeology or to establish paternity.","Several nucleic acid amplification techniques are known based on different mechanisms of action.","One method for the amplification of nucleic add is known as the “Polymerase Chain Reaction” (PCR) is described in European patent applications EP 200362 and EP 201148.The present invention is concerned with a different class of nudeic acid amplification methods namely the “transcription based amplification techniques”.","With these methods multiple RNA copies are obtained from a DNA template that comprises a functional promoter recognized by the RNA polymerase.","Said RNA copies are used as target from which new DNA templates are obtained etc.","Gingeras et al.","in WO88/10315 and Burg et al.","in WO89/1050 have described such methods.","Isothermal transcription based amplification techniques have been described by Davey et al.","in EP 323822 (relating to the NASBA method), by Gingeras et al.","in EP 373960 and by Kacian et al.","in EP 408295.Transcription based amplification reactions may also be performed with thermostable enzymes.","Transcription based amplifications are usually carried out at a temperature around 41 degrees Celsius.","Thermostable enzymes allow the reaction to be carried out at more elevated temperatures.","Such a thermostable method is described in EP 682121 filed in the name of Toyo Boseki KK.","The methods as described in EP 323822, EP 373960 and EP 408295 are isothermal continuous methods.","With these methods four enzyme activities are required to achieve amplification: an RNA dependent DNA polymerase activity, an DNA dependent DNA polymerase activity, an RNase (H) activity and an RNA polymerase activity.","Some of these activities can be combined in one enzyme, so usually only 2 or 3 enzymes are necessary.","Reverse transcriptase such as AMV (Avian Myoblastosis Virus) or MMLV (Moloney Murine Leukemia Virus) reverse transcriptase have both RNA- and DNA dependent DNA polymerase activity but also an inherent RNase H activity.","In addition an RNase may be added to the reaction mixture of a transcription based amplification reaction, such as E. coli RNase H. DNA dependent RNA polymerases synthesize multiple RNA copies from a DNA template including a promoter recognized by the RNA polymerase.","Examples of RNA polymerases are polymerases from E. coli and bacteriophages T7, T3 and SP6.An example of an RNA polymerase commonly used with transcription based amplification methods is T7 polymerase.","Thus the promoter that is incorporated in the template used to produce multiple copies of RNA would then be the T7-promoter.","Usually the template comprising the promoter has to be created starting from the nucleic acid comprising the target sequence.","Said nucleic acid may be present in the starting material that is used as input for the amplification reaction.","The nucleic acid present in the starting material will usually contain the target sequence as part of a much longer sequence.","Additional nucleic acid sequences may be present on both the 3′- and the 5′-end of the target sequence.","The amplification reaction can be started by bringing together this nucleic acid from the starting material, the appropriate enzymes that together provide the above mentioned activities and at least one, but usually two, oligonucleotide(s).","At least one of these oligonucleotides should comprise the sequence of the RNA polymerase promoter.","Transcription based amplification methods are particularly useful if the input material is single stranded RNA, although single or double stranded DNA can likewise be used as input material.","When a transcription based amplification method is practiced on a sample with single stranded RNA with additional sequences on both the 3′-end and the 5′ end of the target sequence a pair of oligonucleotides that is conveniently used with the methods as described in the prior art would consist of: a first oligonucleotide (usually referred to as “promoter-primer” or “forward-primer”) that is capable of hybridizing to the 3′-end of the target sequence, which oligonucleotide has the sequence of a promoter (preferably the T7 promoter) attached to its 5′ end (the hybridizing part of this oligonucleotide has the opposite polarity as the target RNA used as input material).","a second oligonucleotide (usually referred to as “reverse primer”) which comprises the 5′ end of the target sequence (this oligonucleotide has the same polarity as the target RNA).","When such a pair of oligonucleotides, together with all enzymes having the appropriate activities, and a sufficient supply of the necessary ribonucleotides and deoxy-ribonucleotides are put together in one reaction mixture and are kept under the appropriate conditions (that is, under the appropriate buffer conditions and at the appropriate temperature) for a sufficient period of time an isothermal continuous amplification reaction will start.","Many variants of the above theme have been described in the prior art.","A transcription based amplification reaction comprises the synthesis of single stranded RNA transcripts from a template comprising a promoter (e.g.","the T7 promoter) that is recognized by an RNA polymerase (e.g.","T7 RNA polymerase).","A forward primer, comprising the promoter sequence, serves as a primer to initiate the synthesis of a strand of DNA complementary to the target RNA.","The primer will be extended by the activity of RNA dependent DNA polymerase.","The RNA-cDNA hybrid formed will be degraded by RNase H. This enables the hybridization of the specific reverse primer to the cDNA.","Extension of this primer by RNA dependent DNA polymerase up to the 5′ end of the cDNA results in the formation of a double-stranded promoter sequence, whereby the promoter sequence that was part of the forward primer is used as a template.","This double stranded promoter will then be used by the DNA dependent RNA polymerase to produce many new RNA molecules that are complementary to the target RNA.","After this initiation phase, the amplification enters a cyclic phase.","In practice, the whole sequence of events, starting from the single stranded RNA in the sample, will take place as soon as all ingredients are put together, and the mixture is brought to the appropriate temperature for the enzymes to be all active.","The practitioner of the method need not to intervene to accomplish any of these steps.","As explained above, transcription based amplification methods are particularly useful for amplifications that start from single stranded RNA.","The starting material containing the nucleic acid to be amplified may not contain the target nucleic acid as RNA of a defined length.","When a transcription based amplification method is performed on starting material comprising the target sequence only as double stranded DNA, either circular or linear, the DNA would have to be converted to single stranded nucleic acid.","This can be achieved by separating the strands of the double stranded DNA by applying an elevated temperature (up to a 100 degrees Celsius).","The first of the oligonucleotides used as primers in the amplification may than anneal to one of the single strands.","The enzymes used with current transcription based amplification methods cannot withstand such a high temperature and consequently can only be added after the DNA strands have been separated.","When one of the oligonucleotides anneals to a single strand DNA and is elongated, double stranded DNA is created again, and the reaction mixture would have to be subjected to an elevated temperature sufficiently high to melt the double stranded DNA into its separated strands again.","Again the enzymes would be inactivated and new enzymes are to be added after the heat step has been applied.","The second oligonucleotide can now be added and anneal to the strand that was created from the elongated first oligonucleotide in the first step.","As one of the oligonudeotides includes a 5′ promoter sequence of a DNA dependent RNA polymerase (see above), a double stranded DNA template including a double stranded functional promoter is obtained, from which a first step of RNA production can take place.","The resulting RNA transcripts may enter the cyclic phase of the amplification and the process can further be isothermal.","From the above it is evident that starting a transcription based amplification method from double stranded DNA can be a tedious process.","It requires several specific actions to be taken by the practitioner, the sample has to be heated and cooled repeatedly and enzymes have to be replenished after each heating step.","Some research has already gone into the developments of transcription based amplification methods that can start from dsDNA, avoiding the tedious procedure described above to convert the dsDNA into ssRNA that can be used as input for the A rather simple transcription based amplification method for dsDNA has been disclosed in WO9925868.According to the method described in WO9925868 dsDNA in a sample can be amplified by means of a transcription based amplification protocol directly, without any heat treatment step [of over 90 C] at all, or —in a preferred embodiment—with only one initial heating step dsDNA, that is relatively short, is to be preferred in this method.","Actually, the method does not differ essentially from a conventional transcription based amplification protocol to amplify ssRNA.","Alternatively, the double stranded DNA in the starting material can be transcribed into RNA before the start of the amplification.","Such an extra step can be based on an enzyme, for instance E. coli RNA polymerase, that transcribes the double stranded DNA into RNA without the presence of a promoter sequence, also referred to as a polymerase binding site.","Such a process of extra steps to facilitate the amplification of double stranded DNA by transcription based amplification methods has been described in PCT patent application no.","WO9602668.The extra steps described in this procedure do not only include extra handling steps and handling time, but also the use of additional ingredients, i.e.","the E. coli RNA polymerase.","Another way of preparing suitable templates for transcription based amplification methods for dsDNA is described in EP 397269.In this patent a method is described whereby dsDNA is pretreated with a restriction enzyme.","After treatment with the restriction enzyme only one heat separation step is needed to create single stranded DNA (ssDNA).","With this method a forward primer (promoter-primer) is used that has a 3′ part including a sequence that is complementary to the exact 3′ end of one of the single strands of DNA and a 5′ end including a promoter sequence recognized by a RNA polymerase (for example T7 RNA polymerase).","When the promoter-primer is hybridized to the 3′ end of the single strand of DNA a double stranded complex Is formed, of which the 5′ promoter sequence of the forward primer can serve as a template for an elongation reaction starting from the 3′ end of the DNA strand.","Thus, a double stranded promoter is formed by a DNA dependent DNA polymerase and the resulting complex can serve as template for the DNA dependent RNA polymerase to synthesize multiple copies of RNA.","In WO9104340 also several methods are disclosed to start a transcription based amplification reaction for single stranded DNA.","Again, a restriction enzyme may be used to create an appropriate 3′ end on the DNA, which can hybridize with a 3′ sequence of a promoter primer.","In WO9104340 it is disdosed how the defined 3′ end on the ssDNA may be created using a restriction enzyme that cuts ssDNA.","In another embodiment of the same method, a restriction enzyme is used that cuts dsDNA, together with a restriction oligonucleotide that hybridizes to the target ssDNA and thus creates a double stranded piece of DNA that can be cut by the restriction enzyme to create the appropriate 3′ end.","With this method a small piece of the restriction oligonucleotide will remain after the restriction enzyme has cut the double stranded complex.","However, according to the disclosure of WO9104340, the restriction oligonucleotide is apparently chosen in such a way that after digestion, the remaining piece will be to small to stay hybridized to the 3′ end of the ssDNA, and thus will fall of to make room for the promoter oligonucleotide.","However, the pre-treatment with a restriction enzyme as used with the prior art methods, although it may result in a sensitive transcription based assay, require many extra handling steps and handling time.","Hepatitis B virus (HBV) infection in humans is widespread.","The virus that causes hepatitis B appears to infect only humans and chimpanzees.","The hepatitis infection is transmitted by three general mechanisms: (1) by parenteral inoculation of infected blood or body fluids, either in large amounts as in blood transfusions or in minute amounts as through an accidental skinprick; (2) by close family or sexual contact; and (3) by some mothers, who transmit the virus to their new-bom children.","Under natural conditions, HBV is not highly contagious.","Transmission by inhalation occurs rarely, if ever.","The transmission route through contaminated blood or blood products is a major threat to the human health.","Infection with HBV often results in subclinical or acute self-limited liver disease or can result in chronic long-term infection.","Chronic HBV infection elicits a spectrum of disease entities ranging from the most severe form of chronic active hepatitis to less severe chronic persistent hepatitis to the asymptomatic carrier state.","An array of diagnostic assays have recently been developed to aid the clinician in differentiating hepatitis B virus infections from other forms of viral hepatitis (i.e., hepatitis A, hepatitis C or hepatitis E).","However, the ability to distinguish between an acute hepatitis B infection and symptomatic chronic hepatitis B infection is still problematic.","This is especially true since chronic active hepatitis and chronic persistent hepatitis patients often demonstrate a cyclic pattern of hepatitis characterized by acute exacerbation of liver injury alternating with normal liver function.","After infection with HBV, large quantities of the virus and associated particles are present in the serum.","During symptomatic phases of infection, both acute and chronic HBV patients have elevated liver enzyme levels, possess the hepatitis B surface antigen (HBsAg) in their serum, and produce antibodies to the nucleocapsid antigen (HBcAg).","Antibodies specific for the HBsAg or the hepatitis B e antigen (HBeAg) are not detected.","The appearance of antibody to HBsAg is usually not observed until approximately two months following disappearance of circulating HBsAg.","The viral particles present in the serum are known to shed their surface coat exposing the nucleocapsid, known as the core antigen (HBcAg).","Antibody production of HBcAg occurs early in the course of the acute phase of HBV infection and can persist for many years, and chronically infected patients can produce high titers of anti-HBc antibodies.","HBsAg is established as the most important marker of acute or chronic hepatitis B infection, detectable in serum of infected individuals.","HBsAg screening of donor blood for example, is essential to avoid transmission of hepatitis B.","It is clear that sensitivity is of utmost importance in diagnostic HBV assays.","HBV is the smallest DNA virus known; and its genome shows a highly compacted organization.","A unique aspect in the replication cycle of HBV is that a pre-genomic mRNA serves as a template for the synthesis of the first viral DNA strand.","The RNAse H activity of the HBV DNA polymerase subsequently removes the mRNA and the complementary DNA strand is then synthesized, generating a partially double stranded DNA molecule for packaging in virions.","Upon successful virus entry, the partially double stranded DNA molecule is converted into a fully double stranded DNA molecule.","HBV DNA can be detected in the blood of infected hosts who are HBsAg and HBeAg positive in more than 90% of cases.","The state of the art method of measuring the quantity of infectious particles is by measuring the quantity of viral DNA in serum or plasma, because it most reliably reflects the amount of replicating virus.","Several assays are available for this purpose, such as the branched DNA (bDNA) assay (Hendricks D A, Stowe B J, Hoo B S, Kolberg J, Irvine B D, Neuwald P D, Urdea M S, Perrillo R P, 1995.Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay.","Am J Clin Pathol 104: 537-546.","), DNA hybridization assays and quantitative PCR (Pawlotsky J M, Bastie A, Lonjon I, Remire J, Darthuy F, Soussy C J, Dhumeaux D, 1997.What technique should be used for routine detection and quantification of HBV DNA in clinical samples?","Journal of Virological Methods 65: 245-253; Zaaijer H L, ter Borg F, Cuypers H T, Hermus M C, Lelie P N, 1994.Comparison of methods for detection of hepatitis B virus DNA.","J Clin Microbiol 32: 2088-2091.).","Most of these assays, however, have only limited sensitivity.","The present invention is concerned with a transcription based amplification method including a restriction enzyme digestion.","The method enables a sensitive and specific amplification (and subsequent detection) of DNA of the Hepatitis B virus.","With the method of the invention HBV DNA can be amplified and detected in a more efficient way than with prior art transcription based amplification methods in contrast to the prior art methods the use of the restriction enzyme does not complicate the procedures used method of the invention.","The present invention provides a method for the transcription based amplification of a target HBV nucleic acid sequence starting from HBV DNA optionally present in a sample, comprising the steps of, incubating the sample, suspected to contain HBV, in an amplification buffer with one or more restriction enzymes capable of cleaving the DNA at a selected restriction site, said restriction enzyme creating a defined 3′ end on one of the DNA strands, and a promoter-primer, said promoter-primer having a 5′ region comprising the sequence of a promoter recognized by a DNA-dependent RNA polymerase and a 3′ region complementary to the defined 3′ end of the DNA strand, a second primer, having the opposite polarity of the promoter-primer and comprising the 5′ end of the target sequence, and in case of a HBV ssDNA, a restriction primer, maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for a digestion by the restriction enzyme to take place, subjecting the sample to a heat treatment at a temperature and time sufficient to inactivate the restriction enzyme and/or to render at least partially a double strand single stranded, adding the following reagents to the sample: an enzyme having RNA dependent DNA polymerase activity an enzyme having DNA dependent DNA polymerase activity an enzyme having RNase H activity an enzyme having RNA polymerase activity, and maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for the amplification to take place.","The [for an adequate amplification] necessary (appropriate) nucleoside triphosphates may be present already during the incubation step with the restriction enzyme(s), for example as part of the said amplification buffer.","They may, however, be added later on in the process, for example together with the enzymes after the heat treatment.","The person skilled in the art knows the enzymes used for the transcription based amplification method, and the conditions under which the transcription based amplification method is carried out and is aware of all the usual modifications that can be made with regard to optimizing transcription based amplification reactions.","For example, the forward primer, the promoter primer, may comprise a purine region between the promoter sequence on the 5′ end of the primer and the hybridizing sequence on the 3′ end of the primer.","The sequence of the primers is largely determined by the position of the restriction site chosen.","The 3′ end of a forward primer should anneal to the target sequence directly next to the restriction site.","The primer may vary in length as long as it is sufficiently long to hybridize under the conditions used with the amplification reaction.","In general the hybridizing part of the primer consists of about 10 to about 35 nucleotides.","Restriction primers, used in the method according to the invention if the target is HBV ssDNA, require that the overlap they show with the forward primer is minimal and the sequence of the restriction site is incorporated in such a way that the restriction enzyme actually cuts the DNA efficiently.","A restriction enzyme is an enzyme that can cut dsDNA at a selected site (i.e.","a specific nucleotide sequence recognized by the enzyme).","In selecting an appropriate restriction enzyme for the method of the invention care should be taken that a restriction site is chosen that is present in al variants of the HBV DNA (for example, a restriction site that is present in all genotypes of the hepatitis B virus, if the amplification is carried out to detect all viral HBV DNA in the sample).","The restriction site should not be present in the DNA sequence in between the forward and reverse primers used.","The addition of the restriction enzyme results in the creation of a defined 3′ end of the target strand of the DNA, which is then available for binding to the hybridizing part of the promoter primer.","An additional aspect is that, because of the digestion, denaturation of that part of the DNA will be improved and so primer binding will be facilitated.","The promoter oligonucleotide containing the T7-promoter sequence should be designed in such a way that the hybridizing part will interact with the template directly upstream of the restriction site.","The enzyme having DNA dependent DNA polymerase activity (usually a reverse transcriptase such as MMLV-RT or AMV-RT) can extend the 3′ end of the target strand of the DNA created by the digestion with the restriction enzyme, using the primer as template.","A double-stranded T7-promoter sequence will be formed and the production of amplicon RNA can start.","Surprisingly it has been found that a restriction enzyme can be used efficiently in an environment that is suitable for and adapted to a transcription based HBV DNA amplification process.","In other words it has been found that the use of a restriction enzyme to cut the HBV DNA that is used as input material for a transcription based amplification, does not have to lead to complicated, additional handling of the sample.","The use of the restriction enzyme is incorporated into and is part of the steps that are usually already part of the protocol for a transcription based DNA amplification.","All prior art methods describe the use of a restriction enzyme in the preparation of a DNA template for transcription based amplification as a separate pre-treatment prior to the actual transcription based amplification.","Consequently the prior art use of a restriction enzyme in preparing the DNA template resulted in additional handling of the sample, like separate inactivation of the restriction enzyme and separate purification of the DNA.","It complicates the whole amplification procedure, especially an automated process, and increases the risk of contamination.","Although the addition of a restriction oligonucleotide together with a restriction enzyme has already been disclosed in WO9104340, it has not been disdosed prior to the present invention how the use of the restriction enzyme (and oligonucleotide) can efficiently be combined with the transcription based amplification.","It has not been disclosed in the prior art in which way the use of restriction enzyme can be combined with transcription based amplification without additional sample handling and reagent adding steps.","The method of the invention provides this combination without complicating the prior art transcription based DNA amplification process.","The method of the invention hardly differs from a normal transcription based amplification method.","The only additional step to be taken is the “built in incubation” of the sample with the restriction enzyme, which means that the restriction enzyme is used in such a way that the actual handling of the sample does not differ from a conventional/prior art transcription based DNA amplification process.","The preferred restriction enzyme used in the method of the invention is, of course, an enzyme that is relatively stable and retains a high activity under conditions where it is added to a reaction mixture comprising an amplification-buffer (which contains relatively high concentrations of salts).","After the addition of the restriction enzyme the sample needs to be incubated under the appropriate conditions for the enzyme to be active, and for a suitable amount of time.","The sample may be incubated with the restriction enzyme for a relatively short period of time, preferably for about 10-20 minutes and more preferred for about 15 minutes at a temperature of about 35 to about 45° C. and more specifically at about 37-41° C., obviously depending on the nature of the restriction enzyme used.","In fact, this is the only additional measure to be taken, when compared to a conventional transcription based DNA amplification method.","The method of the invention comprises the step of heating the sample after the incubation with the restriction enzyme.","During this heating the restriction enzyme is inactivated and double stranded DNA is rendered single stranded [at least partially].","This heating step is already part of the protocols for carrying out a transcription based amplification method.","These methods involve a heat treatment of the sample after primer-addition, to create optimal circumstances for primer annealing (the nucleic acid is stretched, strands or internal loops of the nucleic acid are separated, and during the cooling down, hybridization of the primers to the template is facilitated.)","The heating after the incubation with the enzyme may be done at a lower temperature of about 50° C. or higher, but is preferably carried out by way of a short incubation (about 5-10 minutes) at a temperature above 90° C. and preferably at 95+/−3° C. Thereafter the sample may be cooled to the appropriate temperature for a transcription based amplification reaction to take place (usually about b 41° C.).","Due to the heating, at least a part of the double stranded DNA is rendered single stranded.","As the primers, especially the promoter oligonucleotide, are already present prior to the heating of the sample, the heat treatment may facilitate primer annealing to DNA as well.","Thus, there is no need to purify the DNA from the sample after it has been subjected to a treatment with the restriction enzyme.","The enzyme is simply inactivated in the heat treatment that was already part of the transcription based amplification procedure.","It has been proven with the method of the invention that this is sufficient to eliminate the risk that the restriction enzyme will interfere with the actual transcription based amplification reaction.","After the heat treatment the additional amplification reagents for the transcription-based amplification are added in the usual way, and the transcription based amplification can be carried out in the usual way known to the skilled person The amplification enzymes are only added after the heat treatment, to prevent degradation of the enzymes during the heat treatment (unless, of course, thermostable enzymes are used).","The major advantage of the method of the invention is that, even though that additional reagents are used (e.g.","the restriction enzyme) this does not result in additional (separate) reaction steps or activities to be carried out.","The fact that no additional handling of the sample is required is especially important because every additional handling of the sample would increase the contamination risk, which is to be avoided at all costs, especially in amplification reactions.","Moreover, if additional sample handling steps were required this would complicate automation of the method.","The method of the invention may also be used for single stranded DNA.","When the DNA is single stranded a restriction oligonucleotide or restriction primer comprising a sequence complementary to the region including the restriction site of the target DNA is added together with the restriction enzyme.","The restriction oligonucleotide [restriction primer] hybridizes with the single stranded DNA and forms a double stranded complex that can be cut with the restriction enzyme.","The addition of yet another reagent (the restriction oligonucleotide) does not result in additional steps to be carried out by the practitioner.","The restriction oligonucleotide can simply be added together with the restriction enzyme and the other oligonucleotides necessary for the amplification.","Thus, there is no need to open the amplification system for yet another addition of reagents.","In a preferred embodiment of the invention, the function of the restriction oligonucleotide may be incorporated in the oligonucleotide that also comprises the sequence of a promoter recognized by a DNA dependent RNA polymerase (the combined promoter and restriction-primer).","In this way only two oligo's are needed for the amplification, a promoter [or forward] primer in which a sequence complementary to the target region including the restriction site has been incorporated and the second [or reverse] primer.","The sequence including the restriction site of this preferred [combined promoter and restriction] primer should preferably be allocated in such a way that: after digestion, the remaining part of the primer will denature from the target during the heating step the remaining part of the hybridizing sequence of the target is long enough for a new combined promoter and restriction primer to bind, Extra nucleotides surrounding the restriction site are included in the hybridization if necessary for the activity of the restriction enzyme.","Thus, a part of the combined promoter and restriction primer will now serve as restriction oligonucleotide; it will anneal to the target DNA, resulting in dsDNA comprising the restriction site recognized by the restriction enzyme.","Subsequently the restriction enzyme will cut the said dsDNA, thus providing the defined 3′ end on the DNA.","Preferably, an at least 1000 fold excess of this combined promoter and restriction primer with respect to the amount of target DNA should be present, as is also usual already for a conventional promoter primer in transcription based amplification reactions.","The method of the invention provides an efficient and sensitive method for the amplification and detection of viral DNA in samples suspected to contain the hepatitis B virus (HBV), especially with the HBV primers and HBV probes as described herein.","Hence, these HBV primers and HBV probes represent another embodiment of the invention, The restriction site that is selected in the method of the invention is preferably one that is conserved among the different genotypes of HBV.","Good results were obtained if the target sequence and restriction site were located in the part of the HBV genome that encodes the surface antigen.","Conserved restriction sites in the HBsAg coding region that are especially useful in the method of the invention (together with the restriction enzymes that cut at these sites) are the XbaI site located at nucleotides 247-252 according to the EcoRI site, the BssSI site located at nucleotides 253-258 according to the EcoRI site and the AvrII site located at nucleotides 178-183 according to the EcoRI site.","The oligonucleotide sequence of the primers is, of course, largely determined by the position of the restriction site chosen.","The primer may vary in length as long as it is sufficiently long to hybridize under the conditions used with the amplification reaction.","In general the hybridizing part of the primer consists of about 10 to about 35 nucleotides and more preferably of about 15 to about 30 nucleotides.","The 5′ end of a forward primer should anneal to the target sequence directly next to the restriction site.","The position of the reverse primer is less critical, preferably it should have the sequence of a part of a conserved region sufficiently remote from the position of the forward primer to allow a probe to hybridize to the amplified target sequence in the region in between the forward and reverse primer region.","The preferred restriction primer is an oligonucleotide that has a minimal overlap with the forward primer, includes the sequence of the restriction site in such a way that the restriction enzyme can actually cut the dsDNA thus formed and is sufficiently long so as to hybridize with the HBV DNA in a sufficient manner.","Oligonucleotide forward primers which are especially useful in combination with the XbaI, AvrII and/or BssSI restriction enzymes are preferred embodiments of the invention.","Specifically, oligonucleotide sequences comprising at least 10 and preferably more than 15 nucleotides [counted from the cleavage site] of the HBV hybridizing part of SEQ ID No 1 [said hybridizing part as such being SEQ ID No 10] in combination with the restriction enzyme XbaI, of the HBV hybridizing part of SEQ ID No 2 [said hybridizing part as such being SEQ ID No 11] in combination with BssSI and/or of the hybridizing part of SEQ ID No 3 [said hybridizing part as such being SEQ ID No 12] in combination with Avril are preferred.","Oligonucleotides which can be used as restriction primers, comprise at least 10 and preferably more than 15 nucleotides “flanking” the HBV DNA restriction site concerned and especially “flanking” the XbaI site located at nucleotides 247-252 according to the EcoRI site, the BssSI site located at nucleotides 253-258 according to the EcoRI site and the AvrII site located at nudeotides 178-183 according to the EcoRI site.","Particularly suitable restriction primers have the oligonucleotide sequences depicted in SEQ ID No.","8 and SEQ ID No 9.Suitable probes for the detection of the amplified HBV target contain from 10 to about 35 and more preferably from about 15 to about 30 nucleotides which hybridize with the amplified HBV target and comprise at least 10 and preferably more than 15 nucleotides of the HBV hybridizing part of SEQ ID No 6 and SEQ ID No 7.","(The HBV hybridizing parts of these sequences are shown in Table 1 and indicated as SEQ ID No 13 and SEQ ID No 14 respectively.","The probes shown in Table 1 are further provided at both ends with non-HBV nucleotides forming entirely or sometimes together with a few HBV nucleotides the “stem” of the probe; said stern being part of the detection system chosen).","Suitable reverse primers contain from about 10 to about 35 and more preferably from about 15 to about 30 nucleotides of a conserved region of HBV.","Preferred reverse primers comprise at least 10 and preferably more than 15 nucleotides of SEQ ID No 4 and SEQ ID No 5.Another embodiment of the invention is a test kit suitable for carrying out the transcription based amplification and detection of HBV DNA according to the invention comprising a restriction enzyme as herein described, a forward primer that corresponds with the cleavage site of the restriction enzyme chosen, as herein explained, and Is provided with a promoter sequence, other reagents for carrying out a transcription based amplification reaction, means for detecting the amplified HBV DNA, and instructions for use.","BRIEF DESCRIPTION OF THE FIGURES FIG.","1: Schematic presentation of DNA NASBA including restriction enzyme digestion.","The restriction enzyme (arrow) is only active during the initiation phase of NASBA.","After digestion, the forward primer is hybridized to the template.","AMV RT will extend the 3′ end of the target strand (black) of the DNA, using the forward primer, including the T7 promoter sequence (dark grey) as template.","The T7 DdRp will recognize the double stranded T7 promoter sequence and RNA amplicon (light grey) production will begin.","The RNA amplicon sequence is complementary to the target DNA strand.","During the cyclic phase, the RNA amplicon will be amplified and detected by molecular beacon technology.","RNase H and the reverse primer are only required during the cyclic phase.","FIG.","2: NASBA of HBV DNA with and without digestion with XbaI in combination with forward primer S-p3.8.After digestion with XbaI, S-p3.8 can be used as template for the extension of target DNA.","Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.","A sample without template (NT) is used as negative control.","FIG.","3: NASBA of HBV DNA with and without digestion with BssSI in combination with forward primer S-p3.10.After digestion with BssSI, S-p3.10 can be used as template for the extension of target DNA.","Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.","A sample without template (NT) is used as negative control.","FIG.","4.NASBA of HBV DNA with and without digestion with XbaI in combination with forward primer S-p3.10.S-p3.10 can not be used as template for the extension of target DNA, after digestion with XbaI.","Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.","A sample without template (NT) is used as negative control.","FIG.","5.NASBA of HBV DNA with and without digestion with BssSI in combination with forward primer S-p3.8.S-p3.8 can be used as template for the extension of target DNA, after digestion with BssSI.","Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.","A sample without template (NT) is used as negative control.","FIG.","6.NASBA of HBV DNA with and without digestion with AvrII in combination with forward primer S-p3.5.S-p3.5 can be used as template for the extension of target DNA, after digestion with AvrII.","Primer S-p4.4 is used as reverse primer and molecular beacon S-WT4 as probe.","A sample without template (NT) is used as negative control.","The invention is further exemplified by the following Examples.","EXAMPLES Example 1 Amplification of HBV DNA Two conserved restriction sites (XbaI and BssSI) are encoded in the conserved region (nt 244 to 285 according to the EcoRI-site) of the S-gen of HBV DNA.","As this part of the S-region can be sinqle-stranded DNA of negative polarity, an oligonucleotide (‘restriction primer’ (RP) complementary to the region including the restriction site sequences was added to create a double-stranded restriction site for all genomic DNAs present.","HBV DNA was isolated from a series of dilution of plasma infected with HBV genotype A of 3×109 geq/ml, using the Nuclisens Extractor (Organon Teknika).","Following the standard procedure as described for RNA isolation (Operator Manual Extractor, 41001-9, rev A, 1999), a 50 I extract is obtained.","Five I of the extract is used peassay.","The restriction enzyme digestion was performed in NASBA buffer (40 mM Tris-HCl pH 8.5,12 mM MgCl2, 70 mM KCl, 15% v/v DMSO, 5 mM DTT, 1 mM each dNTP, 2 mM ATP, 2 mM CTP, 2 mM UTP, 1.5 mM GTP, 0.5 mM ITP, 0.2 μM forward primer (S-p3.8 for XbaI, and S-p3.10 for BssSI, table 1), 0.2 μM reverse primer (S-p4.5, table 1), 0.1 μM molecular beacon probe (S-W12, table 1), 0.17 μM restriction primer (RP-3, table 1)) and 0.2 units restriction enzyme BssSI (New England BioLabs, Inc., Beverly, Mass., USA) or 3.0 units restriction enzyme XbaI (New England BioLabs, Inc., Beverly, Mass., USA).","After incubation of 15 min at 41 C, the restriction enzymes were heatinactivated and the DNA template denatured at 95 C for 5 min.","Hybridization of the primers occured during ccaling down to 41 C for 3 min.","Subsequently, NASBA enzymes (2.1 μg BSA, 0.08 units RNase H, 32 units T7 RNA polymerase and 6.4 units AMV reverse transcriptase) were added, the reaction mixture was mixed by gently tapping and short centrifugation, and the amplification and real-time detection was started.","The reaction mixture was incubated at 41° C. in the NucliSens EasyQ Analyzer (Organon Teknika) for 120 minutes with fluorescence monitoring every minute.","The reactions were excited at 485 nm and the emission signal was measured at 518 nm.","Example 1.1 Amplification of HBV DNA including XbaI Digestion A NASBA assay with and without the treatment with the restriction enzyme XbaI was performed.","The optimal concentration of XbaI was determined under NASBA conditions by digestion of 109 copies of a PCR fragment including the amplicon region of the HBV DNA, and was shown to be 3 units.","S-p3.8 is used as forward primer and can be used as template by AMV RT to extend the template DNA after digestion with XbaI.","Without digestion a sensitivity of 3×106 geq/ml is obtained while after digestion the sensitivity is 3×103 geq/ml, meaning a 1000 fold increase in sensitivity (FIG.","2).","In addition, without digestion the time to positivity (TTP) is about 16 minutes while after XbaI digestion this is about 6 min, meaning a decrease in TTP of about 10 min (FIG.","2).","Both are indications for an improved amplification reaction.","Example 1.2 Amplification of HBV DNA including BssSI Digestion A NASBA reaction, with and without treatment with the restriction enzyme BssSI was performed with the same HBV DNA extract and comparable reaction conditions as described above.","The ontimal concentration of BssSI was determined under NASBA conditions by digestion of 109 copies of a PCR fragment including the amplicon region of the HBV DNA, and was shown to be 0.2 units.","S-p3.10 is used as forward primer and can be used as template by AMV RT to extend the template DNA after digestion with BssSI.","Again significant test improvements were obtained as a result of treatment with the restriction enzyme BssSI (FIG.","3).","Without digestion a sensitivity of only 3×107 geq/ml is obtained while after digestion the sensitivity is 3×104 geq/ml, meaning again a 1000 fold increase in sensitivity (FIG.","3) as a result of the digestion.","In addition, without digestion the time to positivity (TTP) is about 21 minutes while after BssSI digestion this is about 11 min, meaning again a decrease in TUP of about 10 min (FIG.","3).","The results prove that the digestion of HBV DNA with a restriction enzyme prior to the NASBA reaction can considerably improve the amplification and so the detection of a HBV DNA Example 1.3 Amplification of HBV DNA including XbaI digestion-2 To test if the digestion by itself or the combination of the restriction enzymes with the selected primers was the basis for the improved assay results, the assay including the XbaI digestion was repeated with primer S-p3.10 instead of S-p3.8.AMV RT can not use S-p3.10 as template to extend the target sequence after digestion with XbaI.","As can be seen in FIG.","4, only a slight increase in sensitivity (10 fold) and small decrease in TTP (about 5 min, from 21 to 16 min) is obtained after digestion with XbaI in combination with S-p3.10.This indicates that the extension of the template during the initiation of NASBA is responsible for the improved results as obtained with XbaI and primer S-p3.8 and with BssSI and primer Sp3.10.Example 1.4 Amplification of HBV [NA including BssSI digesfion-2 To test if the extension of the target was indeed the basis for the improved assay results, the assay including the BssSI digestion was repeated with primer S-p3.8 instead of S-p3.10.AMV RT can use S-p3.8 as template to extend the target sequence after digestion with BssSI.","However, after digestion with BssSI, only 17 nucleotides are included in hybridization of the primer to the target sequence while normally this is about 20 nucleotides.","Despite this difference, again dear test improvements were obtained after digestion with BssSI (FIG.","5).","A double digestion can be performed with both XbaI and BssSI included in NASBA using the primers S-p3.8 and S-p4.5, restriction primer RT-3 and molecular beacon S-WT2 without loss of amplification efficiency as compared to the single digestion NASBA assays.","Example 1.5 Amplification of HBV DNA including AvrII Digestion The restriction site (AvrII) is encoded in another conserved region (nt 177 to 192 according to the EcoRI-site) of the S-gen of HBV DNA.","Forward primer S-p3.5, reverse primer S-p4.4, molecular beacon S-WT4 and restriction primer RP-1 (table 1) were as used in NASBA.","The AMV RT can extent the target strand of HBV DNA after digestion with AvrII, using S-p3.5 as template.","The same reaction conditions as described above were used.","A NASBA reaction, with and without treatment with the restriction enzyme AvrII was performed with the same HBV DNA extract as described above, using 2 units of AvrII per reaction.","Without digestion, a sensitivity of >108 geq/ml is obtained while after digestion the sensitivity is 1×105 geq/ml, meaning a >103 fold increase in sensitivity (FIG.","6) as a result of the digestion.","Again, these results prove that the digestion of HBV DNA with a restriction enzyme digestion included in the NASBA reaction can considerably improve the amplification of HBV DNA TABLE 1 Primer and probe sequences Primer/Probe Sequence Label S-p3.8 5′AATTCTAATACGACTCACTATAGGG a (SEQ ID No.","1) GACTCGTGGTGGACTTCTCTCA 3′ S-p3.10 5′AATTCTAATACGACTCACTATAGGG (SEQ ID No.","2) agaa GGTGGACTTCTCTCAATTTTC 3′ S-p3.5 5′AATTCTAATACGACTCACTATAGGG (SEQ ID No.","3) aga GGACCCCTGCTCGTGTTACAGGC 3′ S-p4.5 5′GAACCAACAAGAAGATGAGGCA 3′ (SEQ ID No.","4) S-p4.4 5′GGGACTGCGAATTTTGGCCA 3′ (SEQ ID No.","5) S-WT2 5′CGATCG AGGGACTGCGAATTTTGGC FAM (SEQ ID No.","6) CGATCG 3′ S-WT4 5′GGATCCC FAM (SEQ ID No.","7) TIGAAAATTGAGAGAAGTCCACCAC GGGATCC 3′ RP-3 5′AATACCGCAGAGTCTAGACTCGTGG 3′NH2 (SEQ ID No.","8) 3′ Xbal| BssSI RP-1 5′CATCAGGAYTCCTAGGA 3′ 3′NH2 (SEQ ID No.","9) Avrll *The 17-promoter sequence is written in italics, the purine-stretch in lower case, the stem sequence of the probe in underlined italics and the restriction sites are indicated."]],"string":"[\n [\n \"Method for the amplification and detection of hbv dna using a transcription based amplification\",\n \"The present invention provides a method for the transcription based amplification of a target HBV nucleic acid sequence starting from HBV DNA optionally present in a sample, comprising the steps of,—incubating the sample, suspected to contain HBV, in an amplification buffer with one or more restriction enzymes capable of cleaving the HBV DNA at a selected restriction site, said restriction enzyme creating a defined 3′ end of the said HBV DNA strand(s), a promotor-primer, said promotor-primer having a 5′ region comprising the sequence of a promotor recognized by a DNA-dependent RNA polymerase and a 3′ region complementary to the define 3′ end of the DNA strand, a second or reverse primer, having the opposite polarity of the promotor-primer and comprising the 5′ end of the said target sequence, and in case of HBV ssDNA as the target sequence, a restriction primer,—maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for a digestion by the restriction enzyme to take place,—subjecting the sample thus obtained to a heat treatment at a temperature and time sufficient to inactivate the restriction enzyme and/or to render at least partially a double strand single stranded,—adding the following reagents to the sample: an enzyme having RNA dependent DNA polymerase activity, an enzyme having DNA dependent DNA polymerase activity, an enzyme having Rnase H activity, an enzyme having RNA polymerase activity, and—maintaining the thus created reaction mixture under the appropriate conditions to a sufficient amount of time for the amplification to take place.\"\n ],\n [\n \"1.A method for the transcription based amplification of a target HBV nucleic acid sequence starting from HBV DNA optionally present in a sample, said method comprising: -a) incubating the sample, suspected to contain HBV, in an amplification buffer with i) one or more restriction enzymes capable of cleaving the HBV DNA at a selected restriction site, said restriction enzyme creating a defined 3′ end of the said HBV DNA strand(s), ii) a promoter-primer, said promoter-primer having a 5′ region comprising the sequence of a promoter recognized by a DNA-dependent RNA polymerase and a 3′ region complementary to the defined 3′ end of the DNA strand, iii) a second or reverse primer, having the opposite polarity of the promoter-primer and comprising the 5′ end of the said target sequence, and iv) in case of HBV ssDNA as the target sequence, a restriction primer; -b) maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for a digestion by the restriction enzyme to take place; -c) subjecting the sample thus obtained to a heat treatment at a temperature and time sufficient to inactivate the restriction enzyme and/or to render at least partially a double strand single stranded; -d) adding the following reagents to the sample: i) an enzyme having RNA dependent DNA polymerase activity ii) an enzyme having DNA dependent DNA polymerase activity iii) an enzyme having Rnase H activity iv) an enzyme having RNA polymerase activity; and e) maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for the amplification to take place.\",\n \"2.The method according to claim 1, wherein the DNA is double stranded HBV DNA.\",\n \"3.The method according to claim 1, wherein the DNA is single stranded and the promoter primer and the restriction primer are combined in using a combined promoter and restriction primer comprising a sequence complementary to the region including the restriction site of the target ssDNA and the sequence of a promoter recognized by a DNA-dependent RNA polymerase.\",\n \"4.The method according to claim 1 in which nucleoside triphosphates are added to the initial incubation mixture prior to the heat treatment.\",\n \"5.The method according to claim 1 in which a reverse transcriptase is used combining the activities of the enzyme having RNA dependent DNA polymerase activity and the enzyme having DNA dependent DNA activity.\",\n \"6.The method according to claim 1, in which a reverse transcriptase is used having inherent RNase H activity replacing 3 enzymes, namely the enzyme having RNA dependent DNA polymerase activity, the enzyme having DNA dependent DNA activity as well as the enzyme having Rnase H activity.\",\n \"7.The method according to claim 1, in which the incubation temperature is from about 35° C. to about 45° C. 8.The method according to claim 1 in which to the heating step is carried out at a temperature between about 92° C. and about 98° C. 9.The method according to claim 1, wherein a restriction enzyme is used that cuts the HBV DNA at a site that is conserved among the different genotypes of HBV.\",\n \"10.The method according to claim 1, wherein the restriction site is located in the part of the HBV genome that encodes the surface antigen.\",\n \"11.The method according to claim 10, wherein the restriction site is an XbaI site located at nucleotides 247-252 according to the EcoRI site, the BssSI site located at nucleotides 253-258 according to the EcoRI site or an AvrII site located at nucleotides 178-183 according to the EcoRI site, and the restriction enzyme used is the XbaI, BssSI or AvrII restriction enzyme.\",\n \"12.The method according to claim 1, wherein the restriction primer is an oligonucleotide containing from about 10 to about 35 nucleotides which hybridize with the HBV target and comprise at least 10 nucleotides flanking the XbaI site located at nucleotides 247-252 according to the EcoRI site, the BssSI site located at nucleotides 253-258 according to the EcoRI site or the AvrII site located at nucleotides 178-183 according to the EcoRI site.\",\n \"13.The method according to claim 12, wherein the restriction primer has the oligonucleotide sequence of SEQ ID No.\",\n \"8 or SEQ ID No 9.14.The method according to claim 1, wherein the promoter or forward primer is an oligonucleotide containing from about 10 to about 35 nucleotides which hybridize with the HBV target and comprising at least 10 nucleotides, counted from the cleavage site, of the nucleotide sequence of SEQ ID No 10 in combination with the restriction enzyme XbaI, or of SEQ ID No 11 in combination with BssSI or of SEQ ID No 12 in combination with AvrII, linked to a promoter sequence.\",\n \"15.The method according to claim 14, wherein the promoter oligonucleotide has the nucleotide sequence of SEQ ID No 1, SEQ ID No 2, or SEQ ID No.\",\n \"3.16.The method according to claim 1, wherein the amplified HBV nucleic acid is additionally detected using a hybridization probe comprising an oligonucleotide sequence containing from about 10 to about 35 nucleotides which hybridize with the amplified HBV target and comprise at least 10 nucleotides of SEQ ID No.\",\n \"13 or SEQ ID No 14.17.The method according to claim 16, wherein the probe has the oligonucleotide sequence of SEQ ID No 6 or SEQ ID No 7.18.The method according to claim 1 wherein the second or reverse primer is an oligonucleotide containing from about 10 to about 35 nucleotides and comprising at least 10 nucleotides of the nucleotide sequence of SEQ ID No.\",\n \"4 or SEQ ID No.\",\n \"5.19.An oligonucleotide with an nucleotide sequence selected from the group consisting of SEQ ID No 1, SEQ ID No.\",\n \"2, SEQ ID No.\",\n \"3, SEQ ID No.\",\n \"4, SEQ ID No.\",\n \"5, SEQ ID No.\",\n \"6, SEQ ID No.\",\n \"7, SEQ ID No.\",\n \"8, SEQ ID No.\",\n \"9, SEQ ID No.\",\n \"10, SEQ ID No.\",\n \"11, SEQ ID No.\",\n \"12, SEQ ID No.\",\n \"13, and SEQ ID No.\",\n \"14.20.A set of oligonucleotide primers suitable for use in the amplification of HBV nucleic acid according to the method of claim 1 comprising a promoter primer, wherein the promoter primer is an oligonucleotide containing from about 10 to about 35 nucleotides which hybridize with the HBV target and comprising at least 10 nucleotides, counted from the cleavage site, of the nucleotide sequence of SEQ ID No.\",\n \"10 in combination with the restriction enzyme XbaI, or of SEQ ID No.\",\n \"11 in combination with BssSI or of SEQ ID No.\",\n \"12 in combination with AvrII, linked to a promoter sequence and a reverse primer, wherein the reverse primer is an oligonucleotide containing from about 10 to about 35 nucleotides and comprising at least 10 nucleotides of the nucleotide sequence of SEQ ID No.\",\n \"4 or SEQ ID No.\",\n \"5.21.A test kit suitable for carrying out the transcription based amplification and detection of HBV DNA according to claim 1 comprising a restriction enzyme capable of cleaving the HBV DNA at a selected restriction site, said restriction enzyme creating a defined 3′ end of the said HBV DNA strand(s), a forward primer that corresponds with the cleavage site of the restriction enzyme chosen, said forward primer having a 5′ region comprising the sequence of a promoter recognized by a DNA-dependent RNA polymerase and a 3′ region complementary to the defined 3′ end of the DNA strand, and is provided with a promoter sequence, other reagents for carrying out a transcription based amplification reaction, and means for detecting the amplified HBV DNA.\",\n \"22.The method according to claim 1, in which the incubation temperature is from about 37° C. to about 41° C. 23.The method according to claim 1, in which the heating step is carried out at a temperature of about 95° C. 24.The method according to claim 1, wherein the restriction primer is an oligonucleotide containing from about 15 to about 30 nucleotides which hybridize with the HBV target and comprise at least 15 nucleotides flanking the XbaI site located at nucleotides 247-252 according to the EcoRI site, the BssSI site located at nucleotides 253-258 according to the EcoRI site or the AvrII site located at nucleotides 178-183 according to the EcoRI site.\",\n \"25.The method according to claim 1, wherein the promoter or forward primer is an oligonucleotide containing from about 15 to about 30 nucleotides which hybridize with the HBV target and comprising at least 15 nucleotides, counted from the cleavage site, of the nucleotide sequence of SEQ ID No.\",\n \"10 in combination with the restriction enzyme XbaI, or of SEQ ID No.\",\n \"11 in combination with BssSI or of SEQ ID No.\",\n \"12 in combination with AvrII, linked to a promoter sequence.\",\n \"26.The method according to claim 1, wherein the amplified HBV nucleic acid is additionally detected using a hybridization probe comprising an oligonucleotide sequence containing from about 15 to about 30 nucleotides which hybridize with the amplified HBV target and comprise at least 15 nucleotides of SEQ ID No.\",\n \"13 or SEQ ID No.\",\n \"14.27.The method according to claim 1, wherein the second or reverse primer is an oligonucleotide containing from about 15 to about 30 nucleotides and comprising at least 15 nucleotides of the nucleotide sequence of SEQ ID No.\",\n \"4 or SEQ ID No.\",\n \"5.28.A set of oligonucleotide primers comprising a promoter primer, wherein the promoter primer is an oligonucleotide containing from about 15 to about 30 nucleotides which hybridize with the HBV target and comprising at least 15 nucleotides, counted from the cleavage site, of the nucleotide sequence of SEQ ID No.\",\n \"10 in combination with the restriction enzyme XbaI, or of SEQ ID No.\",\n \"11 in combination with BssSI or of SEQ ID No.\",\n \"12 in combination with AvrII, linked to a promoter sequence and a reverse primer, wherein the reverse primer is an oligonucleotide containing from about 15 to about 30 nucleotides and comprising at least 15 nucleotides of the nucleotide sequence of SEQ ID No.\",\n \"4 or SEQ ID No.\",\n \"5.\"\n ],\n [\n \"<SOH> BRIEF DESCRIPTION OF THE FIGURES <EOH>FIG.\",\n \"1 : Schematic presentation of DNA NASBA including restriction enzyme digestion.\",\n \"The restriction enzyme (arrow) is only active during the initiation phase of NASBA.\",\n \"After digestion, the forward primer is hybridized to the template.\",\n \"AMV RT will extend the 3′ end of the target strand (black) of the DNA, using the forward primer, including the T7 promoter sequence (dark grey) as template.\",\n \"The T7 DdRp will recognize the double stranded T7 promoter sequence and RNA amplicon (light grey) production will begin.\",\n \"The RNA amplicon sequence is complementary to the target DNA strand.\",\n \"During the cyclic phase, the RNA amplicon will be amplified and detected by molecular beacon technology.\",\n \"RNase H and the reverse primer are only required during the cyclic phase.\",\n \"FIG.\",\n \"2 : NASBA of HBV DNA with and without digestion with XbaI in combination with forward primer S-p3.8.After digestion with XbaI, S-p3.8 can be used as template for the extension of target DNA.\",\n \"Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.\",\n \"A sample without template (NT) is used as negative control.\",\n \"FIG.\",\n \"3 : NASBA of HBV DNA with and without digestion with BssSI in combination with forward primer S-p3.10.After digestion with BssSI, S-p3.10 can be used as template for the extension of target DNA.\",\n \"Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.\",\n \"A sample without template (NT) is used as negative control.\",\n \"FIG.\",\n \"4 .\",\n \"NASBA of HBV DNA with and without digestion with XbaI in combination with forward primer S-p3.10.S-p3.10 can not be used as template for the extension of target DNA, after digestion with XbaI.\",\n \"Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.\",\n \"A sample without template (NT) is used as negative control.\",\n \"FIG.\",\n \"5 .\",\n \"NASBA of HBV DNA with and without digestion with BssSI in combination with forward primer S-p3.8.S-p3.8 can be used as template for the extension of target DNA, after digestion with BssSI.\",\n \"Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.\",\n \"A sample without template (NT) is used as negative control.\",\n \"FIG.\",\n \"6 .\",\n \"NASBA of HBV DNA with and without digestion with AvrII in combination with forward primer S-p3.5.S-p3.5 can be used as template for the extension of target DNA, after digestion with AvrII.\",\n \"Primer S-p4.4 is used as reverse primer and molecular beacon S-WT4 as probe.\",\n \"A sample without template (NT) is used as negative control.\",\n \"The invention is further exemplified by the following Examples.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"The present invention is directed to a transcription based amplification method for the amplification of HBV DNA.\",\n \"Nucleic acid amplification methods are used in the field of molecular biology and recombinant DNA technology.\",\n \"These methods are used to increase the number of copies of a particular nucleic acid sequence, present in small amounts and often in an environment in which a wide variety of other nucleic acid sequences, both RNA and DNA, are also present.\",\n \"In particular, nucleic acid amplification methods are used to facilitate the detection or quantification of nucleic acid and are important for diagnosing for example infectious diseases, inherited diseases and various types of cancer.\",\n \"Nucleic acid amplification methods have also found their applications in other fields where samples are investigated in which nucleic acid may be present in minute amounts, such as forensic sciences, archeology or to establish paternity.\",\n \"Several nucleic acid amplification techniques are known based on different mechanisms of action.\",\n \"One method for the amplification of nucleic add is known as the “Polymerase Chain Reaction” (PCR) is described in European patent applications EP 200362 and EP 201148.The present invention is concerned with a different class of nudeic acid amplification methods namely the “transcription based amplification techniques”.\",\n \"With these methods multiple RNA copies are obtained from a DNA template that comprises a functional promoter recognized by the RNA polymerase.\",\n \"Said RNA copies are used as target from which new DNA templates are obtained etc.\",\n \"Gingeras et al.\",\n \"in WO88/10315 and Burg et al.\",\n \"in WO89/1050 have described such methods.\",\n \"Isothermal transcription based amplification techniques have been described by Davey et al.\",\n \"in EP 323822 (relating to the NASBA method), by Gingeras et al.\",\n \"in EP 373960 and by Kacian et al.\",\n \"in EP 408295.Transcription based amplification reactions may also be performed with thermostable enzymes.\",\n \"Transcription based amplifications are usually carried out at a temperature around 41 degrees Celsius.\",\n \"Thermostable enzymes allow the reaction to be carried out at more elevated temperatures.\",\n \"Such a thermostable method is described in EP 682121 filed in the name of Toyo Boseki KK.\",\n \"The methods as described in EP 323822, EP 373960 and EP 408295 are isothermal continuous methods.\",\n \"With these methods four enzyme activities are required to achieve amplification: an RNA dependent DNA polymerase activity, an DNA dependent DNA polymerase activity, an RNase (H) activity and an RNA polymerase activity.\",\n \"Some of these activities can be combined in one enzyme, so usually only 2 or 3 enzymes are necessary.\",\n \"Reverse transcriptase such as AMV (Avian Myoblastosis Virus) or MMLV (Moloney Murine Leukemia Virus) reverse transcriptase have both RNA- and DNA dependent DNA polymerase activity but also an inherent RNase H activity.\",\n \"In addition an RNase may be added to the reaction mixture of a transcription based amplification reaction, such as E. coli RNase H. DNA dependent RNA polymerases synthesize multiple RNA copies from a DNA template including a promoter recognized by the RNA polymerase.\",\n \"Examples of RNA polymerases are polymerases from E. coli and bacteriophages T7, T3 and SP6.An example of an RNA polymerase commonly used with transcription based amplification methods is T7 polymerase.\",\n \"Thus the promoter that is incorporated in the template used to produce multiple copies of RNA would then be the T7-promoter.\",\n \"Usually the template comprising the promoter has to be created starting from the nucleic acid comprising the target sequence.\",\n \"Said nucleic acid may be present in the starting material that is used as input for the amplification reaction.\",\n \"The nucleic acid present in the starting material will usually contain the target sequence as part of a much longer sequence.\",\n \"Additional nucleic acid sequences may be present on both the 3′- and the 5′-end of the target sequence.\",\n \"The amplification reaction can be started by bringing together this nucleic acid from the starting material, the appropriate enzymes that together provide the above mentioned activities and at least one, but usually two, oligonucleotide(s).\",\n \"At least one of these oligonucleotides should comprise the sequence of the RNA polymerase promoter.\",\n \"Transcription based amplification methods are particularly useful if the input material is single stranded RNA, although single or double stranded DNA can likewise be used as input material.\",\n \"When a transcription based amplification method is practiced on a sample with single stranded RNA with additional sequences on both the 3′-end and the 5′ end of the target sequence a pair of oligonucleotides that is conveniently used with the methods as described in the prior art would consist of: a first oligonucleotide (usually referred to as “promoter-primer” or “forward-primer”) that is capable of hybridizing to the 3′-end of the target sequence, which oligonucleotide has the sequence of a promoter (preferably the T7 promoter) attached to its 5′ end (the hybridizing part of this oligonucleotide has the opposite polarity as the target RNA used as input material).\",\n \"a second oligonucleotide (usually referred to as “reverse primer”) which comprises the 5′ end of the target sequence (this oligonucleotide has the same polarity as the target RNA).\",\n \"When such a pair of oligonucleotides, together with all enzymes having the appropriate activities, and a sufficient supply of the necessary ribonucleotides and deoxy-ribonucleotides are put together in one reaction mixture and are kept under the appropriate conditions (that is, under the appropriate buffer conditions and at the appropriate temperature) for a sufficient period of time an isothermal continuous amplification reaction will start.\",\n \"Many variants of the above theme have been described in the prior art.\",\n \"A transcription based amplification reaction comprises the synthesis of single stranded RNA transcripts from a template comprising a promoter (e.g.\",\n \"the T7 promoter) that is recognized by an RNA polymerase (e.g.\",\n \"T7 RNA polymerase).\",\n \"A forward primer, comprising the promoter sequence, serves as a primer to initiate the synthesis of a strand of DNA complementary to the target RNA.\",\n \"The primer will be extended by the activity of RNA dependent DNA polymerase.\",\n \"The RNA-cDNA hybrid formed will be degraded by RNase H. This enables the hybridization of the specific reverse primer to the cDNA.\",\n \"Extension of this primer by RNA dependent DNA polymerase up to the 5′ end of the cDNA results in the formation of a double-stranded promoter sequence, whereby the promoter sequence that was part of the forward primer is used as a template.\",\n \"This double stranded promoter will then be used by the DNA dependent RNA polymerase to produce many new RNA molecules that are complementary to the target RNA.\",\n \"After this initiation phase, the amplification enters a cyclic phase.\",\n \"In practice, the whole sequence of events, starting from the single stranded RNA in the sample, will take place as soon as all ingredients are put together, and the mixture is brought to the appropriate temperature for the enzymes to be all active.\",\n \"The practitioner of the method need not to intervene to accomplish any of these steps.\",\n \"As explained above, transcription based amplification methods are particularly useful for amplifications that start from single stranded RNA.\",\n \"The starting material containing the nucleic acid to be amplified may not contain the target nucleic acid as RNA of a defined length.\",\n \"When a transcription based amplification method is performed on starting material comprising the target sequence only as double stranded DNA, either circular or linear, the DNA would have to be converted to single stranded nucleic acid.\",\n \"This can be achieved by separating the strands of the double stranded DNA by applying an elevated temperature (up to a 100 degrees Celsius).\",\n \"The first of the oligonucleotides used as primers in the amplification may than anneal to one of the single strands.\",\n \"The enzymes used with current transcription based amplification methods cannot withstand such a high temperature and consequently can only be added after the DNA strands have been separated.\",\n \"When one of the oligonucleotides anneals to a single strand DNA and is elongated, double stranded DNA is created again, and the reaction mixture would have to be subjected to an elevated temperature sufficiently high to melt the double stranded DNA into its separated strands again.\",\n \"Again the enzymes would be inactivated and new enzymes are to be added after the heat step has been applied.\",\n \"The second oligonucleotide can now be added and anneal to the strand that was created from the elongated first oligonucleotide in the first step.\",\n \"As one of the oligonudeotides includes a 5′ promoter sequence of a DNA dependent RNA polymerase (see above), a double stranded DNA template including a double stranded functional promoter is obtained, from which a first step of RNA production can take place.\",\n \"The resulting RNA transcripts may enter the cyclic phase of the amplification and the process can further be isothermal.\",\n \"From the above it is evident that starting a transcription based amplification method from double stranded DNA can be a tedious process.\",\n \"It requires several specific actions to be taken by the practitioner, the sample has to be heated and cooled repeatedly and enzymes have to be replenished after each heating step.\",\n \"Some research has already gone into the developments of transcription based amplification methods that can start from dsDNA, avoiding the tedious procedure described above to convert the dsDNA into ssRNA that can be used as input for the A rather simple transcription based amplification method for dsDNA has been disclosed in WO9925868.According to the method described in WO9925868 dsDNA in a sample can be amplified by means of a transcription based amplification protocol directly, without any heat treatment step [of over 90 C] at all, or —in a preferred embodiment—with only one initial heating step dsDNA, that is relatively short, is to be preferred in this method.\",\n \"Actually, the method does not differ essentially from a conventional transcription based amplification protocol to amplify ssRNA.\",\n \"Alternatively, the double stranded DNA in the starting material can be transcribed into RNA before the start of the amplification.\",\n \"Such an extra step can be based on an enzyme, for instance E. coli RNA polymerase, that transcribes the double stranded DNA into RNA without the presence of a promoter sequence, also referred to as a polymerase binding site.\",\n \"Such a process of extra steps to facilitate the amplification of double stranded DNA by transcription based amplification methods has been described in PCT patent application no.\",\n \"WO9602668.The extra steps described in this procedure do not only include extra handling steps and handling time, but also the use of additional ingredients, i.e.\",\n \"the E. coli RNA polymerase.\",\n \"Another way of preparing suitable templates for transcription based amplification methods for dsDNA is described in EP 397269.In this patent a method is described whereby dsDNA is pretreated with a restriction enzyme.\",\n \"After treatment with the restriction enzyme only one heat separation step is needed to create single stranded DNA (ssDNA).\",\n \"With this method a forward primer (promoter-primer) is used that has a 3′ part including a sequence that is complementary to the exact 3′ end of one of the single strands of DNA and a 5′ end including a promoter sequence recognized by a RNA polymerase (for example T7 RNA polymerase).\",\n \"When the promoter-primer is hybridized to the 3′ end of the single strand of DNA a double stranded complex Is formed, of which the 5′ promoter sequence of the forward primer can serve as a template for an elongation reaction starting from the 3′ end of the DNA strand.\",\n \"Thus, a double stranded promoter is formed by a DNA dependent DNA polymerase and the resulting complex can serve as template for the DNA dependent RNA polymerase to synthesize multiple copies of RNA.\",\n \"In WO9104340 also several methods are disclosed to start a transcription based amplification reaction for single stranded DNA.\",\n \"Again, a restriction enzyme may be used to create an appropriate 3′ end on the DNA, which can hybridize with a 3′ sequence of a promoter primer.\",\n \"In WO9104340 it is disdosed how the defined 3′ end on the ssDNA may be created using a restriction enzyme that cuts ssDNA.\",\n \"In another embodiment of the same method, a restriction enzyme is used that cuts dsDNA, together with a restriction oligonucleotide that hybridizes to the target ssDNA and thus creates a double stranded piece of DNA that can be cut by the restriction enzyme to create the appropriate 3′ end.\",\n \"With this method a small piece of the restriction oligonucleotide will remain after the restriction enzyme has cut the double stranded complex.\",\n \"However, according to the disclosure of WO9104340, the restriction oligonucleotide is apparently chosen in such a way that after digestion, the remaining piece will be to small to stay hybridized to the 3′ end of the ssDNA, and thus will fall of to make room for the promoter oligonucleotide.\",\n \"However, the pre-treatment with a restriction enzyme as used with the prior art methods, although it may result in a sensitive transcription based assay, require many extra handling steps and handling time.\",\n \"Hepatitis B virus (HBV) infection in humans is widespread.\",\n \"The virus that causes hepatitis B appears to infect only humans and chimpanzees.\",\n \"The hepatitis infection is transmitted by three general mechanisms: (1) by parenteral inoculation of infected blood or body fluids, either in large amounts as in blood transfusions or in minute amounts as through an accidental skinprick; (2) by close family or sexual contact; and (3) by some mothers, who transmit the virus to their new-bom children.\",\n \"Under natural conditions, HBV is not highly contagious.\",\n \"Transmission by inhalation occurs rarely, if ever.\",\n \"The transmission route through contaminated blood or blood products is a major threat to the human health.\",\n \"Infection with HBV often results in subclinical or acute self-limited liver disease or can result in chronic long-term infection.\",\n \"Chronic HBV infection elicits a spectrum of disease entities ranging from the most severe form of chronic active hepatitis to less severe chronic persistent hepatitis to the asymptomatic carrier state.\",\n \"An array of diagnostic assays have recently been developed to aid the clinician in differentiating hepatitis B virus infections from other forms of viral hepatitis (i.e., hepatitis A, hepatitis C or hepatitis E).\",\n \"However, the ability to distinguish between an acute hepatitis B infection and symptomatic chronic hepatitis B infection is still problematic.\",\n \"This is especially true since chronic active hepatitis and chronic persistent hepatitis patients often demonstrate a cyclic pattern of hepatitis characterized by acute exacerbation of liver injury alternating with normal liver function.\",\n \"After infection with HBV, large quantities of the virus and associated particles are present in the serum.\",\n \"During symptomatic phases of infection, both acute and chronic HBV patients have elevated liver enzyme levels, possess the hepatitis B surface antigen (HBsAg) in their serum, and produce antibodies to the nucleocapsid antigen (HBcAg).\",\n \"Antibodies specific for the HBsAg or the hepatitis B e antigen (HBeAg) are not detected.\",\n \"The appearance of antibody to HBsAg is usually not observed until approximately two months following disappearance of circulating HBsAg.\",\n \"The viral particles present in the serum are known to shed their surface coat exposing the nucleocapsid, known as the core antigen (HBcAg).\",\n \"Antibody production of HBcAg occurs early in the course of the acute phase of HBV infection and can persist for many years, and chronically infected patients can produce high titers of anti-HBc antibodies.\",\n \"HBsAg is established as the most important marker of acute or chronic hepatitis B infection, detectable in serum of infected individuals.\",\n \"HBsAg screening of donor blood for example, is essential to avoid transmission of hepatitis B.\",\n \"It is clear that sensitivity is of utmost importance in diagnostic HBV assays.\",\n \"HBV is the smallest DNA virus known; and its genome shows a highly compacted organization.\",\n \"A unique aspect in the replication cycle of HBV is that a pre-genomic mRNA serves as a template for the synthesis of the first viral DNA strand.\",\n \"The RNAse H activity of the HBV DNA polymerase subsequently removes the mRNA and the complementary DNA strand is then synthesized, generating a partially double stranded DNA molecule for packaging in virions.\",\n \"Upon successful virus entry, the partially double stranded DNA molecule is converted into a fully double stranded DNA molecule.\",\n \"HBV DNA can be detected in the blood of infected hosts who are HBsAg and HBeAg positive in more than 90% of cases.\",\n \"The state of the art method of measuring the quantity of infectious particles is by measuring the quantity of viral DNA in serum or plasma, because it most reliably reflects the amount of replicating virus.\",\n \"Several assays are available for this purpose, such as the branched DNA (bDNA) assay (Hendricks D A, Stowe B J, Hoo B S, Kolberg J, Irvine B D, Neuwald P D, Urdea M S, Perrillo R P, 1995.Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay.\",\n \"Am J Clin Pathol 104: 537-546.\",\n \"), DNA hybridization assays and quantitative PCR (Pawlotsky J M, Bastie A, Lonjon I, Remire J, Darthuy F, Soussy C J, Dhumeaux D, 1997.What technique should be used for routine detection and quantification of HBV DNA in clinical samples?\",\n \"Journal of Virological Methods 65: 245-253; Zaaijer H L, ter Borg F, Cuypers H T, Hermus M C, Lelie P N, 1994.Comparison of methods for detection of hepatitis B virus DNA.\",\n \"J Clin Microbiol 32: 2088-2091.).\",\n \"Most of these assays, however, have only limited sensitivity.\",\n \"The present invention is concerned with a transcription based amplification method including a restriction enzyme digestion.\",\n \"The method enables a sensitive and specific amplification (and subsequent detection) of DNA of the Hepatitis B virus.\",\n \"With the method of the invention HBV DNA can be amplified and detected in a more efficient way than with prior art transcription based amplification methods in contrast to the prior art methods the use of the restriction enzyme does not complicate the procedures used method of the invention.\",\n \"The present invention provides a method for the transcription based amplification of a target HBV nucleic acid sequence starting from HBV DNA optionally present in a sample, comprising the steps of, incubating the sample, suspected to contain HBV, in an amplification buffer with one or more restriction enzymes capable of cleaving the DNA at a selected restriction site, said restriction enzyme creating a defined 3′ end on one of the DNA strands, and a promoter-primer, said promoter-primer having a 5′ region comprising the sequence of a promoter recognized by a DNA-dependent RNA polymerase and a 3′ region complementary to the defined 3′ end of the DNA strand, a second primer, having the opposite polarity of the promoter-primer and comprising the 5′ end of the target sequence, and in case of a HBV ssDNA, a restriction primer, maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for a digestion by the restriction enzyme to take place, subjecting the sample to a heat treatment at a temperature and time sufficient to inactivate the restriction enzyme and/or to render at least partially a double strand single stranded, adding the following reagents to the sample: an enzyme having RNA dependent DNA polymerase activity an enzyme having DNA dependent DNA polymerase activity an enzyme having RNase H activity an enzyme having RNA polymerase activity, and maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for the amplification to take place.\",\n \"The [for an adequate amplification] necessary (appropriate) nucleoside triphosphates may be present already during the incubation step with the restriction enzyme(s), for example as part of the said amplification buffer.\",\n \"They may, however, be added later on in the process, for example together with the enzymes after the heat treatment.\",\n \"The person skilled in the art knows the enzymes used for the transcription based amplification method, and the conditions under which the transcription based amplification method is carried out and is aware of all the usual modifications that can be made with regard to optimizing transcription based amplification reactions.\",\n \"For example, the forward primer, the promoter primer, may comprise a purine region between the promoter sequence on the 5′ end of the primer and the hybridizing sequence on the 3′ end of the primer.\",\n \"The sequence of the primers is largely determined by the position of the restriction site chosen.\",\n \"The 3′ end of a forward primer should anneal to the target sequence directly next to the restriction site.\",\n \"The primer may vary in length as long as it is sufficiently long to hybridize under the conditions used with the amplification reaction.\",\n \"In general the hybridizing part of the primer consists of about 10 to about 35 nucleotides.\",\n \"Restriction primers, used in the method according to the invention if the target is HBV ssDNA, require that the overlap they show with the forward primer is minimal and the sequence of the restriction site is incorporated in such a way that the restriction enzyme actually cuts the DNA efficiently.\",\n \"A restriction enzyme is an enzyme that can cut dsDNA at a selected site (i.e.\",\n \"a specific nucleotide sequence recognized by the enzyme).\",\n \"In selecting an appropriate restriction enzyme for the method of the invention care should be taken that a restriction site is chosen that is present in al variants of the HBV DNA (for example, a restriction site that is present in all genotypes of the hepatitis B virus, if the amplification is carried out to detect all viral HBV DNA in the sample).\",\n \"The restriction site should not be present in the DNA sequence in between the forward and reverse primers used.\",\n \"The addition of the restriction enzyme results in the creation of a defined 3′ end of the target strand of the DNA, which is then available for binding to the hybridizing part of the promoter primer.\",\n \"An additional aspect is that, because of the digestion, denaturation of that part of the DNA will be improved and so primer binding will be facilitated.\",\n \"The promoter oligonucleotide containing the T7-promoter sequence should be designed in such a way that the hybridizing part will interact with the template directly upstream of the restriction site.\",\n \"The enzyme having DNA dependent DNA polymerase activity (usually a reverse transcriptase such as MMLV-RT or AMV-RT) can extend the 3′ end of the target strand of the DNA created by the digestion with the restriction enzyme, using the primer as template.\",\n \"A double-stranded T7-promoter sequence will be formed and the production of amplicon RNA can start.\",\n \"Surprisingly it has been found that a restriction enzyme can be used efficiently in an environment that is suitable for and adapted to a transcription based HBV DNA amplification process.\",\n \"In other words it has been found that the use of a restriction enzyme to cut the HBV DNA that is used as input material for a transcription based amplification, does not have to lead to complicated, additional handling of the sample.\",\n \"The use of the restriction enzyme is incorporated into and is part of the steps that are usually already part of the protocol for a transcription based DNA amplification.\",\n \"All prior art methods describe the use of a restriction enzyme in the preparation of a DNA template for transcription based amplification as a separate pre-treatment prior to the actual transcription based amplification.\",\n \"Consequently the prior art use of a restriction enzyme in preparing the DNA template resulted in additional handling of the sample, like separate inactivation of the restriction enzyme and separate purification of the DNA.\",\n \"It complicates the whole amplification procedure, especially an automated process, and increases the risk of contamination.\",\n \"Although the addition of a restriction oligonucleotide together with a restriction enzyme has already been disclosed in WO9104340, it has not been disdosed prior to the present invention how the use of the restriction enzyme (and oligonucleotide) can efficiently be combined with the transcription based amplification.\",\n \"It has not been disclosed in the prior art in which way the use of restriction enzyme can be combined with transcription based amplification without additional sample handling and reagent adding steps.\",\n \"The method of the invention provides this combination without complicating the prior art transcription based DNA amplification process.\",\n \"The method of the invention hardly differs from a normal transcription based amplification method.\",\n \"The only additional step to be taken is the “built in incubation” of the sample with the restriction enzyme, which means that the restriction enzyme is used in such a way that the actual handling of the sample does not differ from a conventional/prior art transcription based DNA amplification process.\",\n \"The preferred restriction enzyme used in the method of the invention is, of course, an enzyme that is relatively stable and retains a high activity under conditions where it is added to a reaction mixture comprising an amplification-buffer (which contains relatively high concentrations of salts).\",\n \"After the addition of the restriction enzyme the sample needs to be incubated under the appropriate conditions for the enzyme to be active, and for a suitable amount of time.\",\n \"The sample may be incubated with the restriction enzyme for a relatively short period of time, preferably for about 10-20 minutes and more preferred for about 15 minutes at a temperature of about 35 to about 45° C. and more specifically at about 37-41° C., obviously depending on the nature of the restriction enzyme used.\",\n \"In fact, this is the only additional measure to be taken, when compared to a conventional transcription based DNA amplification method.\",\n \"The method of the invention comprises the step of heating the sample after the incubation with the restriction enzyme.\",\n \"During this heating the restriction enzyme is inactivated and double stranded DNA is rendered single stranded [at least partially].\",\n \"This heating step is already part of the protocols for carrying out a transcription based amplification method.\",\n \"These methods involve a heat treatment of the sample after primer-addition, to create optimal circumstances for primer annealing (the nucleic acid is stretched, strands or internal loops of the nucleic acid are separated, and during the cooling down, hybridization of the primers to the template is facilitated.)\",\n \"The heating after the incubation with the enzyme may be done at a lower temperature of about 50° C. or higher, but is preferably carried out by way of a short incubation (about 5-10 minutes) at a temperature above 90° C. and preferably at 95+/−3° C. Thereafter the sample may be cooled to the appropriate temperature for a transcription based amplification reaction to take place (usually about b 41° C.).\",\n \"Due to the heating, at least a part of the double stranded DNA is rendered single stranded.\",\n \"As the primers, especially the promoter oligonucleotide, are already present prior to the heating of the sample, the heat treatment may facilitate primer annealing to DNA as well.\",\n \"Thus, there is no need to purify the DNA from the sample after it has been subjected to a treatment with the restriction enzyme.\",\n \"The enzyme is simply inactivated in the heat treatment that was already part of the transcription based amplification procedure.\",\n \"It has been proven with the method of the invention that this is sufficient to eliminate the risk that the restriction enzyme will interfere with the actual transcription based amplification reaction.\",\n \"After the heat treatment the additional amplification reagents for the transcription-based amplification are added in the usual way, and the transcription based amplification can be carried out in the usual way known to the skilled person The amplification enzymes are only added after the heat treatment, to prevent degradation of the enzymes during the heat treatment (unless, of course, thermostable enzymes are used).\",\n \"The major advantage of the method of the invention is that, even though that additional reagents are used (e.g.\",\n \"the restriction enzyme) this does not result in additional (separate) reaction steps or activities to be carried out.\",\n \"The fact that no additional handling of the sample is required is especially important because every additional handling of the sample would increase the contamination risk, which is to be avoided at all costs, especially in amplification reactions.\",\n \"Moreover, if additional sample handling steps were required this would complicate automation of the method.\",\n \"The method of the invention may also be used for single stranded DNA.\",\n \"When the DNA is single stranded a restriction oligonucleotide or restriction primer comprising a sequence complementary to the region including the restriction site of the target DNA is added together with the restriction enzyme.\",\n \"The restriction oligonucleotide [restriction primer] hybridizes with the single stranded DNA and forms a double stranded complex that can be cut with the restriction enzyme.\",\n \"The addition of yet another reagent (the restriction oligonucleotide) does not result in additional steps to be carried out by the practitioner.\",\n \"The restriction oligonucleotide can simply be added together with the restriction enzyme and the other oligonucleotides necessary for the amplification.\",\n \"Thus, there is no need to open the amplification system for yet another addition of reagents.\",\n \"In a preferred embodiment of the invention, the function of the restriction oligonucleotide may be incorporated in the oligonucleotide that also comprises the sequence of a promoter recognized by a DNA dependent RNA polymerase (the combined promoter and restriction-primer).\",\n \"In this way only two oligo's are needed for the amplification, a promoter [or forward] primer in which a sequence complementary to the target region including the restriction site has been incorporated and the second [or reverse] primer.\",\n \"The sequence including the restriction site of this preferred [combined promoter and restriction] primer should preferably be allocated in such a way that: after digestion, the remaining part of the primer will denature from the target during the heating step the remaining part of the hybridizing sequence of the target is long enough for a new combined promoter and restriction primer to bind, Extra nucleotides surrounding the restriction site are included in the hybridization if necessary for the activity of the restriction enzyme.\",\n \"Thus, a part of the combined promoter and restriction primer will now serve as restriction oligonucleotide; it will anneal to the target DNA, resulting in dsDNA comprising the restriction site recognized by the restriction enzyme.\",\n \"Subsequently the restriction enzyme will cut the said dsDNA, thus providing the defined 3′ end on the DNA.\",\n \"Preferably, an at least 1000 fold excess of this combined promoter and restriction primer with respect to the amount of target DNA should be present, as is also usual already for a conventional promoter primer in transcription based amplification reactions.\",\n \"The method of the invention provides an efficient and sensitive method for the amplification and detection of viral DNA in samples suspected to contain the hepatitis B virus (HBV), especially with the HBV primers and HBV probes as described herein.\",\n \"Hence, these HBV primers and HBV probes represent another embodiment of the invention, The restriction site that is selected in the method of the invention is preferably one that is conserved among the different genotypes of HBV.\",\n \"Good results were obtained if the target sequence and restriction site were located in the part of the HBV genome that encodes the surface antigen.\",\n \"Conserved restriction sites in the HBsAg coding region that are especially useful in the method of the invention (together with the restriction enzymes that cut at these sites) are the XbaI site located at nucleotides 247-252 according to the EcoRI site, the BssSI site located at nucleotides 253-258 according to the EcoRI site and the AvrII site located at nucleotides 178-183 according to the EcoRI site.\",\n \"The oligonucleotide sequence of the primers is, of course, largely determined by the position of the restriction site chosen.\",\n \"The primer may vary in length as long as it is sufficiently long to hybridize under the conditions used with the amplification reaction.\",\n \"In general the hybridizing part of the primer consists of about 10 to about 35 nucleotides and more preferably of about 15 to about 30 nucleotides.\",\n \"The 5′ end of a forward primer should anneal to the target sequence directly next to the restriction site.\",\n \"The position of the reverse primer is less critical, preferably it should have the sequence of a part of a conserved region sufficiently remote from the position of the forward primer to allow a probe to hybridize to the amplified target sequence in the region in between the forward and reverse primer region.\",\n \"The preferred restriction primer is an oligonucleotide that has a minimal overlap with the forward primer, includes the sequence of the restriction site in such a way that the restriction enzyme can actually cut the dsDNA thus formed and is sufficiently long so as to hybridize with the HBV DNA in a sufficient manner.\",\n \"Oligonucleotide forward primers which are especially useful in combination with the XbaI, AvrII and/or BssSI restriction enzymes are preferred embodiments of the invention.\",\n \"Specifically, oligonucleotide sequences comprising at least 10 and preferably more than 15 nucleotides [counted from the cleavage site] of the HBV hybridizing part of SEQ ID No 1 [said hybridizing part as such being SEQ ID No 10] in combination with the restriction enzyme XbaI, of the HBV hybridizing part of SEQ ID No 2 [said hybridizing part as such being SEQ ID No 11] in combination with BssSI and/or of the hybridizing part of SEQ ID No 3 [said hybridizing part as such being SEQ ID No 12] in combination with Avril are preferred.\",\n \"Oligonucleotides which can be used as restriction primers, comprise at least 10 and preferably more than 15 nucleotides “flanking” the HBV DNA restriction site concerned and especially “flanking” the XbaI site located at nucleotides 247-252 according to the EcoRI site, the BssSI site located at nucleotides 253-258 according to the EcoRI site and the AvrII site located at nudeotides 178-183 according to the EcoRI site.\",\n \"Particularly suitable restriction primers have the oligonucleotide sequences depicted in SEQ ID No.\",\n \"8 and SEQ ID No 9.Suitable probes for the detection of the amplified HBV target contain from 10 to about 35 and more preferably from about 15 to about 30 nucleotides which hybridize with the amplified HBV target and comprise at least 10 and preferably more than 15 nucleotides of the HBV hybridizing part of SEQ ID No 6 and SEQ ID No 7.\",\n \"(The HBV hybridizing parts of these sequences are shown in Table 1 and indicated as SEQ ID No 13 and SEQ ID No 14 respectively.\",\n \"The probes shown in Table 1 are further provided at both ends with non-HBV nucleotides forming entirely or sometimes together with a few HBV nucleotides the “stem” of the probe; said stern being part of the detection system chosen).\",\n \"Suitable reverse primers contain from about 10 to about 35 and more preferably from about 15 to about 30 nucleotides of a conserved region of HBV.\",\n \"Preferred reverse primers comprise at least 10 and preferably more than 15 nucleotides of SEQ ID No 4 and SEQ ID No 5.Another embodiment of the invention is a test kit suitable for carrying out the transcription based amplification and detection of HBV DNA according to the invention comprising a restriction enzyme as herein described, a forward primer that corresponds with the cleavage site of the restriction enzyme chosen, as herein explained, and Is provided with a promoter sequence, other reagents for carrying out a transcription based amplification reaction, means for detecting the amplified HBV DNA, and instructions for use.\",\n \"BRIEF DESCRIPTION OF THE FIGURES FIG.\",\n \"1: Schematic presentation of DNA NASBA including restriction enzyme digestion.\",\n \"The restriction enzyme (arrow) is only active during the initiation phase of NASBA.\",\n \"After digestion, the forward primer is hybridized to the template.\",\n \"AMV RT will extend the 3′ end of the target strand (black) of the DNA, using the forward primer, including the T7 promoter sequence (dark grey) as template.\",\n \"The T7 DdRp will recognize the double stranded T7 promoter sequence and RNA amplicon (light grey) production will begin.\",\n \"The RNA amplicon sequence is complementary to the target DNA strand.\",\n \"During the cyclic phase, the RNA amplicon will be amplified and detected by molecular beacon technology.\",\n \"RNase H and the reverse primer are only required during the cyclic phase.\",\n \"FIG.\",\n \"2: NASBA of HBV DNA with and without digestion with XbaI in combination with forward primer S-p3.8.After digestion with XbaI, S-p3.8 can be used as template for the extension of target DNA.\",\n \"Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.\",\n \"A sample without template (NT) is used as negative control.\",\n \"FIG.\",\n \"3: NASBA of HBV DNA with and without digestion with BssSI in combination with forward primer S-p3.10.After digestion with BssSI, S-p3.10 can be used as template for the extension of target DNA.\",\n \"Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.\",\n \"A sample without template (NT) is used as negative control.\",\n \"FIG.\",\n \"4.NASBA of HBV DNA with and without digestion with XbaI in combination with forward primer S-p3.10.S-p3.10 can not be used as template for the extension of target DNA, after digestion with XbaI.\",\n \"Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.\",\n \"A sample without template (NT) is used as negative control.\",\n \"FIG.\",\n \"5.NASBA of HBV DNA with and without digestion with BssSI in combination with forward primer S-p3.8.S-p3.8 can be used as template for the extension of target DNA, after digestion with BssSI.\",\n \"Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe.\",\n \"A sample without template (NT) is used as negative control.\",\n \"FIG.\",\n \"6.NASBA of HBV DNA with and without digestion with AvrII in combination with forward primer S-p3.5.S-p3.5 can be used as template for the extension of target DNA, after digestion with AvrII.\",\n \"Primer S-p4.4 is used as reverse primer and molecular beacon S-WT4 as probe.\",\n \"A sample without template (NT) is used as negative control.\",\n \"The invention is further exemplified by the following Examples.\",\n \"EXAMPLES Example 1 Amplification of HBV DNA Two conserved restriction sites (XbaI and BssSI) are encoded in the conserved region (nt 244 to 285 according to the EcoRI-site) of the S-gen of HBV DNA.\",\n \"As this part of the S-region can be sinqle-stranded DNA of negative polarity, an oligonucleotide (‘restriction primer’ (RP) complementary to the region including the restriction site sequences was added to create a double-stranded restriction site for all genomic DNAs present.\",\n \"HBV DNA was isolated from a series of dilution of plasma infected with HBV genotype A of 3×109 geq/ml, using the Nuclisens Extractor (Organon Teknika).\",\n \"Following the standard procedure as described for RNA isolation (Operator Manual Extractor, 41001-9, rev A, 1999), a 50 I extract is obtained.\",\n \"Five I of the extract is used peassay.\",\n \"The restriction enzyme digestion was performed in NASBA buffer (40 mM Tris-HCl pH 8.5,12 mM MgCl2, 70 mM KCl, 15% v/v DMSO, 5 mM DTT, 1 mM each dNTP, 2 mM ATP, 2 mM CTP, 2 mM UTP, 1.5 mM GTP, 0.5 mM ITP, 0.2 μM forward primer (S-p3.8 for XbaI, and S-p3.10 for BssSI, table 1), 0.2 μM reverse primer (S-p4.5, table 1), 0.1 μM molecular beacon probe (S-W12, table 1), 0.17 μM restriction primer (RP-3, table 1)) and 0.2 units restriction enzyme BssSI (New England BioLabs, Inc., Beverly, Mass., USA) or 3.0 units restriction enzyme XbaI (New England BioLabs, Inc., Beverly, Mass., USA).\",\n \"After incubation of 15 min at 41 C, the restriction enzymes were heatinactivated and the DNA template denatured at 95 C for 5 min.\",\n \"Hybridization of the primers occured during ccaling down to 41 C for 3 min.\",\n \"Subsequently, NASBA enzymes (2.1 μg BSA, 0.08 units RNase H, 32 units T7 RNA polymerase and 6.4 units AMV reverse transcriptase) were added, the reaction mixture was mixed by gently tapping and short centrifugation, and the amplification and real-time detection was started.\",\n \"The reaction mixture was incubated at 41° C. in the NucliSens EasyQ Analyzer (Organon Teknika) for 120 minutes with fluorescence monitoring every minute.\",\n \"The reactions were excited at 485 nm and the emission signal was measured at 518 nm.\",\n \"Example 1.1 Amplification of HBV DNA including XbaI Digestion A NASBA assay with and without the treatment with the restriction enzyme XbaI was performed.\",\n \"The optimal concentration of XbaI was determined under NASBA conditions by digestion of 109 copies of a PCR fragment including the amplicon region of the HBV DNA, and was shown to be 3 units.\",\n \"S-p3.8 is used as forward primer and can be used as template by AMV RT to extend the template DNA after digestion with XbaI.\",\n \"Without digestion a sensitivity of 3×106 geq/ml is obtained while after digestion the sensitivity is 3×103 geq/ml, meaning a 1000 fold increase in sensitivity (FIG.\",\n \"2).\",\n \"In addition, without digestion the time to positivity (TTP) is about 16 minutes while after XbaI digestion this is about 6 min, meaning a decrease in TTP of about 10 min (FIG.\",\n \"2).\",\n \"Both are indications for an improved amplification reaction.\",\n \"Example 1.2 Amplification of HBV DNA including BssSI Digestion A NASBA reaction, with and without treatment with the restriction enzyme BssSI was performed with the same HBV DNA extract and comparable reaction conditions as described above.\",\n \"The ontimal concentration of BssSI was determined under NASBA conditions by digestion of 109 copies of a PCR fragment including the amplicon region of the HBV DNA, and was shown to be 0.2 units.\",\n \"S-p3.10 is used as forward primer and can be used as template by AMV RT to extend the template DNA after digestion with BssSI.\",\n \"Again significant test improvements were obtained as a result of treatment with the restriction enzyme BssSI (FIG.\",\n \"3).\",\n \"Without digestion a sensitivity of only 3×107 geq/ml is obtained while after digestion the sensitivity is 3×104 geq/ml, meaning again a 1000 fold increase in sensitivity (FIG.\",\n \"3) as a result of the digestion.\",\n \"In addition, without digestion the time to positivity (TTP) is about 21 minutes while after BssSI digestion this is about 11 min, meaning again a decrease in TUP of about 10 min (FIG.\",\n \"3).\",\n \"The results prove that the digestion of HBV DNA with a restriction enzyme prior to the NASBA reaction can considerably improve the amplification and so the detection of a HBV DNA Example 1.3 Amplification of HBV DNA including XbaI digestion-2 To test if the digestion by itself or the combination of the restriction enzymes with the selected primers was the basis for the improved assay results, the assay including the XbaI digestion was repeated with primer S-p3.10 instead of S-p3.8.AMV RT can not use S-p3.10 as template to extend the target sequence after digestion with XbaI.\",\n \"As can be seen in FIG.\",\n \"4, only a slight increase in sensitivity (10 fold) and small decrease in TTP (about 5 min, from 21 to 16 min) is obtained after digestion with XbaI in combination with S-p3.10.This indicates that the extension of the template during the initiation of NASBA is responsible for the improved results as obtained with XbaI and primer S-p3.8 and with BssSI and primer Sp3.10.Example 1.4 Amplification of HBV [NA including BssSI digesfion-2 To test if the extension of the target was indeed the basis for the improved assay results, the assay including the BssSI digestion was repeated with primer S-p3.8 instead of S-p3.10.AMV RT can use S-p3.8 as template to extend the target sequence after digestion with BssSI.\",\n \"However, after digestion with BssSI, only 17 nucleotides are included in hybridization of the primer to the target sequence while normally this is about 20 nucleotides.\",\n \"Despite this difference, again dear test improvements were obtained after digestion with BssSI (FIG.\",\n \"5).\",\n \"A double digestion can be performed with both XbaI and BssSI included in NASBA using the primers S-p3.8 and S-p4.5, restriction primer RT-3 and molecular beacon S-WT2 without loss of amplification efficiency as compared to the single digestion NASBA assays.\",\n \"Example 1.5 Amplification of HBV DNA including AvrII Digestion The restriction site (AvrII) is encoded in another conserved region (nt 177 to 192 according to the EcoRI-site) of the S-gen of HBV DNA.\",\n \"Forward primer S-p3.5, reverse primer S-p4.4, molecular beacon S-WT4 and restriction primer RP-1 (table 1) were as used in NASBA.\",\n \"The AMV RT can extent the target strand of HBV DNA after digestion with AvrII, using S-p3.5 as template.\",\n \"The same reaction conditions as described above were used.\",\n \"A NASBA reaction, with and without treatment with the restriction enzyme AvrII was performed with the same HBV DNA extract as described above, using 2 units of AvrII per reaction.\",\n \"Without digestion, a sensitivity of >108 geq/ml is obtained while after digestion the sensitivity is 1×105 geq/ml, meaning a >103 fold increase in sensitivity (FIG.\",\n \"6) as a result of the digestion.\",\n \"Again, these results prove that the digestion of HBV DNA with a restriction enzyme digestion included in the NASBA reaction can considerably improve the amplification of HBV DNA TABLE 1 Primer and probe sequences Primer/Probe Sequence Label S-p3.8 5′AATTCTAATACGACTCACTATAGGG a (SEQ ID No.\",\n \"1) GACTCGTGGTGGACTTCTCTCA 3′ S-p3.10 5′AATTCTAATACGACTCACTATAGGG (SEQ ID No.\",\n \"2) agaa GGTGGACTTCTCTCAATTTTC 3′ S-p3.5 5′AATTCTAATACGACTCACTATAGGG (SEQ ID No.\",\n \"3) aga GGACCCCTGCTCGTGTTACAGGC 3′ S-p4.5 5′GAACCAACAAGAAGATGAGGCA 3′ (SEQ ID No.\",\n \"4) S-p4.4 5′GGGACTGCGAATTTTGGCCA 3′ (SEQ ID No.\",\n \"5) S-WT2 5′CGATCG AGGGACTGCGAATTTTGGC FAM (SEQ ID No.\",\n \"6) CGATCG 3′ S-WT4 5′GGATCCC FAM (SEQ ID No.\",\n \"7) TIGAAAATTGAGAGAAGTCCACCAC GGGATCC 3′ RP-3 5′AATACCGCAGAGTCTAGACTCGTGG 3′NH2 (SEQ ID No.\",\n \"8) 3′ Xbal| BssSI RP-1 5′CATCAGGAYTCCTAGGA 3′ 3′NH2 (SEQ ID No.\",\n \"9) Avrll *The 17-promoter sequence is written in italics, the purine-stretch in lower case, the stem sequence of the probe in underlined italics and the restriction sites are indicated.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471073"}}},{"rowIdx":573,"cells":{"text":{"kind":"list like","value":[["Adenosine receptor selective modulators","The invention relates to the compounds of formula (I), to a method for producing the same and to the use thereof as medicaments."],["1.A compound of the formula (I) in which R1, R2 and R3 independently of one another represent (C1-C8)-alkyl which may be substituted up to three times, independently of one another, by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C2-C4)-alkynyl, halogen or (C6-C10)-aryloxy, (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxy-carbonyl or mono- or di-(C1-C4)-alkylamino, (C1-C8)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C6-C10)-aryl, 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, (C6-C10)-aryloxy, halogen, cyano, (C1-C4)-alkoxycarbonyl, amino or mono- or di-(C1-C4)-alkylamino, hydrogen, hydroxyl, halogen, nitro, cyano, or —NH—C(O)—R7, in which R7 represents (C1-C4)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, or R1 and R2 are attached to adjacent phenyl ring atoms and, together with the two ring carbon atoms, form a 5- to 7-membered saturated or partially unsaturated heterocycle having one or two heteroatoms from the group consisting of N, O and/or S which may be substituted by (C1-C4)-alkyl or oxo, R4 represents (C1-C8)-alkyl which may be substituted by hydroxyl, —NH—CO—R8, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C6-C10)-aryl, 5- or 6-membered saturated or partially unsaturated heterocyclyl having up to three heteroatoms from the group consisting of N, O and/or S or 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, or (C3-C7)-cycloalkyl which may be substituted by hydroxyl or (C1-C8)-alkyl, in which R8 represents (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, R5 represents hydrogen or (C1-C4)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy or (C3-C7)-cycloalkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain one or two further heteroatoms from the group consisting of N, O and/or S in the ring and which may be mono- to trisubstituted, independently of one another, by oxo, fluorine, chlorine, bromine, hydroxyl, (C1-C6)-alkyl or (C1-C6)-alkoxy, and R6 represents (C3-C7)-cycloalkyl or (C1-C8)-alkyl, where alkyl may be substituted up to three times, independently of one another, by (C3-C7)-cyclo-alkyl, hydroxyl, —CO—NH—R9, (C1-C4)-alkoxy, (C2-C4)-alkenyl, (C6-C10)-aryl or 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where aryl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, and R9 represents hydrogen, (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, or a salt, a hydrate, a hydrate of a salt or a solvate thereof.","2.A compound of the formula (I) in which R1, R2 and R3 independently of one another represent hydrogen, hydroxyl, (C1-C4)-alkyl, trifluoromethyl, trifluoromethoxy, fluorine, chlorine, (C1-C4)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl or (C3-C6)-cycloalkyl, —NH—C(O)—CH3 or —NH—C(O)—C2H5, or R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O— or —CH2—CH2—O—, R4 represents (C1-C6)-alkyl which may be substituted by hydroxyl, (C1-C4)— alkoxy, (C3-C6)-cycloalkyl, —NH—C(O)—CH3, phenyl, furyl, pyridyl, imidazolyl, thienyl or hexahydropyranyl, or (C3-C6)-cycloalkyl, R5 represents hydrogen or (C1-C4)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy or (C3-C6)-cycloalkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain a further heteroatom from the group consisting of N; O or S in the ring and which may be mono- to trisubstituted, independently of one another, by hydroxyl, (C1-C4)-alkyl or (C1-C4)-alkoxy, and R6 represents (C3-C6)-cycloalkyl, (C1-C6)-alkyl which may be substituted up to two times, independently of one another, by (C3-C6)-cycloalkyl, —CO—NH—R9, hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl, phenyl or 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where phenyl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, and R9 represents hydrogen or (C1-C4)-alkyl, or a salt, a hydrate, a hydrate of a salt or a solvate thereof.","3.A compound of the formula (I) in which R1 and R2 independently of one another represent hydrogen, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or —NH—C(O)—CH3, where the alkoxy radicals for their part may be substituted by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or cyclopropyl, or R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O—, R3 represents hydrogen, R4 represents methyl, ethyl, n-propyl, isopropyl, where the alkyl radicals for their part may be substituted by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, cyclopropyl, —NH—C(O)—CH3, furyl, pyridyl, imidazolyl or hexahydropyranyl, or cyclopropyl, R5 represents hydrogen or methyl, or R4 and R5 together with the nitrogen atom to which they are attached represent pyrrolidinyl, morpholinyl, piperidinyl or 4-hydroxypiperidinyl and R6 represents methyl, ethyl or n-propyl, where the alkyl radicals for their part may be substituted up to two times, independently of one another, by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, imidazolyl, nitrofuranyl, pyridyl, phenyl which for its part may in turn be substituted by cyano, nitro, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or amino, —C(O)—NH2 or —C(O)—NH—CH3, or a salt, a hydrate, a hydrate of a salt or a solvate thereof.","4.A process for preparing compounds of the formula (I) as defined in claim 1, characterized in that compounds of the formula (II) in which R1, R2, R3, R4 and R5 are as defined in claim 1 are reacted with compounds of the formula (III) R6—X (III) in which R6 is as defined in claim 1 and X represents a leaving group.","5.A compound of the formula (I) as defined in claim 1 for the prophylaxis and/or treatment of disorders.","6.A composition, comprising at least one compounds of the formula (I) as defined in claim 1 and at least one further auxiliary.","7.The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of disorders of the cardiovascular system (cardiovascular disorders).","8.The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of disorders of the urogenital system and cancer.","9.The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of inflammatory and neuro-inflammatory disorders, neurodegenerative disorders and pain.","10.The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of disorders of the respiratory tract, of liver fibrosis and liver cirrhosis and diabetes."],["The present invention relates to substituted 2-thio-3,5-dicyano-4-aryl-6-amino-pyridines, to a process for their preparation and to their use as medicaments.","Adenosine, a nucleoside consisting of adenine and D-ribose, is an endogenous factor having cell-protective activity, in particular under cell-damaging conditions with limited oxygen and substrate supply, such as, for example, in the case of ischemia in various organs (for example heart and brain).","Adenosine is formed intracellularly as an intermediate during the degradation of adenosine-5′-monophosphate (AMP) and S-adenosylhomocysteine, but it can be released from the cell, in which case it acts as a hormone-like substance or neurotransmitter by binding to specific receptors.","Under normoxic conditions, the concentration of free adenosine in the extracellular space is very low.","However, under ischemic or hypoxic conditions, the extracellular concentration of adenosine in the affected organs is increased dramatically.","Thus, it is known, for example, that adenosine inhibits platelet aggregation and increases the blood supply to the coronary arteries.","Furthermore, it acts on the heart rate, on the release of neurotransmitters and on lymphocyte differentiation.","The aim of these actions of adenosine is to increase the oxygen supply of the affected organs and/or to reduce the metabolism of these organs in order to adjust the metabolism of the organ to the blood supply of the organ under ischemic or hypoxic conditions.","The action of adenosine is mediated via specific receptors.","To date, subtypes A1, A2a, A2b and A3 are known.","The actions of these adenosine receptors are mediated intracellularly by the messenger cAMP.","In the case of the binding of adenosine to the A2a or A2b receptors, the intracellular cAMP is increased via activation of the membrane-bound adenylate cyclase, whereas binding of adenosine to the A1 or A3 receptors results in a decrease of the intracellular cAMP concentration via inhibition of adenylate cyclase.","According to the invention, “adenosine-receptor-selective ligands” are substances which bind selectively to one or more subtypes of the adenosine receptors, thus either mimicking the action of adenosine (adenosine agonists) or blocking its action (adenosine antagonists).","According to their receptor selectivity, adenosine-receptor-selective ligands can be divided into different categories, for example ligands which bind selectively to the A1 or A2 receptors of adenosine and in the case of the latter also, for example, those which bind selectively to the A2a or the A2b receptors of adenosine.","Also possible are adenosine receptor ligands which bind selectively to a plurality of subtypes of the adenosine receptors, for example ligands which bind selectively to the A1 and the A2, but not to the A3 receptors of adenosine.","The abovementioned receptor selectivity can be determined by the effect of the substances on cell lines which, after stable transfection with the corresponding cDNA, express the receptor subtypes in question (see the publication M. E. Olah, H. Ren, J. Ostrowski, K. A. Jacobson, G. L. Stiles, “Cloning, expression, and characterization of the unique bovine A1 adenosine receptor.","Studies on the ligand binding site by, site-directed mutagenesis.” in J. Biol.","Chem.","267 (1992) pages 10764-10770, the disclosure of which is hereby fully incorporated by way of reference).","The effect of the substances on such cell lines can be monitored by biochemical measurement of the intracellular messenger cAMP (see the publication K. N. Klotz, J. Hessling, J. Hegler, C. Owman, B. Kull, B.","B. Fredholm, M. J. Lohse, “Comparative pharmacology of human adenosine receptor subtypes—characterization of stably transfected receptors in CHO cells” in Naunyn Schmiedebergs Arch.","Pharmacol.","357 (1998) pages 1-9, the disclosure of which is hereby fully incorporated by way of reference).","The “adenosine-receptor-specific” ligands known from the prior art are mainly derivatives based on natural adenosine (S.-A.","Poulsen and R. J. Quinn, “Adenosine receptors: new opportunities for future drugs” in Bioorganic and Medicinal Chemistry 6 (1998) pages 619 to 641; K. J. Broadley, “Drugs modulating adenosine receptors as potential therapeutic agents for cardiovascular diseases” in Exp.","Opin.","Ther.","Patents 10 (2000) pages 1669-1692).","However, most of the adenosine ligands known from the prior art have the disadvantage that their action is not really receptor-specific, that their activity is less than that of natural adenosine or that they have only very weak activity after oral administration.","Thus, owing to the aforementioned disadvantages, they are mainly used only for experimental purposes.","It is an object of the present invention to find or provide pharmacologically active substances suitable for the prophylaxis and/or treatment of various disorders, in particular disorders of the cardiovascular system (cardiovascular disorders), the substances preferably acting as adenosine-receptor-selective ligands.","The present invention relates to compounds of the formula (I) in which R1, R2 and R3 independently of one another represent (C1-C8)-alkyl which may be substituted up to three times, independently of one another, by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C2-C4)-alkynyl, halogen or (C6-C10)-aryloxy, (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxy-carbonyl or mono- or di-(C1-C4)-alkylamino, (C1-C8)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C6-C10)-aryl, 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, (C6-C10)-aryloxy, halogen, cyano, (C1-C4)-alkoxycarbonyl, amino or mono- or di-(C1-C4)-alkylamino, hydrogen, hydroxyl, halogen, nitro, cyano, or —NH—C(O)—R7, in which R7 represents (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, or R1 and R2 are attached to adjacent phenyl ring atoms and, together with the two ring carbon atoms, form a 5- to 7-membered saturated or partially unsaturated heterocycle having one or two heteroatoms from the group consisting of N, O and/or S which may be substituted by (C1-C4)-alkyl or oxo, R4 represents (C1-C8)-alkyl which may be substituted by hydroxyl, —NH—CO—R8, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C6-C10)-aryl, 5- or 6-membered saturated or partially unsaturated heterocyclyl having up to three heteroatoms from the group consisting of N, O and/or S or 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, or (C3-C7)-cycloalkyl which may be substituted by hydroxyl or (C1-C8)-alkyl, in which R8 represents (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, R5 represents hydrogen or (C1-C4)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy or (C3-C7)-cycloalkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain one or two further heteroatoms from the group consisting of N, O and/or S in the ring and which may be mono- to trisubstituted, independently of one another, by oxo, fluorine, chlorine, bromine, hydroxyl, (C1-C6)-alkyl or (C1-C6)-alkoxy, and R6 represents (C3-C7)-cycloalkyl or (C1-C8)-alkyl, where alkyl may be substituted up to three times, independently of one another, by (C3-C7)-cycloalkyl, hydroxyl, —CO—NH—R9, (C1-C4)-alkoxy, (C2-C4)-alkenyl, (C6-C10)-aryl or 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where aryl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, and R9 represents hydrogen, (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, and their salts, hydrates, hydrates of the salts and solvates.","Depending on the substitution pattern, the compounds of the formula (I) can exist in stereoisomeric forms which are either like image and mirror image (enantiomers) or not like image and mirror image (diastereomers).","The invention relates both to the enantiomers or diastereomers and to their respective mixtures.","The racemic forms, like the diastereomers, can be separated in a known manner into the stereo-isomerically uniform components.","Likewise, the present invention also relates to the tautomers of the compounds of the formula (I).","Salts of the compounds of the formula (I) can be physiologically acceptable salts of the compounds according to the invention with mineral acids, carboxylic acids, or sulfonic acids.","Particular preference is given, for example, to salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalene-disulfonic acid, trifluoroacetic acid, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.","Salts which may be mentioned include salts with customary bases, such as, for example, alkali metal salts (for example sodium salts or potassium salts), alkaline earth metal salts (for example calcium salts or magnesium salts) or ammonium salts, derived from ammonia or organic amines, such as, for example, diethylamine, triethylamine, ethyldiisopropylamine, procaine, dibenzylamine, N-methyl-morpholine, dihydroabietylamine, 1-ephenamine or methylpiperidine.","According to the invention, hydrates or solvates are those forms of the compounds of the formula (I) which, in solid or liquid state, form, by hydration with water or coordination with solvent molecules, a molecule compound or a complex.","Examples of hydrates are sesquihydrates, monohydrates, dihydrates or trihydrates.","Likewise, the hydrates or solvates of salts of the compounds according to the invention are also suitable.","Moreover, the invention also includes prodrugs of the compounds according to the invention.","According to the invention, prodrugs are forms of compounds of the formula (I) which for their part may be biologically active or inactive, but which can be converted under physiological conditions (for example metabolically or solvolytically) into the corresponding biologically active form.","In the context of the present invention, the substituents have, unless defined otherwise, the following meanings: Halogen generally represents fluorine, chlorine, bromine or iodine.","Preference is given to fluorine, chlorine or bromine.","Very particularly preferred are fluorine or chlorine.","(C1-C8)-Alkyl, (C1-C6)-alkyl and (C1-C4)-alkyl generally represent a straight-chain or branched alkyl radical having 1 to 8, 1 to 6 and 1 to 4 carbon atoms, respectively.","Preference is given to a straight-chain or branched alkyl radical having 1 to 6 carbon atoms.","Particular preference is given to a straight-chain or branched alkyl radical having 1 to 4 carbon atoms.","Examples which may be mentioned are: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl.","(C2-C4-Alkenyl generally represents a straight-chain or branched alkyl radical having 2 to 4 carbon atoms.","Examples which may be mentioned are: vinyl, allyl, isopropenyl and n-but-2-en-1-yl.","(C2-C4)-Alkynyl generally represents a straight-chain or branched alkynyl radical having 2 to 4 carbon atoms.","Examples which may be mentioned are: ethynyl, n-prop-2-yn-1-yl and n-but-2-yn-1-yl.","(C1-C8)-Alkoxy, (C1-C6)-alkoxy and (C1-C4)-alkoxy generally represent a straight-chain or branched alkoxy radical having 1 to 8, 1 to 6 and 1 to 4 carbon atoms, respectively.","Preference is given to a straight-chain or branched alkoxy radical having 1 to 6 carbon atoms.","Particular preference is given to a straight-chain or branched alkoxy radical having 1 to 4 carbon atoms.","Examples which may be mentioned are: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy.","(C1-C4)-Alkoxycarbonyl generally represents a straight-chain or branched alkoxy radical having 1 to 4 carbon atoms which is attached via a carbonyl group.","Examples which may be mentioned are: methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl and t-butoxycarbonyl.","In the context of the invention, mono- or di-(C1-C4)-alkylamino represents an amino group having one or two identical or different straight-chain or branched alkyl substituents each having 1 to 4 carbon atoms.","Examples which may be mentioned are: methylamino, ethylamino, n-propylamino, isopropylamino, t-butylamino, N,N-dimethylamino, N,N-diethylamino, N-ethyl-N-methylamino, N-methyl-N-n-propylamino, N-isopropyl-N-n-propylamino and N-t-butyl-N-methylamino.","(C3-C7)-Cycloalkyl and (C3-C6)-cycloalkyl generally represent a cyclic alkyl radical having 3 to 7 and 3 to 6 carbon atoms, respectively.","Preference is given to cyclic alkyl radicals having 3 to 6 carbon atoms.","Examples which may be mentioned are: cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.","(C6-C10)-Aryl generally represents an aromatic radical having 6 to 10 carbon atoms.","Preferred aryl radicals are phenyl and naphthyl.","(C6-10)-Aryloxy generally represents an aromatic radical as defined above which is attached via an oxygen atom.","5- to 10-membered heteroaryl having up to 3 heteroatoms from the group consisting of N, O and/or S generally represents a mono- or bicyclic, optionally benzo-fused heteroaromatic which is attached via a ring carbon atom of the heteroaromatic, if appropriate also via a ring nitrogen atom of the heteroaromatic.","Examples which may be mentioned are: pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, thienyl, furyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, thiazolyl, oxazolyl, oxdiazolyl, isoxazolyl, benzofuranyl, benzothienyl or benzimidazolyl.","The corresponding heteroaromatics having fewer heteroatoms, such as, for example, those having one or 2 heteroatoms from the group consisting of N, O and/or S, or those having a smaller ring size, such as, for example, 5- or 6-membered heteroaryl, are derived analogously from this definition.","In general, preference is given to 5- or 6-membered aromatic heterocycles having one or 2 heteroatoms from the group consisting of N, O and/or S. Examples which may be mentioned are: pyridyl, pyrimidyl, pyridazinyl, furyl, imidazolyl or thienyl.","5- to 7-membered heterocycle generally represents a saturated or partially unsaturated, optionally benzo-fused heterocycle having up to 3 heteroatoms from the group consisting of N, O and/or S. Examples which may be mentioned are: tetrahydrofuryl, pyrrolidinyl, pyrrolinyl, dihydropyridinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, hexahydropyranyl.","The corresponding heterocycles having fewer heteroatoms, such as, for example, one or 2 heteroatoms from the group consisting of N, O and/or S, or a smaller ring size, such as, for example, 5- or 6-membered heterocyclyl, are derived analogously from this definition.","Preference is given to saturated heterocycles having up to 2 heteroatoms from the group consisting of N, O and/or S, in particular piperidinyl, piperazinyl, morpholinyl and pyrrolidinyl.","Preference is given to compounds of the formula (I) in which R1, R2 and R3 independently of one another represent hydrogen, hydroxyl, (C1-C4)-alkyl, trifluoromethyl, trifluoromethoxy, fluorine, chlorine, (C1-C4)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl or (C3-C6)-cycloalkyl, —NH—C(O)—CH3 or —NH—C(O)—C2H5, or R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O— or —CH2—CH2—O—, R4 represents (C1-C6)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C6)-cycloalkyl, —NH—C(O)—CH3, phenyl, furyl, pyridyl, imidazolyl, thienyl or hexahydropyranyl, or (C3-C6)-cycloalkyl, R5 represents hydrogen or (C1-C4)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy or (C3-C6)-cycloalkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain a further heteroatom from the group consisting of N, O or S in the ring and which may be mono- to trisubstituted, independently of one another, by hydroxyl, (C1-C4)-alkyl or (C1-C4)-alkoxy, and R6 represents (C3-C6)-cycloalkyl, (C1-C6)-alkyl which may be substituted up to two times, independently of one another, by (C3-C6)-cycloalkyl, —CO—NH—R9, hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl, phenyl or 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where phenyl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, and R9 represents hydrogen or (C1-C4)-alkyl, and their salts, hydrates, hydrates of the salts and solvates.","Particular preference is given to compounds of the formula (I) in which R1 and R2 independently of one another represent hydrogen, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or —NH—C(O)—CH3, where the alkoxy radicals for their part may be substituted by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or cyclopropyl, or R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O—, R3 represents hydrogen, R4 represents methyl, ethyl, n-propyl, isopropyl, where the alkyl radicals for their part may be substituted by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, cyclopropyl, —NH—C(O)—CH3, furyl, pyridyl, imidazolyl or hexahydropyranyl, or cyclopropyl, R5 represents hydrogen or methyl, or R4 and R5 together with the nitrogen atom to which they are attached represent pyrrolidinyl, morpholinyl, piperidinyl or 4-hydroxypiperidinyl and R6 represents methyl, ethyl or n-propyl, where the alkyl radicals for their part may be substituted up to two times, independently of one another, by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, imidazolyl, nitrofuranyl, pyridyl, phenyl which for its part may in turn be substituted by cyano, nitro, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or amino, —C(O)—NH2 or —C(O)—NH—CH3, and their salts, hydrates, hydrates of the salts and solvates.","The general or preferred radical definitions or illustrations given above can be combined with one another as desired, i.e.","including combinations between the respective ranges and preferred ranges.","They apply both to the end products and, correspondingly, to precursors and intermediates.","The present invention furthermore relates to a process for preparing the compounds of the formula (I), characterized in that compounds of the formula (II) in which R1, R2, R3, R4 and R5 are as defined above are reacted with compounds of the formula (III) R6—X (III) in which R6 is as defined above and X represents a suitable leaving group in a solvent, if appropriate in the presence of a base.","The process according to the invention can be illustrated in an exemplary manner by the formula scheme below: Suitable solvents for the process according to the invention are all organic solvents which are inert under the reaction conditions.","These include alcohols, such as methanol, ethanol and isopropanol, ketones, such as acetone and methyl ethyl ketone, acyclic and cyclic ethers, such as diethyl ether and tetrahydrofuran, esters, such as ethyl acetate or butyl acetate, hydrocarbons, such as benzene, xylene, toluene, hexane or cyclohexane, chlorinated hydrocarbons, such as dichloromethane, chlorobenzene or dichloroethane, or other solvents, such as dimethylformamide, acetonitrile, pyridine or dimethyl sulfoxide (DMSO).","Water is another suitable solvent.","It is also possible to use mixtures of the solvents mentioned above.","The preferred solvent is dimethylformamide.","Suitable bases are the customary inorganic or organic bases.","These preferably include alkali metal hydroxides, such as, for example, sodium hydroxide or potassium hydroxide, or alkali metal carbonates, such as sodium carbonate or potassium carbonate, or alkali metal bicarbonates, such as sodium bicarbonate or potassium bicarbonate, or alkali metal alkoxides, such as sodium methoxide or potassium methoxide, sodium ethoxide or potassium ethoxide or potassium tert-butoxide, or else amides, such as sodium amide, lithium bis(trimethylsilyl)amide or lithium diisopropylamide, or organometallic compounds, such as butyllithium or phenyllithium, or else amines, such as triethylamine and pyridine.","Preference is given to the alkali metal carbonates and alkali metal bicarbonates.","Suitable leaving groups X in compounds of the formula (III) are, for example: halogen, in particular chlorine, bromine or iodine, or mesylate, tosylate, triflate or 1-imidazolyl, or else a hydroxyl group activated by a Mitsunobu reaction.","The reaction is generally carried out using an equivalent amount or else an excess of compound (III), preferably in a ratio of from 1 to 4 mol of the compound (III), in particular in a ratio of from 1 to 2 mol of the compound (III), per mole of the compound (II).","Here, the base can be employed in a ratio of from 1 to 10 mol, preferably from 1 to 5 mol, in particular from 1 to 4 mol, per mole of the compounds of the formula (II).","The reaction is generally carried out in a temperature range of from −78° C. to +160° C., preferably in a range of from −78° C. to +40° C., in particular at room temperature.","The reaction can be carried out at atmospheric, elevated or reduced pressure (for example in the range from 0.5 to 5 bar).","In general, the reaction is carried out at atmospheric pressure.","Compounds of the formulae (II) can be prepared by converting compounds of the formula (IV) in which R1, R2 and R3 are as defined above with copper(II) chloride and isoamyl nitrite in a suitable solvent into compounds of the formula (V) in which R1, R2 and R3 are as defined above, then reacting them with compounds of the formula (VI) R4—NH—R5 (VI) in which R4 and R5 are as defined above to give compounds of the formula (VII) in which R1, R2, R3, R4 and R5 are as defined above, and finally converting them with sodium sulfide into compounds of the formula (II).","The preparation of compounds of the formula (II) can be illustrated in an exemplary manner by the formula scheme below: The process step (IV)→(V) is generally carried out in a molar ratio of from 2 to 12 mol of copper(II) chloride and from 2 to 12 mol of isoamyl nitrite per mole of (IV).","Suitable solvents for this process step are all organic solvents which are inert under the reaction conditions.","These include, acyclic and cyclic ethers, such as diethyl ether and tetrahydrofuran, esters, such as ethyl acetate or butyl acetate, hydrocarbons, such as benzene, xylene, toluene, hexane or cyclohexane, chlorinated hydrocarbons, such as dichloromethane, chlorobenzene or dichloroethane, or other solvents, such as dimethylformamide, acetonitrile or pyridine.","It is also possible to use mixtures of the solvents mentioned above.","Preferred solvents are acetonitrile and dimethylformamide.","The reaction is generally carried out in a temperature range of from −78° C. to +180° C., preferably in a range of from +20° C. to +100° C., in particular at from +20 to +60° C. The reaction can be carried out at atmospheric, elevated or reduced pressure (for example in the range from 0.5 to 5 bar).","In general, the reaction is carried out at atmospheric pressure.","The process step (V)+(VI)→(VII) is generally carried out in a molar ratio of from 1 to 8 mol of (VI) per mole of (V).","Suitable solvents are all organic solvents which are inert under the reaction conditions.","These include, alcohols, such as methanol, ethanol and isopropanol, ketones, such as acetone and methyl ethyl ketone, acyclic and cyclic ethers, such as diethyl ether and tetrahydrofuran, esters, such as ethyl acetate or butyl acetate, hydrocarbons, such as benzene, xylene, toluene, hexane or cyclohexane, chlorinated hydrocarbons, such as dichloromethane, chlorobenzene or dichloroethane, or other solvents, such as dimethylformamide, acetonitrile, pyridine or dimethyl sulfoxide (DMSO).","Water is another suitable solvent.","It is also possible to use mixtures of the solvents mentioned above.","The preferred solvent is dimethylformamide.","The reaction is generally carried out in a temperature range of from −78° C. to +180° C., preferably in a range of from +20° C. to +160° C., in particular at from +20 to +40° C. The reaction can be carried out at atmospheric, elevated or reduced pressure (for example in the range from 0.5 to 5 bar).","In general, the reaction is carried out at atmospheric pressure.","The process step (VII)→(II) is generally carried out in a molar ratio of from 1 to 8 [lacuna] of sodium sulfide per mole of (VII).","Suitable solvents are all organic solvents which are inert under the reaction conditions.","These include, alcohols, such as methanol, ethanol and isopropanol, ketones, such as acetone and methyl ethyl ketone, acyclic and cyclic ethers, such as diethyl ether and tetrahydrofuran, esters, such as ethyl acetate or butyl acetate, hydrocarbons, such as benzene, xylene, toluene, hexane or cyclohexane, chlorinated hydrocarbons, such as dichloromethane, chlorobenzene or dichloroethane, or other solvents, such as dimethylformamide, acetonitrile, pyridine or dimethyl sulfoxide (DMSO).","It is also possible to use mixtures of the solvents mentioned above.","The preferred solvent is dimethylformamide.","The reaction is generally carried out in a temperature range of from −78° C. to +180° C., preferably in a range of from +20° C. to +160° C., in particular at from +40 to +100° C. The reaction can be carried out at atmospheric, elevated or reduced pressure (for example in the range from 0.5 to 5 bar).","In general, the reaction is carried out at atmospheric pressure.","The compounds of the formula (IV) are known to the person skilled in the art or can be prepared by customary methods known from the literature.","Reference may be made, in particular, to the following publications, whose respective content is incorporated herein by way of reference: Kambe et al., Synthesis 1981, pages 531-533 Elnagdi et al., Z. Naturforsch.","47b, pages 572-578, (1991) The compounds of the formulae (III) and (VI) are either commercially available, known to the person skilled in the art or preparable by customary methods.","Surprisingly, the compounds of the formula (I) have an unforeseeable useful pharmacological activity spectrum and are therefore suitable in particular for the prophylaxis and/or treatment of disorders.","The compounds of the formula (I) are suitable for the prophylaxis and/or treatment of a number of disorders, such as, for example, in particular disorders of the cardiovascular system (cardiovascular disorders).","In the context of the present invention, cardiovascular disorders are to be understood as meaning, in particular, for example the following disorders: coronary heart disease, hypertension (high blood pressure), restenosis, such as, for example, after balloon dilation of peripheral blood vessels, arteriosclerosis, tachycardia, arrhythmias, peripheral vascular disorders and cardiovascular disorders, stable and unstable angina pectoris and atrial fibrillation.","The compounds of the formula (I) are furthermore also particularly suitable, for example, for reducing the size of the myocardial area affected by an infarct.","The compounds of the formula (I) are furthermore particularly suitable, for example, for the prophylaxis and/or treatment of thromboembolic disorders and ischemias, such as myocardial infarction, stroke and transitory ischemic attacks.","Further areas of indication for which the compounds of the formula (I) are suitable are, for example, in particular the prophylaxis and/or treatment of disorders of the urogenital system, such as, for example, an irritable bladder, erectile dysfunction and female sexual dysfunction, and cancer, but additionally also the prophylaxis and/or treatment of inflammatory disorders, such as, for example, asthma and inflammatory dermatoses, of neuroinflammatory disorders of the central nervous system, such as, for example, disorders after stroke, Alzheimer's disease, and furthermore also neurodegenerative disorders, such as Parkinson's disease, and also pain.","A further area of indication is, for example, in particular the prophylaxis and/or treatment of disorders of the respiratory tract, such as, for example, asthma, chronic bronchitis, pulmonary emphysema, bronchiectases, cystic fibrosis (mucoviscidosis) and pulmonary hypertension.","The compounds of the formula (I) are furthermore also suitable, for example, in particular for the prophylaxis and/or treatment of liver fibrosis and liver cirrhosis.","Finally, the compounds of the formula (I) are in particular also suitable, for example, for the prophylaxis and/or treatment of diabetes, in particular diabetes mellitus.","The present invention also relates to the use of the substances of the formula (I) for preparing medicaments and pharmaceutical compositions for the prophylaxis and/or treatment of the clinical pictures mentioned above.","The present invention furthermore relates to a method for the prophylaxis and/or treatment of the clinical pictures mentioned above using the substances of the formula (I).","The pharmaceutical activity of the compounds of the formula (I) mentioned above can be explained by their activity as selective ligands on individual subtypes or a plurality of subtypes of the adenosine receptors, in particular as selective ligands on adenosine A1, adenosine A2a and/or adenosine A2b receptors, preferably as selective ligands on adenosine A1 and/or adenosine A2b receptors.","In the context of the present invention, adenosine receptor ligands are referred to as being “selective” if, firstly, they are clearly active on one or more adenosine receptor subtypes and, secondly, the activity that can be observed on one or more other adenosine receptor subtypes is considerably weaker, if present at all, where, with respect to the test methods for selectivity of action, reference is made to the test methods described in Section A. II.","One advantage of the compounds of the formula (I) according to the invention is that they are more selective than adenosine receptor ligands of the prior art.","In particular, compounds of the formula (I) in which R6 represents an alkyl radical which is substituted by phenyl, alkoxy or hydroxyl generally act selectively on adenosine A1 receptors.","In particular, compounds of the formula (I) in which R6 represents an alkyl radical which is substituted by —CONH2, imidazole or pyridine generally act selectively on adenosine A1 and adenosine A2b receptors.","The receptor selectivity can be determined by biochemical measurement of the intracellular messenger cAMP in cells which specifically only express one subtype of the adenosine receptors.","Here, what is observed, in the case of A2a and A2b agonists (coupling preferably via Gs proteins) and in the case of A2a and A2b antagonists, is an increase of the intracellular cAMP concentration and a decrease of the intracellular cAMP concentration, respectively, following prestimulation with adenosine or adenosine-like substances (see the publications B. Kull, G. Arslan, C. Nilsson, C. Owman, A. Lorenzen, U. Schwabe, B.","B. Fredholm, “Differences in the order of potency for agonists but not antagonists at human and rat adenosine A2A receptors”, Biochem.","Pharmacol., 57 (1999) pages 65-75; and S. P. Alexander, J. Cooper, J.","Shine, S. J. Hill, “Characterization of the human brain putative A2B adenosine receptor expressed in Chinese hamster ovary (CHO.A2B4) cells”, Br.","J.","Pharmacol., 119 (1996) pages 1286-90, the respective content of which is expressly incorporated herein by way of reference).","Correspondingly, A1 agonists (coupling preferably via Gi proteins) and A1 antagonists result in a decrease and increase, respectively, of the cAMP concentration.","Thus, compounds of the formula (I) which bind selectively to adenosine A1 receptors are preferably suitable for myocard protection and for the prophylaxis and/or treatment of tachycardia, atrial arrhythmias, cardiac insufficiency, myocardial infarction, acute kidney failure, diabetes, pain and for wound healing.","Compounds of the formula (I) which bind selectively to adenosine A2a receptors are preferably suitable for the prophylaxis and/or treatment of thromboembolic disorders, of neurodegenerative disorders such as Parkinson's disease and for wound healing.","Compounds of the formula (I) which bind selectively to adenosine A2b-receptors are preferably suitable for the prophylaxis and/or therapy of liver fibrosis, of myocardial infarction, of neuroinflammatory disorders, of Alzheimer's disease, of urogenital incontinence and of disorders of the respiratory tract, such as, for example, asthma and chronic bronchitis.","The present invention also provides medicaments and pharmaceutical compositions comprising at least one compound of the formula (I), preferably together with one or more pharmacologically acceptable auxiliaries or carriers, and their use for the abovementioned purposes.","Suitable for administering the compounds of the formula (I) are all customary administration forms, i.e.","oral, parenteral, inhalative, nasal, sublingual, rectal, local, such as, for example, in the case of implants or stents, or external, such as, for example, transdermal.","In the case of parenteral administration, particular mention may be made of intravenous, intramuscular and subcutaneous administration, for example as a subcutaneous depot.","Particular preference is given to oral administration.","Here, the active compounds can be administered on their own or in the form of preparations.","Suitable preparations for oral administration are inter alia tablets, capsules, pellets, sugar-coated tablets, pills, granules, solid and liquid aerosols, syrups, emulsions, suspensions and solutions.","Here, the active compound has to be present in such a quantity that a therapeutic effect is obtained.","In general, the active compound can be present in a concentration of from 0.1 to 100% by weight, in particular from 0.5 to 90% by weight, preferably from 5 to 80% by weight, i.e.","the active compound should be present in quantities sufficient to achieve the dosage range mentioned.","To this end, the active compounds can be converted in a manner known per se to the customary preparations.","This is achieved using inert nontoxic pharmaceutically suitable carriers, auxiliaries, solvents, vehicles, emulsifiers and/or dispersants.","Auxiliaries which may be mentioned are, for example: water, nontoxic organic solvents, such as, for example, paraffins, vegetable oils (for example sesame oil), alcohols (for example ethanol, glycerol), glycols (for example polyethylene glycol), solid carriers, such as natural or synthetic ground minerals (for example talc or silicates), sugars (for example lactose), emulsifiers, dispersants (for example polyvinylpyrrolidone) and glidants (for example magnesium sulfate).","In the case of oral administration, tablets may, of course, also contain additives such as sodium citrate, together with adjuvants such as starch, gelatin and the like.","Aqueous preparations for oral administration may furthermore be admixed with flavor enhancers or colorants.","In general, it has been found to be advantageous to administer, in the case of parenteral administration, quantities of from about 0.1 to about 10 000 μg/kg, preferably from about 1 to about 1000 μg/kg, in particular from about 1 μg/kg to about 100 μg/kg, of body weight, to obtain effective results.","In the case of oral administration, the quantity is from about 0.1 to about 10 mg/kg, preferably from about 0.5 to about 5 mg/kg, in particular from about 1 to about 4 mg/kg, of body weight.","In spite of this, it may still be necessary, depending on body weight, administration route, individual response to the active compound, the type of preparation and the time or interval at which administration takes place, to deviate from the quantities mentioned.","The present invention is illustrated by the following examples, although these do not restrict the invention in any way.","A. ASSESSING PHYSIOLOGICAL ACTIVITY I. Detecting the Cardiovascular Effect Langendorff Heart of the Rat: After the thorax has been opened, the heart is removed from anesthetized rats and introduced into a conventional Langendorff apparatus.","The coronary arteries are perfused at constant volume (10 ml/min), and the resulting perfusion pressure is recorded by way of an appropriate pressure sensor.","In this set-up, a decrease in the perfusion pressure corresponds to a relaxation of the coronary arteries.","At the same time, the pressure which the heart develops during each contraction is measured by way of a balloon, which has been introduced into the left ventricle, and a second pressure sensor.","The frequency of the heart, which is beating in isolation, is calculated from the number of contractions per time unit.","II.","Assessing the Receptor Selectivity a) Adenosine A1, A2a, A2b and A3 Receptor Selectivity Cells of the CHO (Chinese Hamster Ovary) permanent line are transfected stably with the cDNA for the adenosine receptor subtypes A1, A2a, A2b and A3.The binding of the substances to the A2a or A2b receptor subtypes is determined by measuring the intracellular cAMP content in these cells using a conventional radioimmunological assay (cAMP RIA).","When the substances act as agonists, the binding of the substances is expressed as an increase in the intracellular content of cAMP.","The adenosine-analogous compound NECA (5-N-ethylcarboxamido-adenosine), which binds to all adenosine receptor subtypes with high affinity but not selectively and possesses an agonistic effect, is used as the reference compound in these experiments (Klotz, K. N., Hessling, J., Hegler, J., Owman, C., Kull, B., Fredholm, B.","B., Lohse, M. J., Comparative pharmacology of human adenosine receptor subtypes—characterization of stably transfected receptors in CHO cells, Naunyn Schmiedebergs Arch Pharmacol, 357 (1998), 1-9).","The adenosine receptors A1 and A3 are coupled to a Gi protein, i.e.","stimulation of these receptors leads to inhibition of the adenylate cyclase and consequently to a lowering of the intracellular cAMP level.","In order to identify A1/A3 receptor agonists, the adenylate cyclase is stimulated with forskolin.","However, an additional stimulation of the A1/A3 receptors inhibits the adenylate cyclase, which means that A1/A3 receptor agonists can be detected by a comparatively low content of cAMP in the cell.","In order to detect an antagonistic effect on adenosine receptors, the recombinant cells which are transfected with the corresponding receptor are prestimulated with NECA and the effect of the substances on reducing the intracellular content of cAMP occasioned by this prestimulation is investigated.","XAC (xanthine amine congener), which binds to all adenosine receptor subtypes with high affinity but not selectively and possesses an antagonistic effect, is used as the reference compound in these experiments (Müller, C. E., Stein, B., Adenosine receptor antagonists: structures and potential therapeutic applications, Current Pharmaceutical Design, 2 (1996) 501-530).","b) Adenosine A1, A2a, A2b Receptor Selectivity Cells of the CHO (Chinese Hamster Ovary) permanent line are transfected stably with the cDNA for the adenosine receptor subtypes A1, A2a and A2b.","The adenosine A1 receptors are coupled to the adenylate cyclase by way of Gi proteins, while the adenosine A2a and A2b receptors are coupled by way of Gs proteins.","In correspondence with this, the formation of cAMP in the cell is inhibited or stimulated, respectively.","After that, expression of the luciferase is modulated by way of a cAMP-dependent promoter.","The luciferase test is optimized, with the aim of high sensitivity and reproducibility, low variance and good suitability for implementation on a robot system, by varying several test parameters, such as cell density, duration of the growth phase and the test incubation, forskolin concentration and medium composition.","The following test protocol is used for pharmacologically characterizing the cells and for the robot-assisted substance test screening: The stock cultures is grown, at 37° C. and under 5% CO2, in DMEM/F12 medium containing 10% FCS (fetal calf serum) and in each case split 1:10 after 2-3 days.","The test cultures are seeded in 384-well plates at the rate of from 1 000 to 3 000 cells per well and grown at 37° C. for approx.","48 hours.","The medium is then replaced with a physiological sodium chloride solution (130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 20 mM HEPES, 1 mM MgCl2.6H2O, 5 mM NaHCO3, pH 7.4).","The substances, which are dissolved in DMSO, are diluted 1:10 three times with this physiological sodium chloride solution and pipetted into the test cultures (maximum final concentration of DMSO in the test mixture: 0.5%).","In this way, final substance concentrations of, for example, from 5 μM to 5 nM are obtained.","10 minutes later, forskolin is added to the A1 cells and all the cultures are subsequently incubated at 37° C. for 4 hours.","After that, 35 μl of a solution which is composed of 50% lysis reagent (30 mM disodium hydrogenphosphate, 10% glycerol, 3% TritonX100, 25 mM Tris HCl, 2 mM dithiothreitol (DTT), pH 7.8) and 50% luciferase substrate solution (2.5 mM ATP, 0.5 mM luciferin, 0.1 mM coenzyme A, 10 mM tricine, 1.35 mM MgSO4, 15 mM DTT, pH 7.8) are added to the test cultures, the plates are shaken for approx.","1 minute and the luciferase activity is measured using a camera system.","B. SYNTHESIS EXAMPLES Example 1 Step 1 2-Chloro-4-phenyl-6-(phenylsulfanyl)-3,5-pyridinedicarbonitrile Under argon, 24.5 g (182 mmol) of anhydrous copper(II) chloride are suspended in 180 ml of acetonitrile, and 21.4 g (182 mmol) of isopentyl nitrite are then added dropwise.","After 20 minutes of stirring at room temperature, 10 g (30.5 mmol) of 2-amino-4-phenyl-6-(phenylsulfanyl)-3,5-pyridinedicarbonitrile [Kambe et al., Synthesis, 531-533 (1981)] are added.","The mixture is stirred at room temperature over the weekend.","For work-up, 150 ml of 1 N hydrochloric acid are added to the mixture, the mixture is extracted 4× with dichloromethane and the organic phase is washed once with saturated sodium chloride solution, dried over magnesium sulfate and concentrated under reduced pressure.","The residue is triturated with a little ethyl acetate and allowed to stand in a fridge for one hour.","The crystals are filtered off with suction and washed with a little cold ethyl acetate and diethyl ether.","Yield: 7.26 g (68.5% of theory) of product Mass spectrum: molar mass required: 347, found [M+H]+=348 Step 2 2-Dimethylamino-4-phenyl-6-phenylsulfanyl-3,5-pyridinedicarbonitrile Under argon, 835 mg (2.4 mmol) of 2-chloro-4-phenyl-6-phenylsulfanyl-3,5-pyridinedicarbonitrile (step 1) are dissolved in 4 ml of absolute dimethylformamide (DMF), 0.330 ml of a 2 molar solution of dimethylamine in tetrahydrofuran (THF) is added and the reaction mixture is stirred at room temperature for 2 hours and then purified by preparative HPLC.","Mobile phase: acetonitrile/water (+0.3% trifluoroacetic acid (TFA)), 10/90 to 90/10; 45 ml/min; column: Kromasil 100 C18, 10 μm, 75×30 mm Detection: 220 nm.","The product fraction is concentrated by evaporation and dried under reduced pressure overnight.","Yield: 280 mg (32.7% of theory) of product Mass spectrum: molar mass required: 356, found [M+H]+=357 Step 3 2-Dimethylamino-4-phenyl-6-sulfanyl-3,5-pyridinedicarbonitrile Under argon, 280 mg (0.79 mmol) of 2-dimethylamino-4-phenyl-6-phenylsulfanyl-3,5-pyridinedicarbonitrile (step 2) are dissolved in 3 ml of abs.","dimethylformamide (DMF), and 204 mg (2.6 mmol) of sodium sulfide are added.","The reaction mixture is stirred at 80° C. for 2 hours.","6 ml of 1 N hydrochloric acid are added to the mixture and the resulting yellow crystals are filtered off with suction, washed with water and petroleum ether/diethyl ether 1/1 and dried under reduced pressure overnight.","Yield: 117 mg (53% of theory) of product Mass spectrum: molar mass required: 280, found [M+H]+=281 Step 4 2-Dimethylamino-6-[(2-hydroxyethyl)sulfanyl]-4-phenyl-3,5-pyridinedicarbonitrile 28 mg (0.1 mmol) of 2-dimethylamino-4-phenyl-6-sulfanyl-3,5-pyridinedicarbonitrile (step 3) are, together with 19 mg (0.15 mmol) of 2-bromoethanol and 34 mg (0.4 mmol) of sodium bicarbonate, shaken in 0.6 ml of dimethylformamide (DMF) for about 16 hours.","After filtration, the reaction solution is purified by preparative HPLC.","Column: Grom-Sil 120 ODS-4HE, 5 μm; Mobile phase: acetonitrile/water+0.3% trifluoroacetic acid (TFA) 10/90 to 90/10; 25 ml/min; Detection: 254 nm.","The product fraction is concentrated by evaporation under reduced pressure.","Yield: 23 mg (71% of theory) of product Mass spectrum: molar mass required: 324, found [M+H]+=325 1H-NMR spectrum [DMSO-d6]: δ=3.35 [6H] s; 3.35 [2H] tr; 3.7 [2H] quartet; 5.0 [1H] tr; 7.55 [5H] m. The compounds listed in the table below (examples 2 to 90) are prepared analogously to the procedures of example 1 given above.","The identity and purity of the compounds is examined by LC-MS. Molar Yield Ex.","mass Found (% of No.","Target structure required [M + H]+ theory) NMR data 2 324 325 54 3 370 371 44 4 354 355 66 5 405 406 54 6 354 355 69 7 401 402 52 8 435 436 48 9 310 311 55 1H-NMR spectrum [DMSO-d6]: δ =3.05[3H] d; 3.35[2H] tr; 3.7[2H]quartet; 5.0[1H] tr; 7.55[5H] m; 8.15[1H] broad.","10 356 357 39 11 340 341 49 12 391 392 37 13 340 341 76 14 386 387 57 1H-NMR spectrum [DMSO-d6]: δ =3.6[4H] m; 4.55[2H] s; 4.8[1H] tr; 7.25-7.6[10H] m; 8.0[1H] broad 15 421 422 58 16 367 368 50 17 350 351 40 18 363 364 22 19 397 398 36 20 431 432 39 21 360 361 63 22 390 391 60 23 346 347 36 24 387 388 31 25 393 394 38 26 406 407 14 27 441 442 72 28 470 471 44 29 471 472 61 30 444 445 73 31 445 446 44 32 411 412 42 33 458 459 56 34 459 460 63 35 411 412 28 36 458 459 72 37 459 460 55 38 484 485 73 39 485 486 53 40 457 458 69 41 455 456 51 42 381 382 41 43 428 429 40 44 429 430 52 45 395 396 46 46 367 368 39 47 380 381 34 48 414 415 39 49 414 415 58 50 381 382 38 51 439 440 56 52 485 486 53 53 455 456 60 54 337 338 71 55 351 352 51 56 470 471 51 57 434 435 41 58 448 449 47 59 488 489 63 60 484 485 48 61 448 449 31 62 474 475 45 63 432 433 68 64 446 447 51 65 444 445 62 66 481 482 38 67 418 419 57 68 417 418 65 1H-NMR spectrum [DMSO-d6]: δ =3.0[3H] d; 3.75[2H] quartet; 4.05[2H] tr; 4.6[2H] s; 4.95[1H] tr; 7.05-7.5[3H] m; 8.15[1H] quartet 69 431 432 33 70 447 448 6 1H-NMR spectrum [DMSO-d6]: δ =3.6[4H] m; 3.7[2H] quartet; 4.1[2H] tr; 4.5[2H] s; 4.8[1H] tr; 4.9[1H] tr; 7.05-7.5[3H] m; 7.95[1H] s broad 71 400 401 101 72 414 415 84 73 430 431 89 74 445 446 112 75 426 427 89 76 441 442 90 77 445 446 93 78 445 446 72 79 355 355 77 1H-NMR spectrum [DMSO-d6]: δ =3.0[3H] d; 3.4[2H] tr; 3.7[2H] quartet; 5.05[1H] tr; 6.15[2H] s; 6.95-7.15[3H] m; 8.1[1H] s broad 80 368 369 82 1H-NMR spectrum [DMSO-d6]: δ =1.2[3H] tr; 3.4[2H] tr; 3.5[2H] quintet; 3.65[2H] quartet; 5.05[1H] tr; 6.15[2H] s; 6.95-7.15[3H] m; 8.2[1H] tr 81 384 385 83 82 398 399 82 83 380 381 88 1H-NMR spectrum [DMSO-d6]: δ =0.65-0.9[4H] m; 2.9[1H] m; 3.4[2H] tr; 3.7[2H] tr; 5.0[1H] s broad; 6.15[2H] s; 6.95-7.15[3H] m; 8.3[1H] s broad 84 401 402 74 1H-NMR spectrum [DMSO-d6]: δ =2.95[3H] s; 4.2[2H] s; 6.1[2H] s; 6.95-7.15[3H] m; 7.3[1H] tr; 7.5[1H] d; 7.75[1H] tr; 8.1[1H] s; 8.5[1H] s 85 368 369 88 86 382 383 88 87 398 399 93 88 412 413 84 89 382 383 85 90 394 395 89"]],"string":"[\n [\n \"Adenosine receptor selective modulators\",\n \"The invention relates to the compounds of formula (I), to a method for producing the same and to the use thereof as medicaments.\"\n ],\n [\n \"1.A compound of the formula (I) in which R1, R2 and R3 independently of one another represent (C1-C8)-alkyl which may be substituted up to three times, independently of one another, by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C2-C4)-alkynyl, halogen or (C6-C10)-aryloxy, (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxy-carbonyl or mono- or di-(C1-C4)-alkylamino, (C1-C8)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C6-C10)-aryl, 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, (C6-C10)-aryloxy, halogen, cyano, (C1-C4)-alkoxycarbonyl, amino or mono- or di-(C1-C4)-alkylamino, hydrogen, hydroxyl, halogen, nitro, cyano, or —NH—C(O)—R7, in which R7 represents (C1-C4)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, or R1 and R2 are attached to adjacent phenyl ring atoms and, together with the two ring carbon atoms, form a 5- to 7-membered saturated or partially unsaturated heterocycle having one or two heteroatoms from the group consisting of N, O and/or S which may be substituted by (C1-C4)-alkyl or oxo, R4 represents (C1-C8)-alkyl which may be substituted by hydroxyl, —NH—CO—R8, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C6-C10)-aryl, 5- or 6-membered saturated or partially unsaturated heterocyclyl having up to three heteroatoms from the group consisting of N, O and/or S or 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, or (C3-C7)-cycloalkyl which may be substituted by hydroxyl or (C1-C8)-alkyl, in which R8 represents (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, R5 represents hydrogen or (C1-C4)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy or (C3-C7)-cycloalkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain one or two further heteroatoms from the group consisting of N, O and/or S in the ring and which may be mono- to trisubstituted, independently of one another, by oxo, fluorine, chlorine, bromine, hydroxyl, (C1-C6)-alkyl or (C1-C6)-alkoxy, and R6 represents (C3-C7)-cycloalkyl or (C1-C8)-alkyl, where alkyl may be substituted up to three times, independently of one another, by (C3-C7)-cyclo-alkyl, hydroxyl, —CO—NH—R9, (C1-C4)-alkoxy, (C2-C4)-alkenyl, (C6-C10)-aryl or 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where aryl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, and R9 represents hydrogen, (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, or a salt, a hydrate, a hydrate of a salt or a solvate thereof.\",\n \"2.A compound of the formula (I) in which R1, R2 and R3 independently of one another represent hydrogen, hydroxyl, (C1-C4)-alkyl, trifluoromethyl, trifluoromethoxy, fluorine, chlorine, (C1-C4)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl or (C3-C6)-cycloalkyl, —NH—C(O)—CH3 or —NH—C(O)—C2H5, or R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O— or —CH2—CH2—O—, R4 represents (C1-C6)-alkyl which may be substituted by hydroxyl, (C1-C4)— alkoxy, (C3-C6)-cycloalkyl, —NH—C(O)—CH3, phenyl, furyl, pyridyl, imidazolyl, thienyl or hexahydropyranyl, or (C3-C6)-cycloalkyl, R5 represents hydrogen or (C1-C4)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy or (C3-C6)-cycloalkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain a further heteroatom from the group consisting of N; O or S in the ring and which may be mono- to trisubstituted, independently of one another, by hydroxyl, (C1-C4)-alkyl or (C1-C4)-alkoxy, and R6 represents (C3-C6)-cycloalkyl, (C1-C6)-alkyl which may be substituted up to two times, independently of one another, by (C3-C6)-cycloalkyl, —CO—NH—R9, hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl, phenyl or 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where phenyl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, and R9 represents hydrogen or (C1-C4)-alkyl, or a salt, a hydrate, a hydrate of a salt or a solvate thereof.\",\n \"3.A compound of the formula (I) in which R1 and R2 independently of one another represent hydrogen, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or —NH—C(O)—CH3, where the alkoxy radicals for their part may be substituted by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or cyclopropyl, or R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O—, R3 represents hydrogen, R4 represents methyl, ethyl, n-propyl, isopropyl, where the alkyl radicals for their part may be substituted by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, cyclopropyl, —NH—C(O)—CH3, furyl, pyridyl, imidazolyl or hexahydropyranyl, or cyclopropyl, R5 represents hydrogen or methyl, or R4 and R5 together with the nitrogen atom to which they are attached represent pyrrolidinyl, morpholinyl, piperidinyl or 4-hydroxypiperidinyl and R6 represents methyl, ethyl or n-propyl, where the alkyl radicals for their part may be substituted up to two times, independently of one another, by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, imidazolyl, nitrofuranyl, pyridyl, phenyl which for its part may in turn be substituted by cyano, nitro, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or amino, —C(O)—NH2 or —C(O)—NH—CH3, or a salt, a hydrate, a hydrate of a salt or a solvate thereof.\",\n \"4.A process for preparing compounds of the formula (I) as defined in claim 1, characterized in that compounds of the formula (II) in which R1, R2, R3, R4 and R5 are as defined in claim 1 are reacted with compounds of the formula (III) R6—X (III) in which R6 is as defined in claim 1 and X represents a leaving group.\",\n \"5.A compound of the formula (I) as defined in claim 1 for the prophylaxis and/or treatment of disorders.\",\n \"6.A composition, comprising at least one compounds of the formula (I) as defined in claim 1 and at least one further auxiliary.\",\n \"7.The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of disorders of the cardiovascular system (cardiovascular disorders).\",\n \"8.The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of disorders of the urogenital system and cancer.\",\n \"9.The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of inflammatory and neuro-inflammatory disorders, neurodegenerative disorders and pain.\",\n \"10.The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of disorders of the respiratory tract, of liver fibrosis and liver cirrhosis and diabetes.\"\n ],\n [\n \"The present invention relates to substituted 2-thio-3,5-dicyano-4-aryl-6-amino-pyridines, to a process for their preparation and to their use as medicaments.\",\n \"Adenosine, a nucleoside consisting of adenine and D-ribose, is an endogenous factor having cell-protective activity, in particular under cell-damaging conditions with limited oxygen and substrate supply, such as, for example, in the case of ischemia in various organs (for example heart and brain).\",\n \"Adenosine is formed intracellularly as an intermediate during the degradation of adenosine-5′-monophosphate (AMP) and S-adenosylhomocysteine, but it can be released from the cell, in which case it acts as a hormone-like substance or neurotransmitter by binding to specific receptors.\",\n \"Under normoxic conditions, the concentration of free adenosine in the extracellular space is very low.\",\n \"However, under ischemic or hypoxic conditions, the extracellular concentration of adenosine in the affected organs is increased dramatically.\",\n \"Thus, it is known, for example, that adenosine inhibits platelet aggregation and increases the blood supply to the coronary arteries.\",\n \"Furthermore, it acts on the heart rate, on the release of neurotransmitters and on lymphocyte differentiation.\",\n \"The aim of these actions of adenosine is to increase the oxygen supply of the affected organs and/or to reduce the metabolism of these organs in order to adjust the metabolism of the organ to the blood supply of the organ under ischemic or hypoxic conditions.\",\n \"The action of adenosine is mediated via specific receptors.\",\n \"To date, subtypes A1, A2a, A2b and A3 are known.\",\n \"The actions of these adenosine receptors are mediated intracellularly by the messenger cAMP.\",\n \"In the case of the binding of adenosine to the A2a or A2b receptors, the intracellular cAMP is increased via activation of the membrane-bound adenylate cyclase, whereas binding of adenosine to the A1 or A3 receptors results in a decrease of the intracellular cAMP concentration via inhibition of adenylate cyclase.\",\n \"According to the invention, “adenosine-receptor-selective ligands” are substances which bind selectively to one or more subtypes of the adenosine receptors, thus either mimicking the action of adenosine (adenosine agonists) or blocking its action (adenosine antagonists).\",\n \"According to their receptor selectivity, adenosine-receptor-selective ligands can be divided into different categories, for example ligands which bind selectively to the A1 or A2 receptors of adenosine and in the case of the latter also, for example, those which bind selectively to the A2a or the A2b receptors of adenosine.\",\n \"Also possible are adenosine receptor ligands which bind selectively to a plurality of subtypes of the adenosine receptors, for example ligands which bind selectively to the A1 and the A2, but not to the A3 receptors of adenosine.\",\n \"The abovementioned receptor selectivity can be determined by the effect of the substances on cell lines which, after stable transfection with the corresponding cDNA, express the receptor subtypes in question (see the publication M. E. Olah, H. Ren, J. Ostrowski, K. A. Jacobson, G. L. Stiles, “Cloning, expression, and characterization of the unique bovine A1 adenosine receptor.\",\n \"Studies on the ligand binding site by, site-directed mutagenesis.” in J. Biol.\",\n \"Chem.\",\n \"267 (1992) pages 10764-10770, the disclosure of which is hereby fully incorporated by way of reference).\",\n \"The effect of the substances on such cell lines can be monitored by biochemical measurement of the intracellular messenger cAMP (see the publication K. N. Klotz, J. Hessling, J. Hegler, C. Owman, B. Kull, B.\",\n \"B. Fredholm, M. J. Lohse, “Comparative pharmacology of human adenosine receptor subtypes—characterization of stably transfected receptors in CHO cells” in Naunyn Schmiedebergs Arch.\",\n \"Pharmacol.\",\n \"357 (1998) pages 1-9, the disclosure of which is hereby fully incorporated by way of reference).\",\n \"The “adenosine-receptor-specific” ligands known from the prior art are mainly derivatives based on natural adenosine (S.-A.\",\n \"Poulsen and R. J. Quinn, “Adenosine receptors: new opportunities for future drugs” in Bioorganic and Medicinal Chemistry 6 (1998) pages 619 to 641; K. J. Broadley, “Drugs modulating adenosine receptors as potential therapeutic agents for cardiovascular diseases” in Exp.\",\n \"Opin.\",\n \"Ther.\",\n \"Patents 10 (2000) pages 1669-1692).\",\n \"However, most of the adenosine ligands known from the prior art have the disadvantage that their action is not really receptor-specific, that their activity is less than that of natural adenosine or that they have only very weak activity after oral administration.\",\n \"Thus, owing to the aforementioned disadvantages, they are mainly used only for experimental purposes.\",\n \"It is an object of the present invention to find or provide pharmacologically active substances suitable for the prophylaxis and/or treatment of various disorders, in particular disorders of the cardiovascular system (cardiovascular disorders), the substances preferably acting as adenosine-receptor-selective ligands.\",\n \"The present invention relates to compounds of the formula (I) in which R1, R2 and R3 independently of one another represent (C1-C8)-alkyl which may be substituted up to three times, independently of one another, by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C2-C4)-alkynyl, halogen or (C6-C10)-aryloxy, (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxy-carbonyl or mono- or di-(C1-C4)-alkylamino, (C1-C8)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C6-C10)-aryl, 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, (C6-C10)-aryloxy, halogen, cyano, (C1-C4)-alkoxycarbonyl, amino or mono- or di-(C1-C4)-alkylamino, hydrogen, hydroxyl, halogen, nitro, cyano, or —NH—C(O)—R7, in which R7 represents (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, or R1 and R2 are attached to adjacent phenyl ring atoms and, together with the two ring carbon atoms, form a 5- to 7-membered saturated or partially unsaturated heterocycle having one or two heteroatoms from the group consisting of N, O and/or S which may be substituted by (C1-C4)-alkyl or oxo, R4 represents (C1-C8)-alkyl which may be substituted by hydroxyl, —NH—CO—R8, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C6-C10)-aryl, 5- or 6-membered saturated or partially unsaturated heterocyclyl having up to three heteroatoms from the group consisting of N, O and/or S or 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, or (C3-C7)-cycloalkyl which may be substituted by hydroxyl or (C1-C8)-alkyl, in which R8 represents (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, R5 represents hydrogen or (C1-C4)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy or (C3-C7)-cycloalkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain one or two further heteroatoms from the group consisting of N, O and/or S in the ring and which may be mono- to trisubstituted, independently of one another, by oxo, fluorine, chlorine, bromine, hydroxyl, (C1-C6)-alkyl or (C1-C6)-alkoxy, and R6 represents (C3-C7)-cycloalkyl or (C1-C8)-alkyl, where alkyl may be substituted up to three times, independently of one another, by (C3-C7)-cycloalkyl, hydroxyl, —CO—NH—R9, (C1-C4)-alkoxy, (C2-C4)-alkenyl, (C6-C10)-aryl or 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where aryl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, and R9 represents hydrogen, (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, and their salts, hydrates, hydrates of the salts and solvates.\",\n \"Depending on the substitution pattern, the compounds of the formula (I) can exist in stereoisomeric forms which are either like image and mirror image (enantiomers) or not like image and mirror image (diastereomers).\",\n \"The invention relates both to the enantiomers or diastereomers and to their respective mixtures.\",\n \"The racemic forms, like the diastereomers, can be separated in a known manner into the stereo-isomerically uniform components.\",\n \"Likewise, the present invention also relates to the tautomers of the compounds of the formula (I).\",\n \"Salts of the compounds of the formula (I) can be physiologically acceptable salts of the compounds according to the invention with mineral acids, carboxylic acids, or sulfonic acids.\",\n \"Particular preference is given, for example, to salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalene-disulfonic acid, trifluoroacetic acid, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.\",\n \"Salts which may be mentioned include salts with customary bases, such as, for example, alkali metal salts (for example sodium salts or potassium salts), alkaline earth metal salts (for example calcium salts or magnesium salts) or ammonium salts, derived from ammonia or organic amines, such as, for example, diethylamine, triethylamine, ethyldiisopropylamine, procaine, dibenzylamine, N-methyl-morpholine, dihydroabietylamine, 1-ephenamine or methylpiperidine.\",\n \"According to the invention, hydrates or solvates are those forms of the compounds of the formula (I) which, in solid or liquid state, form, by hydration with water or coordination with solvent molecules, a molecule compound or a complex.\",\n \"Examples of hydrates are sesquihydrates, monohydrates, dihydrates or trihydrates.\",\n \"Likewise, the hydrates or solvates of salts of the compounds according to the invention are also suitable.\",\n \"Moreover, the invention also includes prodrugs of the compounds according to the invention.\",\n \"According to the invention, prodrugs are forms of compounds of the formula (I) which for their part may be biologically active or inactive, but which can be converted under physiological conditions (for example metabolically or solvolytically) into the corresponding biologically active form.\",\n \"In the context of the present invention, the substituents have, unless defined otherwise, the following meanings: Halogen generally represents fluorine, chlorine, bromine or iodine.\",\n \"Preference is given to fluorine, chlorine or bromine.\",\n \"Very particularly preferred are fluorine or chlorine.\",\n \"(C1-C8)-Alkyl, (C1-C6)-alkyl and (C1-C4)-alkyl generally represent a straight-chain or branched alkyl radical having 1 to 8, 1 to 6 and 1 to 4 carbon atoms, respectively.\",\n \"Preference is given to a straight-chain or branched alkyl radical having 1 to 6 carbon atoms.\",\n \"Particular preference is given to a straight-chain or branched alkyl radical having 1 to 4 carbon atoms.\",\n \"Examples which may be mentioned are: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl.\",\n \"(C2-C4-Alkenyl generally represents a straight-chain or branched alkyl radical having 2 to 4 carbon atoms.\",\n \"Examples which may be mentioned are: vinyl, allyl, isopropenyl and n-but-2-en-1-yl.\",\n \"(C2-C4)-Alkynyl generally represents a straight-chain or branched alkynyl radical having 2 to 4 carbon atoms.\",\n \"Examples which may be mentioned are: ethynyl, n-prop-2-yn-1-yl and n-but-2-yn-1-yl.\",\n \"(C1-C8)-Alkoxy, (C1-C6)-alkoxy and (C1-C4)-alkoxy generally represent a straight-chain or branched alkoxy radical having 1 to 8, 1 to 6 and 1 to 4 carbon atoms, respectively.\",\n \"Preference is given to a straight-chain or branched alkoxy radical having 1 to 6 carbon atoms.\",\n \"Particular preference is given to a straight-chain or branched alkoxy radical having 1 to 4 carbon atoms.\",\n \"Examples which may be mentioned are: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy.\",\n \"(C1-C4)-Alkoxycarbonyl generally represents a straight-chain or branched alkoxy radical having 1 to 4 carbon atoms which is attached via a carbonyl group.\",\n \"Examples which may be mentioned are: methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl and t-butoxycarbonyl.\",\n \"In the context of the invention, mono- or di-(C1-C4)-alkylamino represents an amino group having one or two identical or different straight-chain or branched alkyl substituents each having 1 to 4 carbon atoms.\",\n \"Examples which may be mentioned are: methylamino, ethylamino, n-propylamino, isopropylamino, t-butylamino, N,N-dimethylamino, N,N-diethylamino, N-ethyl-N-methylamino, N-methyl-N-n-propylamino, N-isopropyl-N-n-propylamino and N-t-butyl-N-methylamino.\",\n \"(C3-C7)-Cycloalkyl and (C3-C6)-cycloalkyl generally represent a cyclic alkyl radical having 3 to 7 and 3 to 6 carbon atoms, respectively.\",\n \"Preference is given to cyclic alkyl radicals having 3 to 6 carbon atoms.\",\n \"Examples which may be mentioned are: cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.\",\n \"(C6-C10)-Aryl generally represents an aromatic radical having 6 to 10 carbon atoms.\",\n \"Preferred aryl radicals are phenyl and naphthyl.\",\n \"(C6-10)-Aryloxy generally represents an aromatic radical as defined above which is attached via an oxygen atom.\",\n \"5- to 10-membered heteroaryl having up to 3 heteroatoms from the group consisting of N, O and/or S generally represents a mono- or bicyclic, optionally benzo-fused heteroaromatic which is attached via a ring carbon atom of the heteroaromatic, if appropriate also via a ring nitrogen atom of the heteroaromatic.\",\n \"Examples which may be mentioned are: pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, thienyl, furyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, thiazolyl, oxazolyl, oxdiazolyl, isoxazolyl, benzofuranyl, benzothienyl or benzimidazolyl.\",\n \"The corresponding heteroaromatics having fewer heteroatoms, such as, for example, those having one or 2 heteroatoms from the group consisting of N, O and/or S, or those having a smaller ring size, such as, for example, 5- or 6-membered heteroaryl, are derived analogously from this definition.\",\n \"In general, preference is given to 5- or 6-membered aromatic heterocycles having one or 2 heteroatoms from the group consisting of N, O and/or S. Examples which may be mentioned are: pyridyl, pyrimidyl, pyridazinyl, furyl, imidazolyl or thienyl.\",\n \"5- to 7-membered heterocycle generally represents a saturated or partially unsaturated, optionally benzo-fused heterocycle having up to 3 heteroatoms from the group consisting of N, O and/or S. Examples which may be mentioned are: tetrahydrofuryl, pyrrolidinyl, pyrrolinyl, dihydropyridinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, hexahydropyranyl.\",\n \"The corresponding heterocycles having fewer heteroatoms, such as, for example, one or 2 heteroatoms from the group consisting of N, O and/or S, or a smaller ring size, such as, for example, 5- or 6-membered heterocyclyl, are derived analogously from this definition.\",\n \"Preference is given to saturated heterocycles having up to 2 heteroatoms from the group consisting of N, O and/or S, in particular piperidinyl, piperazinyl, morpholinyl and pyrrolidinyl.\",\n \"Preference is given to compounds of the formula (I) in which R1, R2 and R3 independently of one another represent hydrogen, hydroxyl, (C1-C4)-alkyl, trifluoromethyl, trifluoromethoxy, fluorine, chlorine, (C1-C4)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl or (C3-C6)-cycloalkyl, —NH—C(O)—CH3 or —NH—C(O)—C2H5, or R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O— or —CH2—CH2—O—, R4 represents (C1-C6)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C6)-cycloalkyl, —NH—C(O)—CH3, phenyl, furyl, pyridyl, imidazolyl, thienyl or hexahydropyranyl, or (C3-C6)-cycloalkyl, R5 represents hydrogen or (C1-C4)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy or (C3-C6)-cycloalkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain a further heteroatom from the group consisting of N, O or S in the ring and which may be mono- to trisubstituted, independently of one another, by hydroxyl, (C1-C4)-alkyl or (C1-C4)-alkoxy, and R6 represents (C3-C6)-cycloalkyl, (C1-C6)-alkyl which may be substituted up to two times, independently of one another, by (C3-C6)-cycloalkyl, —CO—NH—R9, hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl, phenyl or 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where phenyl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, and R9 represents hydrogen or (C1-C4)-alkyl, and their salts, hydrates, hydrates of the salts and solvates.\",\n \"Particular preference is given to compounds of the formula (I) in which R1 and R2 independently of one another represent hydrogen, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or —NH—C(O)—CH3, where the alkoxy radicals for their part may be substituted by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or cyclopropyl, or R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O—, R3 represents hydrogen, R4 represents methyl, ethyl, n-propyl, isopropyl, where the alkyl radicals for their part may be substituted by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, cyclopropyl, —NH—C(O)—CH3, furyl, pyridyl, imidazolyl or hexahydropyranyl, or cyclopropyl, R5 represents hydrogen or methyl, or R4 and R5 together with the nitrogen atom to which they are attached represent pyrrolidinyl, morpholinyl, piperidinyl or 4-hydroxypiperidinyl and R6 represents methyl, ethyl or n-propyl, where the alkyl radicals for their part may be substituted up to two times, independently of one another, by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, imidazolyl, nitrofuranyl, pyridyl, phenyl which for its part may in turn be substituted by cyano, nitro, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or amino, —C(O)—NH2 or —C(O)—NH—CH3, and their salts, hydrates, hydrates of the salts and solvates.\",\n \"The general or preferred radical definitions or illustrations given above can be combined with one another as desired, i.e.\",\n \"including combinations between the respective ranges and preferred ranges.\",\n \"They apply both to the end products and, correspondingly, to precursors and intermediates.\",\n \"The present invention furthermore relates to a process for preparing the compounds of the formula (I), characterized in that compounds of the formula (II) in which R1, R2, R3, R4 and R5 are as defined above are reacted with compounds of the formula (III) R6—X (III) in which R6 is as defined above and X represents a suitable leaving group in a solvent, if appropriate in the presence of a base.\",\n \"The process according to the invention can be illustrated in an exemplary manner by the formula scheme below: Suitable solvents for the process according to the invention are all organic solvents which are inert under the reaction conditions.\",\n \"These include alcohols, such as methanol, ethanol and isopropanol, ketones, such as acetone and methyl ethyl ketone, acyclic and cyclic ethers, such as diethyl ether and tetrahydrofuran, esters, such as ethyl acetate or butyl acetate, hydrocarbons, such as benzene, xylene, toluene, hexane or cyclohexane, chlorinated hydrocarbons, such as dichloromethane, chlorobenzene or dichloroethane, or other solvents, such as dimethylformamide, acetonitrile, pyridine or dimethyl sulfoxide (DMSO).\",\n \"Water is another suitable solvent.\",\n \"It is also possible to use mixtures of the solvents mentioned above.\",\n \"The preferred solvent is dimethylformamide.\",\n \"Suitable bases are the customary inorganic or organic bases.\",\n \"These preferably include alkali metal hydroxides, such as, for example, sodium hydroxide or potassium hydroxide, or alkali metal carbonates, such as sodium carbonate or potassium carbonate, or alkali metal bicarbonates, such as sodium bicarbonate or potassium bicarbonate, or alkali metal alkoxides, such as sodium methoxide or potassium methoxide, sodium ethoxide or potassium ethoxide or potassium tert-butoxide, or else amides, such as sodium amide, lithium bis(trimethylsilyl)amide or lithium diisopropylamide, or organometallic compounds, such as butyllithium or phenyllithium, or else amines, such as triethylamine and pyridine.\",\n \"Preference is given to the alkali metal carbonates and alkali metal bicarbonates.\",\n \"Suitable leaving groups X in compounds of the formula (III) are, for example: halogen, in particular chlorine, bromine or iodine, or mesylate, tosylate, triflate or 1-imidazolyl, or else a hydroxyl group activated by a Mitsunobu reaction.\",\n \"The reaction is generally carried out using an equivalent amount or else an excess of compound (III), preferably in a ratio of from 1 to 4 mol of the compound (III), in particular in a ratio of from 1 to 2 mol of the compound (III), per mole of the compound (II).\",\n \"Here, the base can be employed in a ratio of from 1 to 10 mol, preferably from 1 to 5 mol, in particular from 1 to 4 mol, per mole of the compounds of the formula (II).\",\n \"The reaction is generally carried out in a temperature range of from −78° C. to +160° C., preferably in a range of from −78° C. to +40° C., in particular at room temperature.\",\n \"The reaction can be carried out at atmospheric, elevated or reduced pressure (for example in the range from 0.5 to 5 bar).\",\n \"In general, the reaction is carried out at atmospheric pressure.\",\n \"Compounds of the formulae (II) can be prepared by converting compounds of the formula (IV) in which R1, R2 and R3 are as defined above with copper(II) chloride and isoamyl nitrite in a suitable solvent into compounds of the formula (V) in which R1, R2 and R3 are as defined above, then reacting them with compounds of the formula (VI) R4—NH—R5 (VI) in which R4 and R5 are as defined above to give compounds of the formula (VII) in which R1, R2, R3, R4 and R5 are as defined above, and finally converting them with sodium sulfide into compounds of the formula (II).\",\n \"The preparation of compounds of the formula (II) can be illustrated in an exemplary manner by the formula scheme below: The process step (IV)→(V) is generally carried out in a molar ratio of from 2 to 12 mol of copper(II) chloride and from 2 to 12 mol of isoamyl nitrite per mole of (IV).\",\n \"Suitable solvents for this process step are all organic solvents which are inert under the reaction conditions.\",\n \"These include, acyclic and cyclic ethers, such as diethyl ether and tetrahydrofuran, esters, such as ethyl acetate or butyl acetate, hydrocarbons, such as benzene, xylene, toluene, hexane or cyclohexane, chlorinated hydrocarbons, such as dichloromethane, chlorobenzene or dichloroethane, or other solvents, such as dimethylformamide, acetonitrile or pyridine.\",\n \"It is also possible to use mixtures of the solvents mentioned above.\",\n \"Preferred solvents are acetonitrile and dimethylformamide.\",\n \"The reaction is generally carried out in a temperature range of from −78° C. to +180° C., preferably in a range of from +20° C. to +100° C., in particular at from +20 to +60° C. The reaction can be carried out at atmospheric, elevated or reduced pressure (for example in the range from 0.5 to 5 bar).\",\n \"In general, the reaction is carried out at atmospheric pressure.\",\n \"The process step (V)+(VI)→(VII) is generally carried out in a molar ratio of from 1 to 8 mol of (VI) per mole of (V).\",\n \"Suitable solvents are all organic solvents which are inert under the reaction conditions.\",\n \"These include, alcohols, such as methanol, ethanol and isopropanol, ketones, such as acetone and methyl ethyl ketone, acyclic and cyclic ethers, such as diethyl ether and tetrahydrofuran, esters, such as ethyl acetate or butyl acetate, hydrocarbons, such as benzene, xylene, toluene, hexane or cyclohexane, chlorinated hydrocarbons, such as dichloromethane, chlorobenzene or dichloroethane, or other solvents, such as dimethylformamide, acetonitrile, pyridine or dimethyl sulfoxide (DMSO).\",\n \"Water is another suitable solvent.\",\n \"It is also possible to use mixtures of the solvents mentioned above.\",\n \"The preferred solvent is dimethylformamide.\",\n \"The reaction is generally carried out in a temperature range of from −78° C. to +180° C., preferably in a range of from +20° C. to +160° C., in particular at from +20 to +40° C. The reaction can be carried out at atmospheric, elevated or reduced pressure (for example in the range from 0.5 to 5 bar).\",\n \"In general, the reaction is carried out at atmospheric pressure.\",\n \"The process step (VII)→(II) is generally carried out in a molar ratio of from 1 to 8 [lacuna] of sodium sulfide per mole of (VII).\",\n \"Suitable solvents are all organic solvents which are inert under the reaction conditions.\",\n \"These include, alcohols, such as methanol, ethanol and isopropanol, ketones, such as acetone and methyl ethyl ketone, acyclic and cyclic ethers, such as diethyl ether and tetrahydrofuran, esters, such as ethyl acetate or butyl acetate, hydrocarbons, such as benzene, xylene, toluene, hexane or cyclohexane, chlorinated hydrocarbons, such as dichloromethane, chlorobenzene or dichloroethane, or other solvents, such as dimethylformamide, acetonitrile, pyridine or dimethyl sulfoxide (DMSO).\",\n \"It is also possible to use mixtures of the solvents mentioned above.\",\n \"The preferred solvent is dimethylformamide.\",\n \"The reaction is generally carried out in a temperature range of from −78° C. to +180° C., preferably in a range of from +20° C. to +160° C., in particular at from +40 to +100° C. The reaction can be carried out at atmospheric, elevated or reduced pressure (for example in the range from 0.5 to 5 bar).\",\n \"In general, the reaction is carried out at atmospheric pressure.\",\n \"The compounds of the formula (IV) are known to the person skilled in the art or can be prepared by customary methods known from the literature.\",\n \"Reference may be made, in particular, to the following publications, whose respective content is incorporated herein by way of reference: Kambe et al., Synthesis 1981, pages 531-533 Elnagdi et al., Z. Naturforsch.\",\n \"47b, pages 572-578, (1991) The compounds of the formulae (III) and (VI) are either commercially available, known to the person skilled in the art or preparable by customary methods.\",\n \"Surprisingly, the compounds of the formula (I) have an unforeseeable useful pharmacological activity spectrum and are therefore suitable in particular for the prophylaxis and/or treatment of disorders.\",\n \"The compounds of the formula (I) are suitable for the prophylaxis and/or treatment of a number of disorders, such as, for example, in particular disorders of the cardiovascular system (cardiovascular disorders).\",\n \"In the context of the present invention, cardiovascular disorders are to be understood as meaning, in particular, for example the following disorders: coronary heart disease, hypertension (high blood pressure), restenosis, such as, for example, after balloon dilation of peripheral blood vessels, arteriosclerosis, tachycardia, arrhythmias, peripheral vascular disorders and cardiovascular disorders, stable and unstable angina pectoris and atrial fibrillation.\",\n \"The compounds of the formula (I) are furthermore also particularly suitable, for example, for reducing the size of the myocardial area affected by an infarct.\",\n \"The compounds of the formula (I) are furthermore particularly suitable, for example, for the prophylaxis and/or treatment of thromboembolic disorders and ischemias, such as myocardial infarction, stroke and transitory ischemic attacks.\",\n \"Further areas of indication for which the compounds of the formula (I) are suitable are, for example, in particular the prophylaxis and/or treatment of disorders of the urogenital system, such as, for example, an irritable bladder, erectile dysfunction and female sexual dysfunction, and cancer, but additionally also the prophylaxis and/or treatment of inflammatory disorders, such as, for example, asthma and inflammatory dermatoses, of neuroinflammatory disorders of the central nervous system, such as, for example, disorders after stroke, Alzheimer's disease, and furthermore also neurodegenerative disorders, such as Parkinson's disease, and also pain.\",\n \"A further area of indication is, for example, in particular the prophylaxis and/or treatment of disorders of the respiratory tract, such as, for example, asthma, chronic bronchitis, pulmonary emphysema, bronchiectases, cystic fibrosis (mucoviscidosis) and pulmonary hypertension.\",\n \"The compounds of the formula (I) are furthermore also suitable, for example, in particular for the prophylaxis and/or treatment of liver fibrosis and liver cirrhosis.\",\n \"Finally, the compounds of the formula (I) are in particular also suitable, for example, for the prophylaxis and/or treatment of diabetes, in particular diabetes mellitus.\",\n \"The present invention also relates to the use of the substances of the formula (I) for preparing medicaments and pharmaceutical compositions for the prophylaxis and/or treatment of the clinical pictures mentioned above.\",\n \"The present invention furthermore relates to a method for the prophylaxis and/or treatment of the clinical pictures mentioned above using the substances of the formula (I).\",\n \"The pharmaceutical activity of the compounds of the formula (I) mentioned above can be explained by their activity as selective ligands on individual subtypes or a plurality of subtypes of the adenosine receptors, in particular as selective ligands on adenosine A1, adenosine A2a and/or adenosine A2b receptors, preferably as selective ligands on adenosine A1 and/or adenosine A2b receptors.\",\n \"In the context of the present invention, adenosine receptor ligands are referred to as being “selective” if, firstly, they are clearly active on one or more adenosine receptor subtypes and, secondly, the activity that can be observed on one or more other adenosine receptor subtypes is considerably weaker, if present at all, where, with respect to the test methods for selectivity of action, reference is made to the test methods described in Section A. II.\",\n \"One advantage of the compounds of the formula (I) according to the invention is that they are more selective than adenosine receptor ligands of the prior art.\",\n \"In particular, compounds of the formula (I) in which R6 represents an alkyl radical which is substituted by phenyl, alkoxy or hydroxyl generally act selectively on adenosine A1 receptors.\",\n \"In particular, compounds of the formula (I) in which R6 represents an alkyl radical which is substituted by —CONH2, imidazole or pyridine generally act selectively on adenosine A1 and adenosine A2b receptors.\",\n \"The receptor selectivity can be determined by biochemical measurement of the intracellular messenger cAMP in cells which specifically only express one subtype of the adenosine receptors.\",\n \"Here, what is observed, in the case of A2a and A2b agonists (coupling preferably via Gs proteins) and in the case of A2a and A2b antagonists, is an increase of the intracellular cAMP concentration and a decrease of the intracellular cAMP concentration, respectively, following prestimulation with adenosine or adenosine-like substances (see the publications B. Kull, G. Arslan, C. Nilsson, C. Owman, A. Lorenzen, U. Schwabe, B.\",\n \"B. Fredholm, “Differences in the order of potency for agonists but not antagonists at human and rat adenosine A2A receptors”, Biochem.\",\n \"Pharmacol., 57 (1999) pages 65-75; and S. P. Alexander, J. Cooper, J.\",\n \"Shine, S. J. Hill, “Characterization of the human brain putative A2B adenosine receptor expressed in Chinese hamster ovary (CHO.A2B4) cells”, Br.\",\n \"J.\",\n \"Pharmacol., 119 (1996) pages 1286-90, the respective content of which is expressly incorporated herein by way of reference).\",\n \"Correspondingly, A1 agonists (coupling preferably via Gi proteins) and A1 antagonists result in a decrease and increase, respectively, of the cAMP concentration.\",\n \"Thus, compounds of the formula (I) which bind selectively to adenosine A1 receptors are preferably suitable for myocard protection and for the prophylaxis and/or treatment of tachycardia, atrial arrhythmias, cardiac insufficiency, myocardial infarction, acute kidney failure, diabetes, pain and for wound healing.\",\n \"Compounds of the formula (I) which bind selectively to adenosine A2a receptors are preferably suitable for the prophylaxis and/or treatment of thromboembolic disorders, of neurodegenerative disorders such as Parkinson's disease and for wound healing.\",\n \"Compounds of the formula (I) which bind selectively to adenosine A2b-receptors are preferably suitable for the prophylaxis and/or therapy of liver fibrosis, of myocardial infarction, of neuroinflammatory disorders, of Alzheimer's disease, of urogenital incontinence and of disorders of the respiratory tract, such as, for example, asthma and chronic bronchitis.\",\n \"The present invention also provides medicaments and pharmaceutical compositions comprising at least one compound of the formula (I), preferably together with one or more pharmacologically acceptable auxiliaries or carriers, and their use for the abovementioned purposes.\",\n \"Suitable for administering the compounds of the formula (I) are all customary administration forms, i.e.\",\n \"oral, parenteral, inhalative, nasal, sublingual, rectal, local, such as, for example, in the case of implants or stents, or external, such as, for example, transdermal.\",\n \"In the case of parenteral administration, particular mention may be made of intravenous, intramuscular and subcutaneous administration, for example as a subcutaneous depot.\",\n \"Particular preference is given to oral administration.\",\n \"Here, the active compounds can be administered on their own or in the form of preparations.\",\n \"Suitable preparations for oral administration are inter alia tablets, capsules, pellets, sugar-coated tablets, pills, granules, solid and liquid aerosols, syrups, emulsions, suspensions and solutions.\",\n \"Here, the active compound has to be present in such a quantity that a therapeutic effect is obtained.\",\n \"In general, the active compound can be present in a concentration of from 0.1 to 100% by weight, in particular from 0.5 to 90% by weight, preferably from 5 to 80% by weight, i.e.\",\n \"the active compound should be present in quantities sufficient to achieve the dosage range mentioned.\",\n \"To this end, the active compounds can be converted in a manner known per se to the customary preparations.\",\n \"This is achieved using inert nontoxic pharmaceutically suitable carriers, auxiliaries, solvents, vehicles, emulsifiers and/or dispersants.\",\n \"Auxiliaries which may be mentioned are, for example: water, nontoxic organic solvents, such as, for example, paraffins, vegetable oils (for example sesame oil), alcohols (for example ethanol, glycerol), glycols (for example polyethylene glycol), solid carriers, such as natural or synthetic ground minerals (for example talc or silicates), sugars (for example lactose), emulsifiers, dispersants (for example polyvinylpyrrolidone) and glidants (for example magnesium sulfate).\",\n \"In the case of oral administration, tablets may, of course, also contain additives such as sodium citrate, together with adjuvants such as starch, gelatin and the like.\",\n \"Aqueous preparations for oral administration may furthermore be admixed with flavor enhancers or colorants.\",\n \"In general, it has been found to be advantageous to administer, in the case of parenteral administration, quantities of from about 0.1 to about 10 000 μg/kg, preferably from about 1 to about 1000 μg/kg, in particular from about 1 μg/kg to about 100 μg/kg, of body weight, to obtain effective results.\",\n \"In the case of oral administration, the quantity is from about 0.1 to about 10 mg/kg, preferably from about 0.5 to about 5 mg/kg, in particular from about 1 to about 4 mg/kg, of body weight.\",\n \"In spite of this, it may still be necessary, depending on body weight, administration route, individual response to the active compound, the type of preparation and the time or interval at which administration takes place, to deviate from the quantities mentioned.\",\n \"The present invention is illustrated by the following examples, although these do not restrict the invention in any way.\",\n \"A. ASSESSING PHYSIOLOGICAL ACTIVITY I. Detecting the Cardiovascular Effect Langendorff Heart of the Rat: After the thorax has been opened, the heart is removed from anesthetized rats and introduced into a conventional Langendorff apparatus.\",\n \"The coronary arteries are perfused at constant volume (10 ml/min), and the resulting perfusion pressure is recorded by way of an appropriate pressure sensor.\",\n \"In this set-up, a decrease in the perfusion pressure corresponds to a relaxation of the coronary arteries.\",\n \"At the same time, the pressure which the heart develops during each contraction is measured by way of a balloon, which has been introduced into the left ventricle, and a second pressure sensor.\",\n \"The frequency of the heart, which is beating in isolation, is calculated from the number of contractions per time unit.\",\n \"II.\",\n \"Assessing the Receptor Selectivity a) Adenosine A1, A2a, A2b and A3 Receptor Selectivity Cells of the CHO (Chinese Hamster Ovary) permanent line are transfected stably with the cDNA for the adenosine receptor subtypes A1, A2a, A2b and A3.The binding of the substances to the A2a or A2b receptor subtypes is determined by measuring the intracellular cAMP content in these cells using a conventional radioimmunological assay (cAMP RIA).\",\n \"When the substances act as agonists, the binding of the substances is expressed as an increase in the intracellular content of cAMP.\",\n \"The adenosine-analogous compound NECA (5-N-ethylcarboxamido-adenosine), which binds to all adenosine receptor subtypes with high affinity but not selectively and possesses an agonistic effect, is used as the reference compound in these experiments (Klotz, K. N., Hessling, J., Hegler, J., Owman, C., Kull, B., Fredholm, B.\",\n \"B., Lohse, M. J., Comparative pharmacology of human adenosine receptor subtypes—characterization of stably transfected receptors in CHO cells, Naunyn Schmiedebergs Arch Pharmacol, 357 (1998), 1-9).\",\n \"The adenosine receptors A1 and A3 are coupled to a Gi protein, i.e.\",\n \"stimulation of these receptors leads to inhibition of the adenylate cyclase and consequently to a lowering of the intracellular cAMP level.\",\n \"In order to identify A1/A3 receptor agonists, the adenylate cyclase is stimulated with forskolin.\",\n \"However, an additional stimulation of the A1/A3 receptors inhibits the adenylate cyclase, which means that A1/A3 receptor agonists can be detected by a comparatively low content of cAMP in the cell.\",\n \"In order to detect an antagonistic effect on adenosine receptors, the recombinant cells which are transfected with the corresponding receptor are prestimulated with NECA and the effect of the substances on reducing the intracellular content of cAMP occasioned by this prestimulation is investigated.\",\n \"XAC (xanthine amine congener), which binds to all adenosine receptor subtypes with high affinity but not selectively and possesses an antagonistic effect, is used as the reference compound in these experiments (Müller, C. E., Stein, B., Adenosine receptor antagonists: structures and potential therapeutic applications, Current Pharmaceutical Design, 2 (1996) 501-530).\",\n \"b) Adenosine A1, A2a, A2b Receptor Selectivity Cells of the CHO (Chinese Hamster Ovary) permanent line are transfected stably with the cDNA for the adenosine receptor subtypes A1, A2a and A2b.\",\n \"The adenosine A1 receptors are coupled to the adenylate cyclase by way of Gi proteins, while the adenosine A2a and A2b receptors are coupled by way of Gs proteins.\",\n \"In correspondence with this, the formation of cAMP in the cell is inhibited or stimulated, respectively.\",\n \"After that, expression of the luciferase is modulated by way of a cAMP-dependent promoter.\",\n \"The luciferase test is optimized, with the aim of high sensitivity and reproducibility, low variance and good suitability for implementation on a robot system, by varying several test parameters, such as cell density, duration of the growth phase and the test incubation, forskolin concentration and medium composition.\",\n \"The following test protocol is used for pharmacologically characterizing the cells and for the robot-assisted substance test screening: The stock cultures is grown, at 37° C. and under 5% CO2, in DMEM/F12 medium containing 10% FCS (fetal calf serum) and in each case split 1:10 after 2-3 days.\",\n \"The test cultures are seeded in 384-well plates at the rate of from 1 000 to 3 000 cells per well and grown at 37° C. for approx.\",\n \"48 hours.\",\n \"The medium is then replaced with a physiological sodium chloride solution (130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 20 mM HEPES, 1 mM MgCl2.6H2O, 5 mM NaHCO3, pH 7.4).\",\n \"The substances, which are dissolved in DMSO, are diluted 1:10 three times with this physiological sodium chloride solution and pipetted into the test cultures (maximum final concentration of DMSO in the test mixture: 0.5%).\",\n \"In this way, final substance concentrations of, for example, from 5 μM to 5 nM are obtained.\",\n \"10 minutes later, forskolin is added to the A1 cells and all the cultures are subsequently incubated at 37° C. for 4 hours.\",\n \"After that, 35 μl of a solution which is composed of 50% lysis reagent (30 mM disodium hydrogenphosphate, 10% glycerol, 3% TritonX100, 25 mM Tris HCl, 2 mM dithiothreitol (DTT), pH 7.8) and 50% luciferase substrate solution (2.5 mM ATP, 0.5 mM luciferin, 0.1 mM coenzyme A, 10 mM tricine, 1.35 mM MgSO4, 15 mM DTT, pH 7.8) are added to the test cultures, the plates are shaken for approx.\",\n \"1 minute and the luciferase activity is measured using a camera system.\",\n \"B. SYNTHESIS EXAMPLES Example 1 Step 1 2-Chloro-4-phenyl-6-(phenylsulfanyl)-3,5-pyridinedicarbonitrile Under argon, 24.5 g (182 mmol) of anhydrous copper(II) chloride are suspended in 180 ml of acetonitrile, and 21.4 g (182 mmol) of isopentyl nitrite are then added dropwise.\",\n \"After 20 minutes of stirring at room temperature, 10 g (30.5 mmol) of 2-amino-4-phenyl-6-(phenylsulfanyl)-3,5-pyridinedicarbonitrile [Kambe et al., Synthesis, 531-533 (1981)] are added.\",\n \"The mixture is stirred at room temperature over the weekend.\",\n \"For work-up, 150 ml of 1 N hydrochloric acid are added to the mixture, the mixture is extracted 4× with dichloromethane and the organic phase is washed once with saturated sodium chloride solution, dried over magnesium sulfate and concentrated under reduced pressure.\",\n \"The residue is triturated with a little ethyl acetate and allowed to stand in a fridge for one hour.\",\n \"The crystals are filtered off with suction and washed with a little cold ethyl acetate and diethyl ether.\",\n \"Yield: 7.26 g (68.5% of theory) of product Mass spectrum: molar mass required: 347, found [M+H]+=348 Step 2 2-Dimethylamino-4-phenyl-6-phenylsulfanyl-3,5-pyridinedicarbonitrile Under argon, 835 mg (2.4 mmol) of 2-chloro-4-phenyl-6-phenylsulfanyl-3,5-pyridinedicarbonitrile (step 1) are dissolved in 4 ml of absolute dimethylformamide (DMF), 0.330 ml of a 2 molar solution of dimethylamine in tetrahydrofuran (THF) is added and the reaction mixture is stirred at room temperature for 2 hours and then purified by preparative HPLC.\",\n \"Mobile phase: acetonitrile/water (+0.3% trifluoroacetic acid (TFA)), 10/90 to 90/10; 45 ml/min; column: Kromasil 100 C18, 10 μm, 75×30 mm Detection: 220 nm.\",\n \"The product fraction is concentrated by evaporation and dried under reduced pressure overnight.\",\n \"Yield: 280 mg (32.7% of theory) of product Mass spectrum: molar mass required: 356, found [M+H]+=357 Step 3 2-Dimethylamino-4-phenyl-6-sulfanyl-3,5-pyridinedicarbonitrile Under argon, 280 mg (0.79 mmol) of 2-dimethylamino-4-phenyl-6-phenylsulfanyl-3,5-pyridinedicarbonitrile (step 2) are dissolved in 3 ml of abs.\",\n \"dimethylformamide (DMF), and 204 mg (2.6 mmol) of sodium sulfide are added.\",\n \"The reaction mixture is stirred at 80° C. for 2 hours.\",\n \"6 ml of 1 N hydrochloric acid are added to the mixture and the resulting yellow crystals are filtered off with suction, washed with water and petroleum ether/diethyl ether 1/1 and dried under reduced pressure overnight.\",\n \"Yield: 117 mg (53% of theory) of product Mass spectrum: molar mass required: 280, found [M+H]+=281 Step 4 2-Dimethylamino-6-[(2-hydroxyethyl)sulfanyl]-4-phenyl-3,5-pyridinedicarbonitrile 28 mg (0.1 mmol) of 2-dimethylamino-4-phenyl-6-sulfanyl-3,5-pyridinedicarbonitrile (step 3) are, together with 19 mg (0.15 mmol) of 2-bromoethanol and 34 mg (0.4 mmol) of sodium bicarbonate, shaken in 0.6 ml of dimethylformamide (DMF) for about 16 hours.\",\n \"After filtration, the reaction solution is purified by preparative HPLC.\",\n \"Column: Grom-Sil 120 ODS-4HE, 5 μm; Mobile phase: acetonitrile/water+0.3% trifluoroacetic acid (TFA) 10/90 to 90/10; 25 ml/min; Detection: 254 nm.\",\n \"The product fraction is concentrated by evaporation under reduced pressure.\",\n \"Yield: 23 mg (71% of theory) of product Mass spectrum: molar mass required: 324, found [M+H]+=325 1H-NMR spectrum [DMSO-d6]: δ=3.35 [6H] s; 3.35 [2H] tr; 3.7 [2H] quartet; 5.0 [1H] tr; 7.55 [5H] m. The compounds listed in the table below (examples 2 to 90) are prepared analogously to the procedures of example 1 given above.\",\n \"The identity and purity of the compounds is examined by LC-MS. Molar Yield Ex.\",\n \"mass Found (% of No.\",\n \"Target structure required [M + H]+ theory) NMR data 2 324 325 54 3 370 371 44 4 354 355 66 5 405 406 54 6 354 355 69 7 401 402 52 8 435 436 48 9 310 311 55 1H-NMR spectrum [DMSO-d6]: δ =3.05[3H] d; 3.35[2H] tr; 3.7[2H]quartet; 5.0[1H] tr; 7.55[5H] m; 8.15[1H] broad.\",\n \"10 356 357 39 11 340 341 49 12 391 392 37 13 340 341 76 14 386 387 57 1H-NMR spectrum [DMSO-d6]: δ =3.6[4H] m; 4.55[2H] s; 4.8[1H] tr; 7.25-7.6[10H] m; 8.0[1H] broad 15 421 422 58 16 367 368 50 17 350 351 40 18 363 364 22 19 397 398 36 20 431 432 39 21 360 361 63 22 390 391 60 23 346 347 36 24 387 388 31 25 393 394 38 26 406 407 14 27 441 442 72 28 470 471 44 29 471 472 61 30 444 445 73 31 445 446 44 32 411 412 42 33 458 459 56 34 459 460 63 35 411 412 28 36 458 459 72 37 459 460 55 38 484 485 73 39 485 486 53 40 457 458 69 41 455 456 51 42 381 382 41 43 428 429 40 44 429 430 52 45 395 396 46 46 367 368 39 47 380 381 34 48 414 415 39 49 414 415 58 50 381 382 38 51 439 440 56 52 485 486 53 53 455 456 60 54 337 338 71 55 351 352 51 56 470 471 51 57 434 435 41 58 448 449 47 59 488 489 63 60 484 485 48 61 448 449 31 62 474 475 45 63 432 433 68 64 446 447 51 65 444 445 62 66 481 482 38 67 418 419 57 68 417 418 65 1H-NMR spectrum [DMSO-d6]: δ =3.0[3H] d; 3.75[2H] quartet; 4.05[2H] tr; 4.6[2H] s; 4.95[1H] tr; 7.05-7.5[3H] m; 8.15[1H] quartet 69 431 432 33 70 447 448 6 1H-NMR spectrum [DMSO-d6]: δ =3.6[4H] m; 3.7[2H] quartet; 4.1[2H] tr; 4.5[2H] s; 4.8[1H] tr; 4.9[1H] tr; 7.05-7.5[3H] m; 7.95[1H] s broad 71 400 401 101 72 414 415 84 73 430 431 89 74 445 446 112 75 426 427 89 76 441 442 90 77 445 446 93 78 445 446 72 79 355 355 77 1H-NMR spectrum [DMSO-d6]: δ =3.0[3H] d; 3.4[2H] tr; 3.7[2H] quartet; 5.05[1H] tr; 6.15[2H] s; 6.95-7.15[3H] m; 8.1[1H] s broad 80 368 369 82 1H-NMR spectrum [DMSO-d6]: δ =1.2[3H] tr; 3.4[2H] tr; 3.5[2H] quintet; 3.65[2H] quartet; 5.05[1H] tr; 6.15[2H] s; 6.95-7.15[3H] m; 8.2[1H] tr 81 384 385 83 82 398 399 82 83 380 381 88 1H-NMR spectrum [DMSO-d6]: δ =0.65-0.9[4H] m; 2.9[1H] m; 3.4[2H] tr; 3.7[2H] tr; 5.0[1H] s broad; 6.15[2H] s; 6.95-7.15[3H] m; 8.3[1H] s broad 84 401 402 74 1H-NMR spectrum [DMSO-d6]: δ =2.95[3H] s; 4.2[2H] s; 6.1[2H] s; 6.95-7.15[3H] m; 7.3[1H] tr; 7.5[1H] d; 7.75[1H] tr; 8.1[1H] s; 8.5[1H] s 85 368 369 88 86 382 383 88 87 398 399 93 88 412 413 84 89 382 383 85 90 394 395 89\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471079"}}},{"rowIdx":574,"cells":{"text":{"kind":"list like","value":[["Helicomimetics and stabilized lxxll peptidomimetics","This invention pertains to the design and synthesis of molecules that can act as protein mimics.","In particular this disclosure teaches the preparation of short, cyclic peptide sequences that can adopt a helical conformation and display a particular arrangement of amino acid side chains oriented in a specific arrangement to serve as a pharmacophore.","A ring, formed by a disulfide bridge between pairs of cysteine residues, maintains the helical structure.","When the cysteines are arranged in a pattern of i to i+3 as illustrated in FIG.","1, and when the first cysteine is of the D-configuration, and the second cysteine is of the L-configuration, the helical arrangement is especially stabilized.","A preferred version of this invention involves a pentapeptide sequence of general structure known as the NR Box, stabilized by a side chain to side chain disulfide bridge formed from the two cysteines."],["1.A helicomimetic compound for stabilizing the alpha helical structure of a protein fragment which can serve as an agonist or antagonist of protein-protein interactions, comprising: a compound consisting of the structure R1-(Xn)-D-Cys-Y-Y-L-Cys-(Xn)-R2, where R1 consists of H, an alkyl, aryl, acetyl, formyl, or other blocking or solubilizing group such as a polyethylene glycol (PEG) or other polyether moiety, linked to the N-terminal nitrogen through a carbon-nitrogen bond; said X comprises one or more natural or unusual amino acids, linked together in a chain from 0 to n in length; said Y comprises a selected natural or unnatural amino acid, usually of the L-configuration, and with two such amino acids that need not be identical, separating the pairs of cysteines to form an i to i+3 type of disulfide bridged unit; and R2 comprises a selected OH, NH2, NHR, or, OR other blocking or solubilizing group such as polyethylene glycol (peg) or other polyether moiety, linked to the c-terminal carbonyl through an oxygen or carbon or nitrogen linkage, such as an amide group.","2.A compound comprising a structure, wherein, R1 is (Xm)-D-Cys-Y-Y-L-Cys-(Xn)-R2, wherein Y-Y is selected from the group consisting of Ile and Leu; said Xm comprises one or more natural or unusual amino acids, linked together in a chain from 0 to n in length; and said Xn is selected from the group consisting of Leu-Leu, or Leu-Leu-Xm.","3.A compound comprising the structure R1-(Xm)-D-Aaa-Y-Y-L-Aaa-(Xn)-R2, wherein said Aaa comprises a cysteine, homocysteine, penicillamine, or other amino acid with a thiol side chain suitable for the formation of disulfide bridges; said Xm comprises one or more natural or unusual amino acids, linked together in a chain from 0 to n in length; said Y-Y is selected from the group consisting of Ile and Leu; said Xn is selected from the group consisting of Leu-Leu, Leu-Leu-Xm, and combinations thereof.","4.The compound of claim 1, wherein said compound is combined with a selected bioconjugate carrier to increase the the solubility, transport and delivery of said helicomimetic for use as a drug.","5.The compound of claim 2, wherein said compound is combined with a selected bioconjugate carrier to increase the the solubility, transport and delivery of said helicomimetic for use as a drug.","6.The compound of claim 3, wherein said compound is combined with a selected bioconjugate carrier to increase the the solubility, transport and delivery of said helicomimetic for use as a drug.","7.The compound of claim 1, wherein the compound is linked to a bioconjugate selected from a group consisting of a polyethylene glycol, an alkylated C-sugar, a transport peptide, and combinations thereof.","8.The compound of claim 2, wherein the compound is linked to a bioconjugate selected from a group consisting of a polyethylene glycol, an alkylated C-sugar, a transport peptide, and combinations thereof.","9.The compound of claim 3, wherein the compound is linked to a bioconjugate selected from a group consisting of a polyethylene glycol, an alkylated C-sugar, a transport peptide, and combinations thereof.","10.The compound of claim 1, said compound containing an amide bond replacement selected from a group consisting of a singly bonded carbon-carbon, double bonded carbon-carbon, a carbon-sulfur, a carbon-nitrogen between pairs of amino acids in order to render said compound more resistant to enyzmatic degradation.","11.The compound of claim 2, said compound containing an amide bond replacement selected from a group consisting of a singly bonded carbon-carbon, a double bonded carbon-carbon, a carbon-sulfur, a carbon-nitrogen between pairs of amino acids in order to render said compound more resistant to enyzinatic degradation.","12.The compound of claim 3, said compound containing an amide bond replacement selected from a group consisting of a singly bonded carbon-carbon, a double bonded carbon-carbon, a carbon-sulfur, a carbon-nitrogen between pairs of amino acids in order to render said compound more resistant to enyzmatic degradation."],["<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to the design and synthesis of a new class of stabilized peptide structures that are useful as mimics of the alpha helical structure ubiquitous in proteins.","2.Description of the Related Art Steroids, along with other lipophilic hormones such as the retinoids and vitamins, bind to members of the nuclear-receptor superfamily.","These ligands modify the DNA-binding and transcriptional properties of these receptors, resulting in the activation or repression of target genes.","Ligand binding induces conformational changes in nuclear receptors and promotes their association with a diverse group of nuclear proteins, including SRC-1/p160, TIF-2/GRIP-1 and CBP/p300 which function as co-activators of transcription.","A short sequence motif LXXLL (where L is leucine and X is any amino acid) present in RIP-140, SRC-1 and CBP is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors.","The ability of coactivators to bind the estrogen receptor and enhance its transcriptional activity is dependent upon the integrity of the LXXLL motifs and on key hydrophobic residues in a conserved helix (helix 12) of the estrogen receptor that are required for its ligand-induced activation function.","Thus the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins (Heery, 1997).","These compounds are intended as protein mimics and thus could find numerous applications, and especially for inhibition of protein-protein interactions where at least one of the proteins displays a helical segment as a prominent feature in terms of its tight binding to another protein.","Where the protein-protein interaction is critical to a biological function, as is often the case, then by retarding or preventing this interaction through the intervention of the helicomimetic (helix protein mimic), this compound can serve as a useful drug candidate in the event of a pathologic process such as cancer or stroke, or other instances such as transcription mediated by nuclear receptors and their cognate macromolecules."],["<SOH> SUMMARY OF THE INVENTION <EOH>This invention includes helix stabilized compounds that contain the so-called NR Box, found in a large number of Nuclear Receptor Coactivator Proteins.","The NR Box sequence, consisting of Leu-Xxx-Yyy-Leu-Leu within a longer peptide, is found in both coactivator proteins and also in certain nuclear receptors themselves.","In the case of the Androgen Receptor, this sequence is varied to include Phe-Xxx-Yyy-Leu-Phe and Phe-Xxx-Yyy-Leu-Trp, where Xxx and Yyy typically consist of two out of a rather large and diverse choice among the 20 common or natural amino acids.","By preparing synthetic variants of relatively short peptides and peptide mimics that contain the crucial hydrophobic amino acids, e.g., leucines, that maintain contact with the nuclear receptor, it is possible to prevent binding of the coactivator proteins to the nuclear receptors.","This intervention prevents the receptor from its normal subsequent step of binding to DNA and thus also prevents the transcription of the DNA segment known os the Estrogen Response Element.","This intervention has a similar pattern with respect to the other types of nuclear receptors, such as the androgen, progestin, glucocorticoid, mineralocorticoid, and the growing number of orphan receptors that have been shown to function in a biochemically equivalent manner.","Thus, by the prevention of the normal receptor-coactivator interactions, we will be able to control such effects as DNA transcription and the related downstream events that are affected, such as cell growth and division.","Thus these molecules represent a novel approach to the control of such diseases as breast cancer and prostate cancer.","These forms of cancer are currently treated with such agents as tamoxifen and raloxifene that function as estrogen antagonists.","But tamoxifen and raloxifene have been shown to bind to the normal steroid binding site, and not to the site occupied by the helical peptide LXXLL.","Thus our peptides represent a novel and distinct approach toward the treatment of cancer through a new form of inhibition of nuclear receptor action.","In order for LXXLL peptides to bind to the receptor, it is believed essential that they do so in the form of an alpha helix conformation.","Shorter linear peptides tend to adont random or β-sheet structures rather than helices.","Various strategies have been used to induce helix folding including incorporation of α-alkyl amino acid residues such as Aib (aminoisobutyric acid) or Deg (diethylglycine).","This approach may lead to unacceptably high hydrophobic character when matched with an LXXLL sequence.","Other options include helix end capping and dipole stabilization, primarily useful for longer sequences.","The peptides of the instant invention are preferable to LXXLL linear sequences for several reasons.","The first is that we and others have shown that short, linear peptides are not able to inhibit coactivator binding, at least to the extent our bioassays reflect this activity.","Second, by including pairs of cysteine residues within the sequence, we are able to enhance the helical character of these peptides.","The preferred method for doing so involves the incorporation of one D-cysteine in the sequence and one L-cysteine.","It is important to note that other workers have generally found that this type of side chain to side chain cyclization does not yield a strongly helical sequence.","Our studies have also demonstrated this trend.","But our work also demonstrates that the cysteine bridges do in fact help stabilize this helical tendency when the cyclic peptide comes into contact with the nuclear receptor.","This has been shown most clearly by a study of the interaction of one of our peptides (that one known as PERM-1 for peptidomimetic estrogen receptor modulator −1) with the ligand binding domain of the estrogen receptor (see the Figure showing the X-ray crystal structure by N. Chirgadze and coworkers).","This finding is significant since it shows the ability of our synthetic peptide to adopt a clear helical conformation in the presence of its partner and to form a strong and stable interaction with the receptor.","Additionally, our peptides can easily be made selective to one or another of the multiple nuclear receptors by changing the structure of the amino acids in the flanking regions.","Thus, as seen in Table I entitled “Peptide Analogs and their Ki values against ER alpha and ER beta”, it is apparent that by changes in amino acid composition, we are able to increase the binding toward ER alpha to a significant degree.","Furthermore, this selectivity in preferred binding to a receptor is in most cases predictable through an examination of both the sequences of amino acids found in various naturally occurring coactivator proteins as well as by an examination of the receptor residues found in close proximity to the LXXLL binding sites.","This is an important attribute of our cyclic peptide analogs since it means that we may retain the preferred small, cyclic helix-forming nature of our peptides and yet still embody the selectivity and specificity important to any useful drug.","For instance a preferred embodiment of a compound of the present invention comprises of the structure R1-(Xn)-D-Cys-Y-Y-L-Cys-(Xn)-R2, where R1 consists of H, an alkyl, aryl, acetyl, formyl, or other blocking or solubilizing group such as a polyethylene glycol (PEG) or other polyether moiety, linked to the N-terminal nitrogen through a carbon-nitrogen bond.","Moreover, X consists of one or more natural or unusual amino acids, linked together in a chain from 0 to n in length, and Y consists of any natural or unnatural amino acid, usually of the L-configuration, and with two such amino acids that need not be identical, separating the pairs of cysteines to form an i to i+3 type of disulfide bridged unit.","R2 consists of an OH, NH2, NHR, OR, or other blocking or solubilizing group such as polyethylene glycol (PEG) or other polyether moiety linked to the C-terminal carbonyl through an oxygen or carbon or nitrogen linkage, such as an amide group."],["This application is a United States non-provisional application which claims the benefit of U.S.","Provisional Application Ser.","No.","60/274,846 filed on Mar.","9, 2001, the teachings of which are hereby incorporated by reference.","BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to the design and synthesis of a new class of stabilized peptide structures that are useful as mimics of the alpha helical structure ubiquitous in proteins.","2.Description of the Related Art Steroids, along with other lipophilic hormones such as the retinoids and vitamins, bind to members of the nuclear-receptor superfamily.","These ligands modify the DNA-binding and transcriptional properties of these receptors, resulting in the activation or repression of target genes.","Ligand binding induces conformational changes in nuclear receptors and promotes their association with a diverse group of nuclear proteins, including SRC-1/p160, TIF-2/GRIP-1 and CBP/p300 which function as co-activators of transcription.","A short sequence motif LXXLL (where L is leucine and X is any amino acid) present in RIP-140, SRC-1 and CBP is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors.","The ability of coactivators to bind the estrogen receptor and enhance its transcriptional activity is dependent upon the integrity of the LXXLL motifs and on key hydrophobic residues in a conserved helix (helix 12) of the estrogen receptor that are required for its ligand-induced activation function.","Thus the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins (Heery, 1997).","These compounds are intended as protein mimics and thus could find numerous applications, and especially for inhibition of protein-protein interactions where at least one of the proteins displays a helical segment as a prominent feature in terms of its tight binding to another protein.","Where the protein-protein interaction is critical to a biological function, as is often the case, then by retarding or preventing this interaction through the intervention of the helicomimetic (helix protein mimic), this compound can serve as a useful drug candidate in the event of a pathologic process such as cancer or stroke, or other instances such as transcription mediated by nuclear receptors and their cognate macromolecules.","SUMMARY OF THE INVENTION This invention includes helix stabilized compounds that contain the so-called NR Box, found in a large number of Nuclear Receptor Coactivator Proteins.","The NR Box sequence, consisting of Leu-Xxx-Yyy-Leu-Leu within a longer peptide, is found in both coactivator proteins and also in certain nuclear receptors themselves.","In the case of the Androgen Receptor, this sequence is varied to include Phe-Xxx-Yyy-Leu-Phe and Phe-Xxx-Yyy-Leu-Trp, where Xxx and Yyy typically consist of two out of a rather large and diverse choice among the 20 common or natural amino acids.","By preparing synthetic variants of relatively short peptides and peptide mimics that contain the crucial hydrophobic amino acids, e.g., leucines, that maintain contact with the nuclear receptor, it is possible to prevent binding of the coactivator proteins to the nuclear receptors.","This intervention prevents the receptor from its normal subsequent step of binding to DNA and thus also prevents the transcription of the DNA segment known os the Estrogen Response Element.","This intervention has a similar pattern with respect to the other types of nuclear receptors, such as the androgen, progestin, glucocorticoid, mineralocorticoid, and the growing number of orphan receptors that have been shown to function in a biochemically equivalent manner.","Thus, by the prevention of the normal receptor-coactivator interactions, we will be able to control such effects as DNA transcription and the related downstream events that are affected, such as cell growth and division.","Thus these molecules represent a novel approach to the control of such diseases as breast cancer and prostate cancer.","These forms of cancer are currently treated with such agents as tamoxifen and raloxifene that function as estrogen antagonists.","But tamoxifen and raloxifene have been shown to bind to the normal steroid binding site, and not to the site occupied by the helical peptide LXXLL.","Thus our peptides represent a novel and distinct approach toward the treatment of cancer through a new form of inhibition of nuclear receptor action.","In order for LXXLL peptides to bind to the receptor, it is believed essential that they do so in the form of an alpha helix conformation.","Shorter linear peptides tend to adont random or β-sheet structures rather than helices.","Various strategies have been used to induce helix folding including incorporation of α-alkyl amino acid residues such as Aib (aminoisobutyric acid) or Deg (diethylglycine).","This approach may lead to unacceptably high hydrophobic character when matched with an LXXLL sequence.","Other options include helix end capping and dipole stabilization, primarily useful for longer sequences.","The peptides of the instant invention are preferable to LXXLL linear sequences for several reasons.","The first is that we and others have shown that short, linear peptides are not able to inhibit coactivator binding, at least to the extent our bioassays reflect this activity.","Second, by including pairs of cysteine residues within the sequence, we are able to enhance the helical character of these peptides.","The preferred method for doing so involves the incorporation of one D-cysteine in the sequence and one L-cysteine.","It is important to note that other workers have generally found that this type of side chain to side chain cyclization does not yield a strongly helical sequence.","Our studies have also demonstrated this trend.","But our work also demonstrates that the cysteine bridges do in fact help stabilize this helical tendency when the cyclic peptide comes into contact with the nuclear receptor.","This has been shown most clearly by a study of the interaction of one of our peptides (that one known as PERM-1 for peptidomimetic estrogen receptor modulator −1) with the ligand binding domain of the estrogen receptor (see the Figure showing the X-ray crystal structure by N. Chirgadze and coworkers).","This finding is significant since it shows the ability of our synthetic peptide to adopt a clear helical conformation in the presence of its partner and to form a strong and stable interaction with the receptor.","Additionally, our peptides can easily be made selective to one or another of the multiple nuclear receptors by changing the structure of the amino acids in the flanking regions.","Thus, as seen in Table I entitled “Peptide Analogs and their Ki values against ER alpha and ER beta”, it is apparent that by changes in amino acid composition, we are able to increase the binding toward ER alpha to a significant degree.","Furthermore, this selectivity in preferred binding to a receptor is in most cases predictable through an examination of both the sequences of amino acids found in various naturally occurring coactivator proteins as well as by an examination of the receptor residues found in close proximity to the LXXLL binding sites.","This is an important attribute of our cyclic peptide analogs since it means that we may retain the preferred small, cyclic helix-forming nature of our peptides and yet still embody the selectivity and specificity important to any useful drug.","For instance a preferred embodiment of a compound of the present invention comprises of the structure R1-(Xn)-D-Cys-Y-Y-L-Cys-(Xn)-R2, where R1 consists of H, an alkyl, aryl, acetyl, formyl, or other blocking or solubilizing group such as a polyethylene glycol (PEG) or other polyether moiety, linked to the N-terminal nitrogen through a carbon-nitrogen bond.","Moreover, X consists of one or more natural or unusual amino acids, linked together in a chain from 0 to n in length, and Y consists of any natural or unnatural amino acid, usually of the L-configuration, and with two such amino acids that need not be identical, separating the pairs of cysteines to form an i to i+3 type of disulfide bridged unit.","R2 consists of an OH, NH2, NHR, OR, or other blocking or solubilizing group such as polyethylene glycol (PEG) or other polyether moiety linked to the C-terminal carbonyl through an oxygen or carbon or nitrogen linkage, such as an amide group.","BRIEF DESCRIPTION OF THE DRAWINGS A better understanding of the present invention will be had upon reference to the following description in conjunction with the accompanying drawings in which like numerals refer to like parts throughout the several views and wherein: FIG.","1 is a structure of a side chain linked amide (a) and disulfide (b) bridges at (i,i+4) and (i, i+3) positions, respectively; FIG.","2 is color a molecular modeling rendition of the structure of a helicomimetic peptide bound to a nuclear receptor; and FIG.","3 is a black and white photocopy of FIG.","2.DESCRIPTION OF THE PREFERRED EMBODIMENT The preferred compound of this invention involves a cyclic peptide containing the LXXLL sequence.","The cycle is formed through a side chain to side chain ring involving a monosulfide or disulfide bridge between pairs of cysteines, penicillamines, homocysteines, combinations of the foregoing, or other pairs of amino acids in which the side chains are linked with either one or two sulfur atoms.","In a preferred embodiment, the peptide cycle is formed with a D-cysteine at the −2 position and an L-cysteine at the first Xxx residue to produce an i to i+3 ring.","With this partial structure, such as -D-Cys-Ile-Leu-Cys-Arg-Leu-Leu-, the flanking residues attached at the N-terminal side of the D-Cys and at the C-terminal side of the Leu provide selectivity as inhibitors against one of several nuclear receptors.","For example, in the case of the compound known as PERM-1, the Ki value against ER beta is approximately 390 nM, while its value against ER alpha is 25 nM.","Thus this compound exhibits selectivity against the ER alpha receptor.","The compounds may also be modified by the attachment of elements designed to stabilize the structure, and to enhance bioavailability.","For example, the N- and/or C-termini may be attached to polyethylene glycol (PEG) fragments, designed to enhance penetration through lipid membranes.","Alternatively, other types of solubility enhancing bioconjugates may be used to assist in membrane permeability.","Other modifiers can also be attached.","For example, the TAT and related hydrophilic peptide sequences, derived originally from the HIV virus, have been demonstrated to assist in the delivery of peptides and other therapeutic agents into cells.","These sequences, along with those known as antennapedia peptides, would be expected to provide a similar benefit for the delivery of these nuclear receptor antagonists into the cell and eventually to the nuclear compartment.","Another approach that has shown promise in enhancing peptide bioavailability is the replacement of one or more amide bonds with various backbone replacements.","These may include pseudopeptides, with CH2S, CH2NH.","CH═(CH, or N-methyl amide bond modifications, or can involve the substitution of an amino acid with more conformationally constrained variants such as alpha methyl and beta methyl substitutions.","The replacement of cysteine by penicillamine (beta, beta-dimethyl cysteine) has been previously mentioned.","This modification is able to reduce the flexibility of the disulfide ring and can enhance stability, potency, and selectivity, as has been documented in the case of the mu selective opioid analog known as DPDPE.","The major therapeutic benefit gained by the design and synthesis of coactivator antagonists is a more effective control of steroid receptor mediated transcriptional processes.","Thus in diseases such as breast cancer or prostate cancer, certain hormone dependent tumors grow through the uncontrolled steroid-mediated transcription within the malignant cells.","Effective therapeutics may me used in the assessment, treatment, and prevention of cancer.","It is also important that these agents be selective so that undesired cell proliferation is prevented but such other benefits of estrogenic agonists such as prevention of osteoporosis should not be compromised.","Control of transcription may be desired in order to help overcome one or more genetic malfunctions in an individual.","It may also be anticipated that these novel antagonists can serve as diagnostic agents and as effective inhibitors in the case of various types of orphan nuclear receptors, whose functions have yet to be determined (Burris and McCabe, 2000).","Experimental (Peptide Synthesis) The linear and cyclic peptides were synthesized using Boc-based Merrifield solid phase peptide synthesis using a anhydrous hydrogen fluoride for cleavage from the methylbenzhydryamine resin support to provide the targeted peptide amides.","Scheme 1 summarizes the approach used for two cyclic variants.","A lactam bridge between Glu, Lys was formed on the resin following base-mediated cleavage of the fluorenylmethyl-class protecting groups.","In contrast, disulfide bridge formation was performed off-resin, with Tam's DMSO oxidation procedure providing the best results when using heated sulfoxide reagent.","Products were analyzed by CD, NMR spectroscopy, reversed phase high pressure liquid chromatography, and thin layer chromatography, and the expected structures confirmed with MALDI-TOF mass spectrometry.","Biological Activity Data The synthetic helicormimetic peptides designed as antagonists of the estrogen receptor-coactivator interactions were tested in a competition binding assay (Lilly Research Labs) against a model linear peptide sequence.","Activities are reported in the Table below in Ki values, with two of the best analogs labeled as PERM-1 and PERM-2, or Peptidomimetic Estrogen Receptor Modulators.","TABLE I Peptide Analogs and their Ki values against ER alpha and ER beta ER α ER β Title Sequence MW μM μM AML-I-22 H-Leu-Glu-Gln-Leu-Leu-OH 614.3 N/A 201.2 AML-I-31 Mannosylacetvl-Leu-Glu-Gln-Leu-Leu-OH 818 N/A 339.4 AML-I-48/4 H-Lys-cyclo (D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 PERM-1 1085 0.025 0.39 AML-I-61/2 H-Lys-Lys-Ile-Leu-His-Arg-Leu-Leu-Gln-NH2 1147 0.17 2.8 AML-I-59/6 K-Lys-cyclo(Glu-Ile-Leu-Arg-Lys)-Leu-Leu-Gln-NH2 1120.7 0.22 4.8 AML-I-71/2 Ac-Lys-cyclo(D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 1127 0.12 7.7 AML-I-86/1 Gu-Lys-cyclo-(D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 1183 0.14 0.6 AML-I-83/4 Aib-Lys-cyclo(D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 1172 0.13 1.4 AML-I-89/2 H-Lys-His-Lys-Ile-Leu-His-Arg-Leu-Leu-Gln-Asp-Ser-Ser-OH 1573.9 0.38 6.9 AKG-I-28 H-D-Lys-cyclo (D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 1085 0.22 1.9 AKG-I-39 H-Lys-cyclo (Ala-Ile-Leu-Ala)- Arg-Leu-Leu-Gln-NH2, 1053 1.18 15.4 Lanthionine AKG-I-40 H-D-Lys-cyclo (AIa-Ile-Leu-Ala)- Arg-Leu-Leu-Gln-NH2, 1053 3.95 13.5 Lanthionine AKG-I-46 H-Lys-Leu-Leu-cyclo(D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln- 1311 0.398 2.0 NH2 AKG-I-48 K-Lys-cyclo(Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 1085 0.416 1.8 AKG-I-50 H-Arg-cyclo(D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 PERM-2 1113 0.011 0.077 AKG-I-59 H-Lys-cyclo(Cys-Leu-Ile-D-Cys)-Arg-Leu-Leu-Gln-NH2 1085 2.1 17.0 AKG-I-60 H-Lys-cyclo(Cys-Ile-Leu-D-Cys)-Arg-Leu-Leu-Gln-NH2 1085 2.4 7.2 AKG-I-61 H-Lys-cyclo(D-Cys-Ile-Leu-D-Cys)-Arg-Leu-Leu-Gln-NH2 1085 0.928 3.9 AKG-I-63 H-Lys-cyclo(Cys-Leu-D-Cys)-Arg-Leu-Leu-Gln-NH2 972 2.2 7.9 AKG-I-64 H-Lys-cyclo(D-Cys-Leu-D-Cys)-Arg-Leu-Leu-Gln-NH2 972 1.8 5.2 AKG-II-1 H-Arg-cyclo (D-Cys-Leu-Ile-Cys)-Arg-Leu-Leu-Gln-NH2 1113 .013 .216 AKG-II-3 H-Arg-cyclo (D-Cys-Ile-Leu-HomoCys)-Arg-Leu-Leu-Gln-NH2 1127 .013 .214 AKG-II-4 H-Arg-cyclo (DHomoCys-lIe-Leu-Cys)- Arg-Leu-Leu-Gln-NH2 1127 .035 .591 AKG-II-8 H-Arg-cyclo (Cys-Ile-Leu-Arg-Cys)-Leu-Leu-Gln-NH2 1113 .174 1.16 AKG-II-9 H-Arg-cyclo (D-Pen-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 1141 .168 .933 AKG-II-10 H-Arg-cyclo (D-Cys-Ile-Leu-Pen)-Arg-Leu-Leu-Gln-NH2 1141 .088 1.91 AKG-II-11 H-Arg-cyclo(D-Pen-Ile-Leu-Pen)-Arg-Leu-Leu-Gln-NH2 1169 .078 3.97 SRC-1 NR2 LTERHKILHRLLQEGSPSD 0.39 — Short linear peptides that contain the LXXLL sequence, such as Leu-Asn-Gln-Leu-Leu, do not display any inhibitory activity with respect to the desired effect of inhibiting the binding of the estrogen receptors to the helical segment of coactivator proteins.","As seen in the Table, compounds that contain a D-Cys, L-Cys pairing are especially active with respect to binding inhibition.","A report by Geistlinger ad Guy teaches of the inhibition of the interaction between thyroid hormone and its interaction using side chain to side chain linked peptides.","The ring in this example is formed through an amide linkage between a lysine residue and a glutamic acid residue.","But this report does not include any examples of disulfide bridges nor of the preference for a D-cysteine and L-cysteine pairing, nor does it include any examples of receptors other than the thyroid nuclear receptor.","X-Ray Structure of a Helicomimetic Peptide Bound to the Estrogen Receptor Dimer Ligand Binding Domain (LBD) The conditions for the cyrstallization of the peptide protein complex as shown in FIG.","1 are describes as follows: Cloning of the Gene Expression and Purification of Human ER LBD.","The ER LBD gene was overexpressed in E. coli and purified by Pan Vera, Inc. Crystallization.","Diffraction-quality crystals of the ER LBD complex were grown by the vapor diffusion technique at 294K using a Hampton (Hampton Research Inc.,) crystallization screen.","Crystals belong to orthorhombic space group C2221, with unit cell parameters a=53.8 Å, b=102.4 Å, c=195.3 Å.","There are two molecules of the complex per asymmetric unit, with a Vm value (Matthews 1968) of 2.28 Å3/Da that corresponds to a solvent content of approximately 46% in both cases.","The 17-estradiol and PERM-1 (peptide) in 2-3 fold of excess of the protein were used for co-crystallization.","Data Collection, Structure Solution, and Crystallographic Refinement.","The diffraction data resolution of (resolution of 2.7 Å; Rmerge=0.114, and completeness of 95%) were collected using a MarCCD (refmar) detector on IMCA (Industrial Macromolecular Crystallography Association) beam line BM-17 at the APS (Advanced Photon Source, Argonne National Laboratories) at 100K, using 15-20% glycerol as a cryoprotectant.","The diffraction data were reduced using HKL2000 (Otwinowski and Minor 1997) and the intensities were scaled with SCALEPACK.","Crystal structure was determined by the method of molecular replacement using the AMORE program suite (CCP4; Collaborative Computing Project #4 1994).","The crystal structure was refined against data between 20-2.7 Å using a maximum likely-hood algorithm as incorporated in the program CNX2000 (Badger et al., 1999) (Rwork=0.203, Rfree=0.258, RMS=0.007 Å) (Brünger 1992).","The program suite QUANTA 98 (Molecular Simulation Inc., San Diego, Calif.) was used for visual inspection and manual corrections between rounds of refinement.","An analysis of the geometry showed all parameters were within the values expected for a model at this resolution.","All residues were found in the most favorable and additionally allowed regions of a Ramachandran plot.","The omitted unbiased electron density map was used for positioning 17β-estradiol and the PERM-1 peptide.","Specific compositions, methods, or embodiments discussed are intended to be only illustrative of the invention disclosed by this specification.","Variation on these compositions, methods, or embodiments are readily apparent to a person of skill in the art based upon the teachings of this specification and are therefore intended to be included as part of the inventions disclosed herein.","Reference to documents made in the specification is intended to result in such patents or literature cited are expressly incorporated herein by reference, including any patents or other literature references cited within such documents as if fully set forth in this specification.","The foregoing detailed description is given primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom, for modification will become obvious to those skilled in the art upon reading this disclosure and may be made upon departing from the spirit of the invention and scope of the appended claims.","Accordingly, this invention is not intended to be limited by the specific exemplifications presented hereinabove.","Rather, what is intended to be covered is within the spirit and scope of the appended claims."]],"string":"[\n [\n \"Helicomimetics and stabilized lxxll peptidomimetics\",\n \"This invention pertains to the design and synthesis of molecules that can act as protein mimics.\",\n \"In particular this disclosure teaches the preparation of short, cyclic peptide sequences that can adopt a helical conformation and display a particular arrangement of amino acid side chains oriented in a specific arrangement to serve as a pharmacophore.\",\n \"A ring, formed by a disulfide bridge between pairs of cysteine residues, maintains the helical structure.\",\n \"When the cysteines are arranged in a pattern of i to i+3 as illustrated in FIG.\",\n \"1, and when the first cysteine is of the D-configuration, and the second cysteine is of the L-configuration, the helical arrangement is especially stabilized.\",\n \"A preferred version of this invention involves a pentapeptide sequence of general structure known as the NR Box, stabilized by a side chain to side chain disulfide bridge formed from the two cysteines.\"\n ],\n [\n \"1.A helicomimetic compound for stabilizing the alpha helical structure of a protein fragment which can serve as an agonist or antagonist of protein-protein interactions, comprising: a compound consisting of the structure R1-(Xn)-D-Cys-Y-Y-L-Cys-(Xn)-R2, where R1 consists of H, an alkyl, aryl, acetyl, formyl, or other blocking or solubilizing group such as a polyethylene glycol (PEG) or other polyether moiety, linked to the N-terminal nitrogen through a carbon-nitrogen bond; said X comprises one or more natural or unusual amino acids, linked together in a chain from 0 to n in length; said Y comprises a selected natural or unnatural amino acid, usually of the L-configuration, and with two such amino acids that need not be identical, separating the pairs of cysteines to form an i to i+3 type of disulfide bridged unit; and R2 comprises a selected OH, NH2, NHR, or, OR other blocking or solubilizing group such as polyethylene glycol (peg) or other polyether moiety, linked to the c-terminal carbonyl through an oxygen or carbon or nitrogen linkage, such as an amide group.\",\n \"2.A compound comprising a structure, wherein, R1 is (Xm)-D-Cys-Y-Y-L-Cys-(Xn)-R2, wherein Y-Y is selected from the group consisting of Ile and Leu; said Xm comprises one or more natural or unusual amino acids, linked together in a chain from 0 to n in length; and said Xn is selected from the group consisting of Leu-Leu, or Leu-Leu-Xm.\",\n \"3.A compound comprising the structure R1-(Xm)-D-Aaa-Y-Y-L-Aaa-(Xn)-R2, wherein said Aaa comprises a cysteine, homocysteine, penicillamine, or other amino acid with a thiol side chain suitable for the formation of disulfide bridges; said Xm comprises one or more natural or unusual amino acids, linked together in a chain from 0 to n in length; said Y-Y is selected from the group consisting of Ile and Leu; said Xn is selected from the group consisting of Leu-Leu, Leu-Leu-Xm, and combinations thereof.\",\n \"4.The compound of claim 1, wherein said compound is combined with a selected bioconjugate carrier to increase the the solubility, transport and delivery of said helicomimetic for use as a drug.\",\n \"5.The compound of claim 2, wherein said compound is combined with a selected bioconjugate carrier to increase the the solubility, transport and delivery of said helicomimetic for use as a drug.\",\n \"6.The compound of claim 3, wherein said compound is combined with a selected bioconjugate carrier to increase the the solubility, transport and delivery of said helicomimetic for use as a drug.\",\n \"7.The compound of claim 1, wherein the compound is linked to a bioconjugate selected from a group consisting of a polyethylene glycol, an alkylated C-sugar, a transport peptide, and combinations thereof.\",\n \"8.The compound of claim 2, wherein the compound is linked to a bioconjugate selected from a group consisting of a polyethylene glycol, an alkylated C-sugar, a transport peptide, and combinations thereof.\",\n \"9.The compound of claim 3, wherein the compound is linked to a bioconjugate selected from a group consisting of a polyethylene glycol, an alkylated C-sugar, a transport peptide, and combinations thereof.\",\n \"10.The compound of claim 1, said compound containing an amide bond replacement selected from a group consisting of a singly bonded carbon-carbon, double bonded carbon-carbon, a carbon-sulfur, a carbon-nitrogen between pairs of amino acids in order to render said compound more resistant to enyzmatic degradation.\",\n \"11.The compound of claim 2, said compound containing an amide bond replacement selected from a group consisting of a singly bonded carbon-carbon, a double bonded carbon-carbon, a carbon-sulfur, a carbon-nitrogen between pairs of amino acids in order to render said compound more resistant to enyzinatic degradation.\",\n \"12.The compound of claim 3, said compound containing an amide bond replacement selected from a group consisting of a singly bonded carbon-carbon, a double bonded carbon-carbon, a carbon-sulfur, a carbon-nitrogen between pairs of amino acids in order to render said compound more resistant to enyzmatic degradation.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to the design and synthesis of a new class of stabilized peptide structures that are useful as mimics of the alpha helical structure ubiquitous in proteins.\",\n \"2.Description of the Related Art Steroids, along with other lipophilic hormones such as the retinoids and vitamins, bind to members of the nuclear-receptor superfamily.\",\n \"These ligands modify the DNA-binding and transcriptional properties of these receptors, resulting in the activation or repression of target genes.\",\n \"Ligand binding induces conformational changes in nuclear receptors and promotes their association with a diverse group of nuclear proteins, including SRC-1/p160, TIF-2/GRIP-1 and CBP/p300 which function as co-activators of transcription.\",\n \"A short sequence motif LXXLL (where L is leucine and X is any amino acid) present in RIP-140, SRC-1 and CBP is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors.\",\n \"The ability of coactivators to bind the estrogen receptor and enhance its transcriptional activity is dependent upon the integrity of the LXXLL motifs and on key hydrophobic residues in a conserved helix (helix 12) of the estrogen receptor that are required for its ligand-induced activation function.\",\n \"Thus the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins (Heery, 1997).\",\n \"These compounds are intended as protein mimics and thus could find numerous applications, and especially for inhibition of protein-protein interactions where at least one of the proteins displays a helical segment as a prominent feature in terms of its tight binding to another protein.\",\n \"Where the protein-protein interaction is critical to a biological function, as is often the case, then by retarding or preventing this interaction through the intervention of the helicomimetic (helix protein mimic), this compound can serve as a useful drug candidate in the event of a pathologic process such as cancer or stroke, or other instances such as transcription mediated by nuclear receptors and their cognate macromolecules.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>This invention includes helix stabilized compounds that contain the so-called NR Box, found in a large number of Nuclear Receptor Coactivator Proteins.\",\n \"The NR Box sequence, consisting of Leu-Xxx-Yyy-Leu-Leu within a longer peptide, is found in both coactivator proteins and also in certain nuclear receptors themselves.\",\n \"In the case of the Androgen Receptor, this sequence is varied to include Phe-Xxx-Yyy-Leu-Phe and Phe-Xxx-Yyy-Leu-Trp, where Xxx and Yyy typically consist of two out of a rather large and diverse choice among the 20 common or natural amino acids.\",\n \"By preparing synthetic variants of relatively short peptides and peptide mimics that contain the crucial hydrophobic amino acids, e.g., leucines, that maintain contact with the nuclear receptor, it is possible to prevent binding of the coactivator proteins to the nuclear receptors.\",\n \"This intervention prevents the receptor from its normal subsequent step of binding to DNA and thus also prevents the transcription of the DNA segment known os the Estrogen Response Element.\",\n \"This intervention has a similar pattern with respect to the other types of nuclear receptors, such as the androgen, progestin, glucocorticoid, mineralocorticoid, and the growing number of orphan receptors that have been shown to function in a biochemically equivalent manner.\",\n \"Thus, by the prevention of the normal receptor-coactivator interactions, we will be able to control such effects as DNA transcription and the related downstream events that are affected, such as cell growth and division.\",\n \"Thus these molecules represent a novel approach to the control of such diseases as breast cancer and prostate cancer.\",\n \"These forms of cancer are currently treated with such agents as tamoxifen and raloxifene that function as estrogen antagonists.\",\n \"But tamoxifen and raloxifene have been shown to bind to the normal steroid binding site, and not to the site occupied by the helical peptide LXXLL.\",\n \"Thus our peptides represent a novel and distinct approach toward the treatment of cancer through a new form of inhibition of nuclear receptor action.\",\n \"In order for LXXLL peptides to bind to the receptor, it is believed essential that they do so in the form of an alpha helix conformation.\",\n \"Shorter linear peptides tend to adont random or β-sheet structures rather than helices.\",\n \"Various strategies have been used to induce helix folding including incorporation of α-alkyl amino acid residues such as Aib (aminoisobutyric acid) or Deg (diethylglycine).\",\n \"This approach may lead to unacceptably high hydrophobic character when matched with an LXXLL sequence.\",\n \"Other options include helix end capping and dipole stabilization, primarily useful for longer sequences.\",\n \"The peptides of the instant invention are preferable to LXXLL linear sequences for several reasons.\",\n \"The first is that we and others have shown that short, linear peptides are not able to inhibit coactivator binding, at least to the extent our bioassays reflect this activity.\",\n \"Second, by including pairs of cysteine residues within the sequence, we are able to enhance the helical character of these peptides.\",\n \"The preferred method for doing so involves the incorporation of one D-cysteine in the sequence and one L-cysteine.\",\n \"It is important to note that other workers have generally found that this type of side chain to side chain cyclization does not yield a strongly helical sequence.\",\n \"Our studies have also demonstrated this trend.\",\n \"But our work also demonstrates that the cysteine bridges do in fact help stabilize this helical tendency when the cyclic peptide comes into contact with the nuclear receptor.\",\n \"This has been shown most clearly by a study of the interaction of one of our peptides (that one known as PERM-1 for peptidomimetic estrogen receptor modulator −1) with the ligand binding domain of the estrogen receptor (see the Figure showing the X-ray crystal structure by N. Chirgadze and coworkers).\",\n \"This finding is significant since it shows the ability of our synthetic peptide to adopt a clear helical conformation in the presence of its partner and to form a strong and stable interaction with the receptor.\",\n \"Additionally, our peptides can easily be made selective to one or another of the multiple nuclear receptors by changing the structure of the amino acids in the flanking regions.\",\n \"Thus, as seen in Table I entitled “Peptide Analogs and their Ki values against ER alpha and ER beta”, it is apparent that by changes in amino acid composition, we are able to increase the binding toward ER alpha to a significant degree.\",\n \"Furthermore, this selectivity in preferred binding to a receptor is in most cases predictable through an examination of both the sequences of amino acids found in various naturally occurring coactivator proteins as well as by an examination of the receptor residues found in close proximity to the LXXLL binding sites.\",\n \"This is an important attribute of our cyclic peptide analogs since it means that we may retain the preferred small, cyclic helix-forming nature of our peptides and yet still embody the selectivity and specificity important to any useful drug.\",\n \"For instance a preferred embodiment of a compound of the present invention comprises of the structure R1-(Xn)-D-Cys-Y-Y-L-Cys-(Xn)-R2, where R1 consists of H, an alkyl, aryl, acetyl, formyl, or other blocking or solubilizing group such as a polyethylene glycol (PEG) or other polyether moiety, linked to the N-terminal nitrogen through a carbon-nitrogen bond.\",\n \"Moreover, X consists of one or more natural or unusual amino acids, linked together in a chain from 0 to n in length, and Y consists of any natural or unnatural amino acid, usually of the L-configuration, and with two such amino acids that need not be identical, separating the pairs of cysteines to form an i to i+3 type of disulfide bridged unit.\",\n \"R2 consists of an OH, NH2, NHR, OR, or other blocking or solubilizing group such as polyethylene glycol (PEG) or other polyether moiety linked to the C-terminal carbonyl through an oxygen or carbon or nitrogen linkage, such as an amide group.\"\n ],\n [\n \"This application is a United States non-provisional application which claims the benefit of U.S.\",\n \"Provisional Application Ser.\",\n \"No.\",\n \"60/274,846 filed on Mar.\",\n \"9, 2001, the teachings of which are hereby incorporated by reference.\",\n \"BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to the design and synthesis of a new class of stabilized peptide structures that are useful as mimics of the alpha helical structure ubiquitous in proteins.\",\n \"2.Description of the Related Art Steroids, along with other lipophilic hormones such as the retinoids and vitamins, bind to members of the nuclear-receptor superfamily.\",\n \"These ligands modify the DNA-binding and transcriptional properties of these receptors, resulting in the activation or repression of target genes.\",\n \"Ligand binding induces conformational changes in nuclear receptors and promotes their association with a diverse group of nuclear proteins, including SRC-1/p160, TIF-2/GRIP-1 and CBP/p300 which function as co-activators of transcription.\",\n \"A short sequence motif LXXLL (where L is leucine and X is any amino acid) present in RIP-140, SRC-1 and CBP is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors.\",\n \"The ability of coactivators to bind the estrogen receptor and enhance its transcriptional activity is dependent upon the integrity of the LXXLL motifs and on key hydrophobic residues in a conserved helix (helix 12) of the estrogen receptor that are required for its ligand-induced activation function.\",\n \"Thus the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins (Heery, 1997).\",\n \"These compounds are intended as protein mimics and thus could find numerous applications, and especially for inhibition of protein-protein interactions where at least one of the proteins displays a helical segment as a prominent feature in terms of its tight binding to another protein.\",\n \"Where the protein-protein interaction is critical to a biological function, as is often the case, then by retarding or preventing this interaction through the intervention of the helicomimetic (helix protein mimic), this compound can serve as a useful drug candidate in the event of a pathologic process such as cancer or stroke, or other instances such as transcription mediated by nuclear receptors and their cognate macromolecules.\",\n \"SUMMARY OF THE INVENTION This invention includes helix stabilized compounds that contain the so-called NR Box, found in a large number of Nuclear Receptor Coactivator Proteins.\",\n \"The NR Box sequence, consisting of Leu-Xxx-Yyy-Leu-Leu within a longer peptide, is found in both coactivator proteins and also in certain nuclear receptors themselves.\",\n \"In the case of the Androgen Receptor, this sequence is varied to include Phe-Xxx-Yyy-Leu-Phe and Phe-Xxx-Yyy-Leu-Trp, where Xxx and Yyy typically consist of two out of a rather large and diverse choice among the 20 common or natural amino acids.\",\n \"By preparing synthetic variants of relatively short peptides and peptide mimics that contain the crucial hydrophobic amino acids, e.g., leucines, that maintain contact with the nuclear receptor, it is possible to prevent binding of the coactivator proteins to the nuclear receptors.\",\n \"This intervention prevents the receptor from its normal subsequent step of binding to DNA and thus also prevents the transcription of the DNA segment known os the Estrogen Response Element.\",\n \"This intervention has a similar pattern with respect to the other types of nuclear receptors, such as the androgen, progestin, glucocorticoid, mineralocorticoid, and the growing number of orphan receptors that have been shown to function in a biochemically equivalent manner.\",\n \"Thus, by the prevention of the normal receptor-coactivator interactions, we will be able to control such effects as DNA transcription and the related downstream events that are affected, such as cell growth and division.\",\n \"Thus these molecules represent a novel approach to the control of such diseases as breast cancer and prostate cancer.\",\n \"These forms of cancer are currently treated with such agents as tamoxifen and raloxifene that function as estrogen antagonists.\",\n \"But tamoxifen and raloxifene have been shown to bind to the normal steroid binding site, and not to the site occupied by the helical peptide LXXLL.\",\n \"Thus our peptides represent a novel and distinct approach toward the treatment of cancer through a new form of inhibition of nuclear receptor action.\",\n \"In order for LXXLL peptides to bind to the receptor, it is believed essential that they do so in the form of an alpha helix conformation.\",\n \"Shorter linear peptides tend to adont random or β-sheet structures rather than helices.\",\n \"Various strategies have been used to induce helix folding including incorporation of α-alkyl amino acid residues such as Aib (aminoisobutyric acid) or Deg (diethylglycine).\",\n \"This approach may lead to unacceptably high hydrophobic character when matched with an LXXLL sequence.\",\n \"Other options include helix end capping and dipole stabilization, primarily useful for longer sequences.\",\n \"The peptides of the instant invention are preferable to LXXLL linear sequences for several reasons.\",\n \"The first is that we and others have shown that short, linear peptides are not able to inhibit coactivator binding, at least to the extent our bioassays reflect this activity.\",\n \"Second, by including pairs of cysteine residues within the sequence, we are able to enhance the helical character of these peptides.\",\n \"The preferred method for doing so involves the incorporation of one D-cysteine in the sequence and one L-cysteine.\",\n \"It is important to note that other workers have generally found that this type of side chain to side chain cyclization does not yield a strongly helical sequence.\",\n \"Our studies have also demonstrated this trend.\",\n \"But our work also demonstrates that the cysteine bridges do in fact help stabilize this helical tendency when the cyclic peptide comes into contact with the nuclear receptor.\",\n \"This has been shown most clearly by a study of the interaction of one of our peptides (that one known as PERM-1 for peptidomimetic estrogen receptor modulator −1) with the ligand binding domain of the estrogen receptor (see the Figure showing the X-ray crystal structure by N. Chirgadze and coworkers).\",\n \"This finding is significant since it shows the ability of our synthetic peptide to adopt a clear helical conformation in the presence of its partner and to form a strong and stable interaction with the receptor.\",\n \"Additionally, our peptides can easily be made selective to one or another of the multiple nuclear receptors by changing the structure of the amino acids in the flanking regions.\",\n \"Thus, as seen in Table I entitled “Peptide Analogs and their Ki values against ER alpha and ER beta”, it is apparent that by changes in amino acid composition, we are able to increase the binding toward ER alpha to a significant degree.\",\n \"Furthermore, this selectivity in preferred binding to a receptor is in most cases predictable through an examination of both the sequences of amino acids found in various naturally occurring coactivator proteins as well as by an examination of the receptor residues found in close proximity to the LXXLL binding sites.\",\n \"This is an important attribute of our cyclic peptide analogs since it means that we may retain the preferred small, cyclic helix-forming nature of our peptides and yet still embody the selectivity and specificity important to any useful drug.\",\n \"For instance a preferred embodiment of a compound of the present invention comprises of the structure R1-(Xn)-D-Cys-Y-Y-L-Cys-(Xn)-R2, where R1 consists of H, an alkyl, aryl, acetyl, formyl, or other blocking or solubilizing group such as a polyethylene glycol (PEG) or other polyether moiety, linked to the N-terminal nitrogen through a carbon-nitrogen bond.\",\n \"Moreover, X consists of one or more natural or unusual amino acids, linked together in a chain from 0 to n in length, and Y consists of any natural or unnatural amino acid, usually of the L-configuration, and with two such amino acids that need not be identical, separating the pairs of cysteines to form an i to i+3 type of disulfide bridged unit.\",\n \"R2 consists of an OH, NH2, NHR, OR, or other blocking or solubilizing group such as polyethylene glycol (PEG) or other polyether moiety linked to the C-terminal carbonyl through an oxygen or carbon or nitrogen linkage, such as an amide group.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS A better understanding of the present invention will be had upon reference to the following description in conjunction with the accompanying drawings in which like numerals refer to like parts throughout the several views and wherein: FIG.\",\n \"1 is a structure of a side chain linked amide (a) and disulfide (b) bridges at (i,i+4) and (i, i+3) positions, respectively; FIG.\",\n \"2 is color a molecular modeling rendition of the structure of a helicomimetic peptide bound to a nuclear receptor; and FIG.\",\n \"3 is a black and white photocopy of FIG.\",\n \"2.DESCRIPTION OF THE PREFERRED EMBODIMENT The preferred compound of this invention involves a cyclic peptide containing the LXXLL sequence.\",\n \"The cycle is formed through a side chain to side chain ring involving a monosulfide or disulfide bridge between pairs of cysteines, penicillamines, homocysteines, combinations of the foregoing, or other pairs of amino acids in which the side chains are linked with either one or two sulfur atoms.\",\n \"In a preferred embodiment, the peptide cycle is formed with a D-cysteine at the −2 position and an L-cysteine at the first Xxx residue to produce an i to i+3 ring.\",\n \"With this partial structure, such as -D-Cys-Ile-Leu-Cys-Arg-Leu-Leu-, the flanking residues attached at the N-terminal side of the D-Cys and at the C-terminal side of the Leu provide selectivity as inhibitors against one of several nuclear receptors.\",\n \"For example, in the case of the compound known as PERM-1, the Ki value against ER beta is approximately 390 nM, while its value against ER alpha is 25 nM.\",\n \"Thus this compound exhibits selectivity against the ER alpha receptor.\",\n \"The compounds may also be modified by the attachment of elements designed to stabilize the structure, and to enhance bioavailability.\",\n \"For example, the N- and/or C-termini may be attached to polyethylene glycol (PEG) fragments, designed to enhance penetration through lipid membranes.\",\n \"Alternatively, other types of solubility enhancing bioconjugates may be used to assist in membrane permeability.\",\n \"Other modifiers can also be attached.\",\n \"For example, the TAT and related hydrophilic peptide sequences, derived originally from the HIV virus, have been demonstrated to assist in the delivery of peptides and other therapeutic agents into cells.\",\n \"These sequences, along with those known as antennapedia peptides, would be expected to provide a similar benefit for the delivery of these nuclear receptor antagonists into the cell and eventually to the nuclear compartment.\",\n \"Another approach that has shown promise in enhancing peptide bioavailability is the replacement of one or more amide bonds with various backbone replacements.\",\n \"These may include pseudopeptides, with CH2S, CH2NH.\",\n \"CH═(CH, or N-methyl amide bond modifications, or can involve the substitution of an amino acid with more conformationally constrained variants such as alpha methyl and beta methyl substitutions.\",\n \"The replacement of cysteine by penicillamine (beta, beta-dimethyl cysteine) has been previously mentioned.\",\n \"This modification is able to reduce the flexibility of the disulfide ring and can enhance stability, potency, and selectivity, as has been documented in the case of the mu selective opioid analog known as DPDPE.\",\n \"The major therapeutic benefit gained by the design and synthesis of coactivator antagonists is a more effective control of steroid receptor mediated transcriptional processes.\",\n \"Thus in diseases such as breast cancer or prostate cancer, certain hormone dependent tumors grow through the uncontrolled steroid-mediated transcription within the malignant cells.\",\n \"Effective therapeutics may me used in the assessment, treatment, and prevention of cancer.\",\n \"It is also important that these agents be selective so that undesired cell proliferation is prevented but such other benefits of estrogenic agonists such as prevention of osteoporosis should not be compromised.\",\n \"Control of transcription may be desired in order to help overcome one or more genetic malfunctions in an individual.\",\n \"It may also be anticipated that these novel antagonists can serve as diagnostic agents and as effective inhibitors in the case of various types of orphan nuclear receptors, whose functions have yet to be determined (Burris and McCabe, 2000).\",\n \"Experimental (Peptide Synthesis) The linear and cyclic peptides were synthesized using Boc-based Merrifield solid phase peptide synthesis using a anhydrous hydrogen fluoride for cleavage from the methylbenzhydryamine resin support to provide the targeted peptide amides.\",\n \"Scheme 1 summarizes the approach used for two cyclic variants.\",\n \"A lactam bridge between Glu, Lys was formed on the resin following base-mediated cleavage of the fluorenylmethyl-class protecting groups.\",\n \"In contrast, disulfide bridge formation was performed off-resin, with Tam's DMSO oxidation procedure providing the best results when using heated sulfoxide reagent.\",\n \"Products were analyzed by CD, NMR spectroscopy, reversed phase high pressure liquid chromatography, and thin layer chromatography, and the expected structures confirmed with MALDI-TOF mass spectrometry.\",\n \"Biological Activity Data The synthetic helicormimetic peptides designed as antagonists of the estrogen receptor-coactivator interactions were tested in a competition binding assay (Lilly Research Labs) against a model linear peptide sequence.\",\n \"Activities are reported in the Table below in Ki values, with two of the best analogs labeled as PERM-1 and PERM-2, or Peptidomimetic Estrogen Receptor Modulators.\",\n \"TABLE I Peptide Analogs and their Ki values against ER alpha and ER beta ER α ER β Title Sequence MW μM μM AML-I-22 H-Leu-Glu-Gln-Leu-Leu-OH 614.3 N/A 201.2 AML-I-31 Mannosylacetvl-Leu-Glu-Gln-Leu-Leu-OH 818 N/A 339.4 AML-I-48/4 H-Lys-cyclo (D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 PERM-1 1085 0.025 0.39 AML-I-61/2 H-Lys-Lys-Ile-Leu-His-Arg-Leu-Leu-Gln-NH2 1147 0.17 2.8 AML-I-59/6 K-Lys-cyclo(Glu-Ile-Leu-Arg-Lys)-Leu-Leu-Gln-NH2 1120.7 0.22 4.8 AML-I-71/2 Ac-Lys-cyclo(D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 1127 0.12 7.7 AML-I-86/1 Gu-Lys-cyclo-(D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 1183 0.14 0.6 AML-I-83/4 Aib-Lys-cyclo(D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 1172 0.13 1.4 AML-I-89/2 H-Lys-His-Lys-Ile-Leu-His-Arg-Leu-Leu-Gln-Asp-Ser-Ser-OH 1573.9 0.38 6.9 AKG-I-28 H-D-Lys-cyclo (D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 1085 0.22 1.9 AKG-I-39 H-Lys-cyclo (Ala-Ile-Leu-Ala)- Arg-Leu-Leu-Gln-NH2, 1053 1.18 15.4 Lanthionine AKG-I-40 H-D-Lys-cyclo (AIa-Ile-Leu-Ala)- Arg-Leu-Leu-Gln-NH2, 1053 3.95 13.5 Lanthionine AKG-I-46 H-Lys-Leu-Leu-cyclo(D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln- 1311 0.398 2.0 NH2 AKG-I-48 K-Lys-cyclo(Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 1085 0.416 1.8 AKG-I-50 H-Arg-cyclo(D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 PERM-2 1113 0.011 0.077 AKG-I-59 H-Lys-cyclo(Cys-Leu-Ile-D-Cys)-Arg-Leu-Leu-Gln-NH2 1085 2.1 17.0 AKG-I-60 H-Lys-cyclo(Cys-Ile-Leu-D-Cys)-Arg-Leu-Leu-Gln-NH2 1085 2.4 7.2 AKG-I-61 H-Lys-cyclo(D-Cys-Ile-Leu-D-Cys)-Arg-Leu-Leu-Gln-NH2 1085 0.928 3.9 AKG-I-63 H-Lys-cyclo(Cys-Leu-D-Cys)-Arg-Leu-Leu-Gln-NH2 972 2.2 7.9 AKG-I-64 H-Lys-cyclo(D-Cys-Leu-D-Cys)-Arg-Leu-Leu-Gln-NH2 972 1.8 5.2 AKG-II-1 H-Arg-cyclo (D-Cys-Leu-Ile-Cys)-Arg-Leu-Leu-Gln-NH2 1113 .013 .216 AKG-II-3 H-Arg-cyclo (D-Cys-Ile-Leu-HomoCys)-Arg-Leu-Leu-Gln-NH2 1127 .013 .214 AKG-II-4 H-Arg-cyclo (DHomoCys-lIe-Leu-Cys)- Arg-Leu-Leu-Gln-NH2 1127 .035 .591 AKG-II-8 H-Arg-cyclo (Cys-Ile-Leu-Arg-Cys)-Leu-Leu-Gln-NH2 1113 .174 1.16 AKG-II-9 H-Arg-cyclo (D-Pen-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 1141 .168 .933 AKG-II-10 H-Arg-cyclo (D-Cys-Ile-Leu-Pen)-Arg-Leu-Leu-Gln-NH2 1141 .088 1.91 AKG-II-11 H-Arg-cyclo(D-Pen-Ile-Leu-Pen)-Arg-Leu-Leu-Gln-NH2 1169 .078 3.97 SRC-1 NR2 LTERHKILHRLLQEGSPSD 0.39 — Short linear peptides that contain the LXXLL sequence, such as Leu-Asn-Gln-Leu-Leu, do not display any inhibitory activity with respect to the desired effect of inhibiting the binding of the estrogen receptors to the helical segment of coactivator proteins.\",\n \"As seen in the Table, compounds that contain a D-Cys, L-Cys pairing are especially active with respect to binding inhibition.\",\n \"A report by Geistlinger ad Guy teaches of the inhibition of the interaction between thyroid hormone and its interaction using side chain to side chain linked peptides.\",\n \"The ring in this example is formed through an amide linkage between a lysine residue and a glutamic acid residue.\",\n \"But this report does not include any examples of disulfide bridges nor of the preference for a D-cysteine and L-cysteine pairing, nor does it include any examples of receptors other than the thyroid nuclear receptor.\",\n \"X-Ray Structure of a Helicomimetic Peptide Bound to the Estrogen Receptor Dimer Ligand Binding Domain (LBD) The conditions for the cyrstallization of the peptide protein complex as shown in FIG.\",\n \"1 are describes as follows: Cloning of the Gene Expression and Purification of Human ER LBD.\",\n \"The ER LBD gene was overexpressed in E. coli and purified by Pan Vera, Inc. Crystallization.\",\n \"Diffraction-quality crystals of the ER LBD complex were grown by the vapor diffusion technique at 294K using a Hampton (Hampton Research Inc.,) crystallization screen.\",\n \"Crystals belong to orthorhombic space group C2221, with unit cell parameters a=53.8 Å, b=102.4 Å, c=195.3 Å.\",\n \"There are two molecules of the complex per asymmetric unit, with a Vm value (Matthews 1968) of 2.28 Å3/Da that corresponds to a solvent content of approximately 46% in both cases.\",\n \"The 17-estradiol and PERM-1 (peptide) in 2-3 fold of excess of the protein were used for co-crystallization.\",\n \"Data Collection, Structure Solution, and Crystallographic Refinement.\",\n \"The diffraction data resolution of (resolution of 2.7 Å; Rmerge=0.114, and completeness of 95%) were collected using a MarCCD (refmar) detector on IMCA (Industrial Macromolecular Crystallography Association) beam line BM-17 at the APS (Advanced Photon Source, Argonne National Laboratories) at 100K, using 15-20% glycerol as a cryoprotectant.\",\n \"The diffraction data were reduced using HKL2000 (Otwinowski and Minor 1997) and the intensities were scaled with SCALEPACK.\",\n \"Crystal structure was determined by the method of molecular replacement using the AMORE program suite (CCP4; Collaborative Computing Project #4 1994).\",\n \"The crystal structure was refined against data between 20-2.7 Å using a maximum likely-hood algorithm as incorporated in the program CNX2000 (Badger et al., 1999) (Rwork=0.203, Rfree=0.258, RMS=0.007 Å) (Brünger 1992).\",\n \"The program suite QUANTA 98 (Molecular Simulation Inc., San Diego, Calif.) was used for visual inspection and manual corrections between rounds of refinement.\",\n \"An analysis of the geometry showed all parameters were within the values expected for a model at this resolution.\",\n \"All residues were found in the most favorable and additionally allowed regions of a Ramachandran plot.\",\n \"The omitted unbiased electron density map was used for positioning 17β-estradiol and the PERM-1 peptide.\",\n \"Specific compositions, methods, or embodiments discussed are intended to be only illustrative of the invention disclosed by this specification.\",\n \"Variation on these compositions, methods, or embodiments are readily apparent to a person of skill in the art based upon the teachings of this specification and are therefore intended to be included as part of the inventions disclosed herein.\",\n \"Reference to documents made in the specification is intended to result in such patents or literature cited are expressly incorporated herein by reference, including any patents or other literature references cited within such documents as if fully set forth in this specification.\",\n \"The foregoing detailed description is given primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom, for modification will become obvious to those skilled in the art upon reading this disclosure and may be made upon departing from the spirit of the invention and scope of the appended claims.\",\n \"Accordingly, this invention is not intended to be limited by the specific exemplifications presented hereinabove.\",\n \"Rather, what is intended to be covered is within the spirit and scope of the appended claims.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471120"}}},{"rowIdx":575,"cells":{"text":{"kind":"list like","value":[["Novel fungicide compositions based on pyridylmethylbenzamide and propamocarb derivative","1) Fungicidal compositions comprising: a) at least one pyridylmethylbenzamide derivative of formula (I): in which the various radicals are as defined in the description, and b) at least one compound (II), which is propamocarb.","2) Method of curatively or preventively combating phytopathogenic fungi in crops, characterized in that an effective, nonphytotoxic amount of one of these fungicidal compositions is applied to the aerial parts of the plants."],["1.Fungicidal compositions comprising: a) at least one pyridylmethylbenzamide derivative of formula (I) in which: R1 is selected from a hydrogen atom, an optionally substituted alkyl radical and an optionally substituted acyl radical; R2 is selected from a hydrogen atom and an optionally substituted alkyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a hydroxyl radical, a cyano radical, a nitro radical, an —SF5 radical, a trialkylsilyl radical, an optionally substituted amino radical, an acyl radical, and a group E, OE or SE in which E is selected from an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl and heterocyclyl radical, each of which can be optionally substituted; c represents 0, 1, 2, 3 or 4; q represents 0, 1, 2, 3 or 4; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field; and b) at least one compound (II), which is propamocarb.","2.Fungicidal compositions according to claim 1, characterized in that the compound (I) possesses one of the following characteristics, taken individually or in combination: R1 and R2, which are identical or different, are selected independently from a hydrogen atom and an optionally substituted alkyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a hydroxyl radical, a nitro radical, an optionally substituted amino radical, an acyl radical, and a group E, OE or SE in which E is selected from an alkyl, cycloalkyl, phenyl and heterocyclyl radical, each of which can be optionally substituted; c represents 0, 1, 2 or 3; q represents 0, 1, 2 or 3; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.","3.Fungicidal compositions according to claim 1, characterized in that the compound (I) possesses one of the following characteristics, taken individually or in combination: R1 and R2, which are identical or different, are selected independently from a hydrogen atom and a methyl or ethyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a nitro radical, an optionally substituted amino radical and an alkyl, cycloalkyl, phenyl or heterocyclyl radical, each of which can be optionally substituted; c represents 1 or 2; q represents 1 or 2; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.","4.Fungicidal compositions according to claim 1, characterized in that the compound (I) possesses one of the following characteristics: R1 and R2 each represent a hydrogen atom; R3 and R4, which are identical or different, are selected independently from a halogen atom, a nitro radical, an alkyl radical and a trifluoromethyl radical; c and q represent, independently of one another, 2; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.","5.Fungicidal compositions according to claim 1, characterized in that the compound of formula (I) is selected from: 2,6-dichloro-N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}benzamide, N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}-2-fluoro-6-nitrobenzamide, and N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}-2-methyl-6-nitrobenzamide, and their possible tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.","6.Fungicidal compositions according to claim 1, characterized in that the compound (D is selected from 2,6-dichloro-N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}benzamide, N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}-2-fluoro-6-nitrobenzamide, and N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl} -2-methyl-6-nitrobenzamide and the compound (II) is propamocarb.","7.Fungicidal compositions according to claim 6, characterized in that the compound (I) is 2,6-dichloro-N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}benzamide and in that the compound (II) is propamocarb.","8.Fungicidal compositions according to claim 1, characterized in that they comprise at least one compound of formula (I) and at least one compound (II), the compound (I)/compound (II) ratio being between 1/500 and 1/1.9.Fungicidal compositions according to claim 8, characterized in that the compound (I)/compound (II) ratio is between 1/200 and 1/5.10.Fungicidal compositions according to claim 9, characterized in that the compound (I)/compound (II) ratio is between 1/150 and 1/10.11.Fungicidal compositions according to claim 1, characterized in that the compound (I)/compound (II) ratio is chosen so as to produce a synergistic effect.","12.Synergistic fungicidal compositions according to claim 11, characterized in that the compound (I)/compound (II) ratio is between 1/200 and 1/5.13.Synergistic fungicidal compositions according to claim 12, characterized in that the compound (I)/compound (II) ratio is between 1/140 and 1/10.14.Fungicidal compositions according to claim 1, characterized in that they comprise, in addition to compounds (I) and (II), an agriculturally suitable inert vehicle and optionally an agriculturally suitable surfactant.","15.Fungicidal compositions according to claim 1, characterized in that they contain from 0.5 to 99% of the combination of compound (I) and compound (II).","16.Method of curatively or preventively combating phytopathogenic fungi in crops, characterized in that an effective (agronomically effective), nonphytotoxic amount of a fungicidal composition according to claim 1 is applied to the soil in which the plants are growing or are liable to grow, to the leaves and/or the fruits of the plants or to the seeds of the plants.","17.Method according to claim 16, characterized in that the fungicidal composition is applied by spraying a liquid onto the aerial parts of the crops to be treated.","18.Method according to claim 16, characterized in that the amount of fungicidal composition corresponds to a dose of compound (I) and of compound (II) between approximately 1 g/ha and approximately 1 000 g/ha.","19.Method according to claim 16, characterized in that the treated crop is tomato, potato or grapevine.","20.Method according to claim 19, characterized in that the phytopathogenic fungus treated is downy mildew in tomatoes, potatoes and grapevines.","21.Product comprising a compound of formula (I) and a compound of formula (II) as a combined preparation for simultaneous, separate or sequential use in the control of phytopathogenic fungi in crops in one locus.","22.Method according to claim 17, characterized in that the amount of fungicidal composition corresponds to a dose of compound (I) and of compound (II) between approximately 1 g/ha and approximately 1 000 g/ha."],["The present invention relates to novel fungicidal compositions comprising at least one pyridylmethylbenzamide derivative and propamocarb or one of its salts and being intended in particular for crop protection.","It likewise relates to a method of protecting crops against fungal disease by applying these compositions.","European patent application EP-A-1 056 723 in particular discloses compounds of the pyridylmethylbenzamide type which have a fungicidal action and allow prevention of the growth and development of phytopathogenic fungi which attack, or are likely to attack, crops.","Propamocarb, moreover, is a widely known fungicidal active substance which is sold in particular under the name Previcur®.","Propamocarb, in the form of the salt with hydrochloric acid, is described for example in patents DE 1567169 and DE 1643040 and also in “The Pesticide Manual”, a World Compendium, 11th edition, C. D. S. Tomlin, British Crop Protection Council, pages 1015-17, No.","599.However, it is always desirable to improve the products which can be used by the farmer to combat fungal diseases in crops, and especially to combat downy mildews.","It is also always desirable to reduce the amounts of chemicals dispersed into the environment in order to combat fungal infestations in crops, particularly by reducing the rates at which the products are applied.","It is always desirable, finally, to increase the number of antifungal products available to the farmer, allowing him or her to select the product most suitable for the particular application.","One aim of the invention is therefore to provide a novel fungicidal composition which is of use in solving the problems set out above.","Another aim of the invention is to provide a novel fungicidal composition which is of use in the preventive and curative treatment of fungal diseases in crops such as, for example, solanums, cereals and grapevines.","Another aim of the invention is to provide a novel fungicidal composition exhibiting improved efficacy against downy mildew, powdery mildew, rust and botrytis in cereals, solanums and grapevines.","Another aim of the invention is to provide a novel fungicidal composition exhibiting improved efficacy against downy mildew and/or powdery mildew and/or botrytis in grapevines.","It has now been found that these aims can be achieved in whole or in part by virtue of the fungicidal compositions of the present invention.","The present invention therefore provides firstly fungicidal compositions comprising: a) at least one pyridylmethylbenzamide derivative of formula (I) in which: R1 is selected from a hydrogen atom, an optionally substituted alkyl radical and an optionally substituted acyl radical; R2 is selected from a hydrogen atom and an optionally substituted alkyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a hydroxyl radical, a cyano radical, a nitro radical, an —SF5 radical, a trialkylsilyl radical, an optionally substituted amino radical, an acyl radical, and a group E, OE or SE in which E is selected from an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl and heterocyclyl radical, each of which can be optionally substituted; c represents 0, 1, 2, 3 or 4; q represents 0, 1, 2, 3 or 4; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field; and b) at least one compound (II), which is propamocarb.","In the definitions of the compounds of formula (I) set out above, the various radicals and chemical terms employed, unless specified otherwise, have the following meanings: “alkyl” or “alkyl-” denotes a saturated linear or branched hydrocarbon radical containing from 1 to 6 carbon atoms; “alkenyl” denotes a linear or branched hydrocarbon radical containing from 1 to 6 carbon atoms and an unsaturation in the form of a double bond; “alkynyl” denotes a linear or branched hydrocarbon radical containing from 1 to 6 carbon atoms and an unsaturation in the form of a triple bond; “alkoxy” denotes an alkyl-oxy radical; “acyl” denotes the formyl radical or an alkoxycarbonyl radical; “cycloalkyl” denotes a saturated cyclic hydrocarbon radical containing from 3 to 8 carbon atoms; “aryl” denotes a phenyl or naphthyl radical; “heterocyclyl” denotes an unsaturated or fully or partly saturated cyclic radical containing from 3 to 8 atoms selected from carbon, nitrogen, sulphur and oxygen nonlimiting examples including pyridyl, pyridinyl, quinolyl, furyl, thienyl, pyrrolyl and oxazolinyl; the term “optionally substituted” signifies that the radicals thus qualified may be substituted by one or more radicals selected from chlorine, bromine, fluorine, iodine, alkyl, alkoxy, hydroxyl, nitro, amino; cyano and acyl.","The compounds of formula (I) are described for example in patent application EP-A-1 056 723 and preference among them will be given to the compounds possessing one of the following characteristics, taken individually or in combination: R1 and R2, which are identical or different, are selected independently from a hydrogen atom and an optionally substituted alkyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a hydroxyl radical, a nitro radical, an optionally substituted amino radical, an acyl radical, and a group E, OE or SE in which E is selected from an alkyl, cycloalkyl, phenyl and heterocyclyl radical, each of which can be optionally substituted; c represents 0, 1, 2 or 3; q represents 0, 1, 2 or 3; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.","Among the compounds of formula (I) further preference will be given to the compounds possessing the following characteristics, taken individually or in combination: R1 and R2, which are identical or different, are selected independently from a hydrogen atom and a methyl or ethyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a nitro radical, an optionally substituted amino radical and an alkyl, cycloalkyl, phenyl or heterocyclyl radical, each of which can be optionally substituted; c represents 1 or 2; q represents 1 or 2; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.","More particularly, the compounds of formula (I) possess the following characteristics: R1 and R2 each represent a hydrogen atom; R3 and R4, which are identical or different, are selected independently from a halogen atom, a nitro radical, an alkyl radical and a trifluoromethyl radical; c and q represent, independently of one another, 2; and their possible tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.","By way of example, the following compounds of formula (I) are very particularly preferred within the context of the present invention: Compound (Ia): 2,6-dichloro-N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}benzamide; Compound (Ib): N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}-2-fluoro-6-nitrobenzamide; Compound (Ic): N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}-2-methyl-6-nitrobenzamide; and their possible tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.","The compound (II) defined above is probamocarb, namely propyl 3-(dimethylamino)propylcarbamate, which is described for example in “The Pesticide Manual”, 11th edition, C D S Tomlin, British Crop Protection Council, pages 1015-1017, No.","599.Advantageously, the propamocarb used in the compositions of the present invention will be in the form of the hydrochloride, i.e.","the addition salt of propamocarb with hydrochloric acid, known commercially under the name Previcur®.","The propamocarb which is useful for the compositions of the present invention can also be used in the form of the salt with fosetyl, as described in patent application EP-A-1 056 755 or else in patent application WO-A-98/44801.Advantageously, the compositions of the present invention comprise compound (Ia) or compound (Ib) or compound (Ic) with compound (II).","The preferred compositions of the present invention comprise compound (Ia) with compound (II).","The present invention accordingly provides fungicidal compositions comprising at least one pyridylmethylbenzamide derivative of formula (I) as defined above and at least one compound (II) as defined above, the compound (I)/compound (II) ratio being between 1/500 and 1/1, preferably between 1/200 and 1/5, more preferably between 1/150 and 1/10.The compound (I)/compound (II) ratio is defined as being the ratio by weight of these two compounds.","The same is true of any ratio of two chemical compounds mentioned subsequently in the present text, unless a different definition of this ratio is expressly indicated.","It will be appreciated that the said fungicidal compositions may comprise a single compound (I) or more than one such compound and/or a single compound (II) or more than one such compound, and also one or more other fungicidal, herbicidal, insecticidal and/or plant growth regulator compounds, depending on the use for which the said compositions are intended.","Accordingly, the fungicidal compositions of the present invention may further comprise, for example, one or more other active fungicidal substances selected from acibenzolar-S-methyl, azoxystrobin, benalaxyl, benomyl, blasticidin-S, bromuconazole, captafol, captan, carbendazim, carboxin, carpropamid, chlorothalonil, fungicidal compositions based on copper, copper derivatives such as copper hydroxide and copper oxychloride, cyazofamid, cymoxanil, cyproconazole, cyprodinyl, dichloran, diclocymet, diethofencarb, difenoconazole, diflumetorim, dimethomorph, diniconazole, discostrobin, dodemorph, dodine, edifenphos, epoxyconazole, ethaboxam, ethirimol, famoxadone, fenamidone, fenarimol, fenbuconazole, fenhexamide, fenpiclonil, fenpropidine, fenpropimorph, ferimzone, fluazinam, fludioxonil, flumetover, fluquinconazole, flusilazole, flusulfamide, flutolanil, flutriafol, folpel, furalaxyl, furametpyr, guazatine, hexaconazole, hymexazol, imizalil, iprobenphos, iprodione, isoprothiolane, kasugamycin, kresoxim-methyl, mancozeb, maneb, mefenoxam, mepanipyrim, metalaxyl and its enantiomeric forms such as metalaxyl-M, metconazole, metiram-zinc, metominostrobin, oxadixyl, pefurazoate, penconazole, pencycuron, phosphorous acid and its derivatives such as fosetyl-Al, phthalide, picoxystrobin, probenazole, prochloraz, procymidone, propiconazole, pyraclostrobin, pyrimethanil, pyroquilon, quinoxyfen, silthiofam, simeconazole, spiroxamine, tebuconazole, tetraconazole, thiabendazole, thifluzamide, thiophanate, for example thiophanate-methyl, thiram, triadimefon, triadimenol, tricyclazole, tridemorph, trifloxystrobin, triticonazole, valinamide derivatives such as, for example, iprovalicarb, vinclozolin, zineb and zoxamide.","The invention further provides a method of curatively or preventively combating phytopathogenic fungi in crops, characterized in that an (agronomically) effective and nonphytotoxic amount of a fungicidal composition of the invention is applied to the soil in which the plants are growing or are liable to grow, to the leaves and/or the fruits of the plants or to the seeds of the plants.","The compositions of the invention are advantageous for combating fungal diseases in numerous crops such as, for example, cereals, vegetables, solanums, market-garden crops, grapevines, fruits in general, and especially for combating downy mildew, damping off, septoria disease and Pythium sp.","in these crops.","The compositions of the invention may also be used for combating other phytopathogenic diseases in crops that are well known to the person skilled in the art to whom the compounds of formula (I) and the compounds (II) are available.","These compounds generally improve markedly the respective isolated actions of compound (I) and of compound (II) for a certain number of fungi which are particularly harmful in crops, particularly for solanums (tomatoes, potatoes, etc.","), more particularly for downy mildew in potatoes and downy mildew in tomatoes, while preserving an absence of phytotoxicity with regard to these crops.","There is therefore an improvement in the spectrum of activity and a possibility of reducing the respective dose of each active substance used, the latter quality being particularly important for ecological reasons which are easy to comprehend.","In the compositions of the invention, the compound (I)/compound (II) ratio is advantageously selected so as to produce a synergistic effect.","The term “synergistic effect” refers in particular to the effect defined by S. R. Colby in an article titled “Calculating synergistic and antagonistic responses of herbicide combinations”, Weeds, (1967), 15, pages 20-22.The latter article makes use of the formula: E = X + Y - X Y 100 in which E represents the expected percentage inhibition of the disease for the combination of the two fungicides at defined doses (for example equal respectively to x and y), X is the observed percentage inhibition of the disease by compound (I) at a defined dose (equal to x), Y is the observed percentage inhibition of the disease by compound (II) at a defined dose (equal to y).","When the observed percentage inhibition of the combination is greater than E, there is a synergistic effect.","A synergistic effect is also understood to be that defined by applying the method of Tammes, “Isoboles, a graphic representation of synergism in pesticides”, Netherlands Journal of Plant Pathology, 70, (1964), pages 73-80.The abovementioned ranges for the compound (I)/compound (II) ratio are by no means limitative of the scope of the invention but rather are cited by way of indication, it being well within the abilities of the person skilled in the art to carry out complementary tests in order to find other values for the dosage ratio of these two compounds at which a synergistic effect is observed.","Accordingly, the compositions of the invention comprising compound (I) and compound (II) allow quite remarkable synergistic properties to be observed.","According to one variant of the compositions of the invention, the compound (I)/compound (II) ratio is advantageously between 1/200 and 1/5, preferably between 1/140 and 1/10.The invention therefore also comprises methods of treating plants to counter phytopathogenic diseases, characterized in that a composition comprising at least one compound of formula (I) with at least one compound (II) is applied.","It is also possible to apply a composition comprising both active substances or else two compositions each comprising one of the two active substances, either simultaneously or in succession so as to have the conjoint effect.","These compositions embrace not only compositions which are ready to be applied to the crop to be treated by means of an appropriate device, such as a sprayer, but also commercial concentrated compositions which have to be diluted before application to the crop.","The present invention provides a method of combating a wide variety of phytopathogenic diseases in crops, particularly for combating septoria disease and downy mildew.","These diseases can be combated by direct foliar application.","The present invention therefore provides a method of curatively or preventively combating phytopathogenic diseases in crops which comprises treating the said crop (for example by application or by administration) with an effective, nonphytotoxic amount of a combination as defined above.","By treatment of the crop is meant application or administration of a fungicidal composition as described above to the aerial parts of the crops or to the soil in which they are growing, which are infested or liable to be infested by a phytopathogenic disease, such as downy mildew or septoria disease, for example.","By treatment of the crop is also meant the treatment of reproduction products of the crop, such as seeds or tubers for example.","The compositions described below are generally used for application to growing plants or to loci on which crops are grown, or for the dressing or coating of seeds.","Among the means which are suitable for applying the compositions of the invention mention may be made of the use of powders, foliar sprays, granules, mists or foams, or else means in the form of suspensions of finely divided or encapsulated compositions; for the treatment of soils or roots with liquid imbibitions, powders, granules, fumes or foams; for application to the seeds of plants, the use, as seed dressing or seed coating agents, of powders or of liquid slurries.","The compositions of the invention are, appropriately, applied to the vegetation and in particular to the leaves infested with the phytopathogenic fungi.","Another method of applying the compounds or compositions of the invention is to add a formulation comprising the active substances to the irrigation water.","This irrigation may be an irrigation using sprinklers.","The formulations which are suitable for applying the compositions of the invention comprise formulations suitable for use in the form, for example, of sprays, powders, granules, mists, foams, emulsions or others.","In practice, for combating phytopathogenic diseases in crops, one method, for example, consists in applying an effective amount of a composition of the invention to the plants or to the medium in which they are growing.","For a method of this kind, the active substances are generally applied to the locus itself in which the infestation is to be controlled, the said application taking place at an effective dose of between approximately 1 g and approximately 1 000 g of active substances in total per hectare of locus treated.","Under ideal conditions, depending on the nature of the phytopathogenic fungus to be treated, a lower dose may provide adequate protection.","Conversely, under poor climatic conditions, resistance or other factors may require higher doses of active substance.","The effective working doses of the combinations employed in the invention may vary within wide proportions, according in particular to the nature of the phytopathogenic fungi to be eliminated or the degree of infestation, for example, of the plants by these fungi.","The optimum dose commonly depends on a number of factors, for example on the type of phytopathogenic fungus to be treated, on the type or level of development of the infested plant, on the density of vegetation, or else on the method of application.","More preferably, an effective dose of active substances (I) and (II) is between approximately 5 g/ha and approximately 700 g/ha.","For their use in practice, the compositions of the invention can be used alone and may also advantageously be used in formulations comprising one or other of the active substances or else both together, in association or combination with one or more other, compatible components which are, for example, solid or liquid fillers or diluents, adjuvants, surfactants, or equivalents which are appropriate for the desired use and acceptable for agricultural uses.","The formulations may be of any type which is known in the sector and suitable for application in all types of plantings or crops.","These formulations, which can be prepared in any manner known in this sector, also form part of the invention.","The formulations may also comprise other types of ingredients such as protective colloids, adhesives, thickeners, thixotropic agents, penetrants, oils for spraying, stabilizers, preservatives (especially antimould agents), sequestrants, or others, and other active ingredients which are known to possess pesticidal properties (especially fungicidal, insecticidal, acaricidal or nematicidal properties) or to possess plant growth regulator properties.","More generally, the compounds used in the invention may be combined with any solid or liquid additives which correspond to the normal formulation techniques.","In general, the formulations of the invention commonly contain from approximately 0.05% to approximately 99% (by weight) of one or more compositions of the invention, from approximately 1% to approximately 95% of one or more solid or liquid fillers, and optionally from approximately 0.1% to approximately 50% of one or more other, compatible compounds, such as surfactants or others.","In the present specification, the term “filler” signifies an organic or inorganic, natural or synthetic component with which the active components are combined in order to facilitate their application to, for example, the plants, seeds, or soil.","Consequently, this filler is generally inert and it must be acceptable (for example, acceptable for agronomic use, especially for treating plants).","The filler can be solid, examples being clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers (for example ammonium salts), natural soil minerals, such as kaolins, clays, talc, lime, quartz, attapulgite, montmorillonite, bentonite or diatomaceous earths, or synthetic minerals, such as silica, alumina, or silicates, especially aluminium silicates or magnesium silicates.","Solid fillers which are suitable for granules are as follows: natural rocks, crushed or broken, such as calcite, marble, pumice, sepiolite and dolomite; synthetic granules of organic or inorganic flours; granules of organic material such as sawdust, coconut shell, corn ear or corn husk or tobacco stem; kieselguhr, tricalcium phosphate, powdered cork, or adsorbent carbon black; water-soluble polymers, resins, waxes; or solid fertilizers.","If desired, such compositions may comprise one or more compatible agents such as wetting agents, dispersants, emulsifiers or colorants which, when solid, may also serve as diluents.","The fillers may also be liquid, examples including the following: water, alcohols, especially butanol or glycol, and their ethers or esters, especially methyl glycol acetate; ketones, especially acetone, cyclohexanone, methyl ethyl ketone, methyl isobutyl ketone or isophorone; petroleum fractions such as paraffinic or aromatic hydrocarbons, especially xylenes or alkylnaphthalenes; mineral oils or plant oils; chlorinated aliphatic hydrocarbons, especially trichloroethane or methylene chloride; chlorinated aromatic hydrocarbons, especially chlorobenzenes; water-soluble or highly polar solvents such as dimethylformamide, dimethyl sulphoxide, N,N-dimethylacetamide and N-methylpyrrolidone; N-octylpyrrolidone, liquefied gases; or others, either separately or in a mixture.","The surfactant may be an emulsifier, a dispersant or a wetting agent, of ionic or nonionic type, or a mixture of these surfactants.","Among them use is made, for example, of salts of polyacrylic acids, salts of lignosulphonic acids, salts of phenolsulphonic or naphthalenesulphonic acids, polycondensates of ethylene oxide with fatty alcohols or fatty acids or fatty esters or fatty amines, substituted phenols (especially alkylphenols or arylphenols), ester salts of sulphosuccinic acid, taurine derivatives (especially alkyl taurates), phosphoric esters of alcohols or of polycondensates of ethylene oxide with phenols, esters of fatty acids with polyols, or sulphate, sulphonate or phosphate functional derivatives of the above-described compounds.","The presence of at least one surfactant is generally essential when the active substances and/or the inert filler are only sparingly soluble or insoluble in water and when the filler of the said composition to be applied is water.","The formulations of the invention may further comprise other additives such as adhesives or colorants.","Adhesives such as carboxymethylcellulose, or synthetic or natural polymers in the form of powders, granules or matrices, such as gum arabic, latex, polyvinylpyrrolidone, polyvinyl alcohol or polyvinyl acetate, natural phospholipids, such as cephalins or lecithins, or synthetic phospholipids can be used in the formulations.","It is possible to use colorants such as inorganic pigments, such as the following, for example: iron oxides, titanium oxides, or Prussian blue; organic dyes, such as those of the alizarin, azo or metal phthalocyanine type; or trace elements such as salts of iron, manganese, boron, copper, cobalt, molybdenum or zinc.","The formulations comprising the compositions of the invention that are employed for combating phytopathogenic fungi in crops may also comprise stabilizers, other fungicidal agents, insecticides, acaricides, nematicides, anthelmintics or anticoccidials, bactericides, attractants or repellents or pheromones for arthropods or vertebrates, deodorizers, flavourings or colorants.","These substances may be selected purposely for improving the intensity, persistence, safety or spectrum of action on the phytopathogenic fungi in the crops or for rendering the composition capable of accomplishing other useful functions for the treated surfaces.","For their use in agriculture, the compositions of the invention are accordingly formulated in various liquid or solid forms.","As solid formulations, mention may be made of powders for dusting (with an active substance content which can be up to 100%) and granules, especially those obtained by extrusion, spraying, compacting, impregnation of a granulated support, or granulation from a powder (the active substance content of these granules being between 0.5 and 80% in the latter cases).","The fungicidal compositions of the invention may also be used in the form of powders for dusting; it is also possible to use formulations comprising 50 g of active substances and 950 g of talc; it is also possible to use formulations containing 20 g of active substances, 10 g of finely divided silica and 970 g of talc; these constituents are mixed and ground and the mixture is applied by dusting.","As formulations which are liquid or which are intended to constitute liquid compositions at the time of application, mention may be made of solutions, especially water-soluble concentrates, emulsifiable concentrates, emulsions, concentrated suspensions and wettable powders (or sprayable powder).","The concentrated suspensions which can be applied by spraying are prepared so as to obtain a stable fluid product which does not settle and which leads to effective bioavailability of the active substances.","These suspensions commonly contain from 5% to 75% of active substances, preferably from 10% to 25%, from 0.5 to 75% of surfactants, preferably from 5% to 50%, from 0 to 10% of appropriate additives, such as thickeners which are mineral or organic in origin, antifoams, corrosion inhibitors, adhesives, preservatives, such as Proxel GXL® for example, antifreezes and, as vehicle, water or an organic liquid in which the active substances are sparingly soluble or insoluble: certain organic solids or mineral salts may be dissolved in the vehicle in order to help prevent sedimentation or as antifreezes for the water.","In certain cases, and especially for formulations intended for treating seeds, one or more colorants may be added.","For foliar application, the choice of surfactants is essential in order to ensure effective bioavailability of the active substances; accordingly, use will be made preferably of a combination of a hydrophilic-type surfactant (HLB>10) and of a lipophilic-type surfactant (HLB<5).","Such combinations of surfactants are described for example in the as-yet unpublished French patent application No.","00 04015.By way of example, here are 3 possible formulations of the suspension concentrate type, adapted for different crops: EXAMPLE SC 1 (in g/kg) This example is more suitable for monocotyledonous crops (cereals, rice, etc.)","active substances 150 hydrophilic-type surfactant (for example 300 Rhodasurf 860P) lipophilic-type surfactant (for example 150 Plurafac LF 700) ethoxylated tristyrylphenol phosphate 50 antifoam 5 propylene glycol 30 Aerosil 200 20 Attagel 50 40 water (qs 1 kg) 255 EXAMPLE SC2 (in g/kg) This example is more suitable for dicotyledonous crops (grapevines, fruit trees, etc.)","active substances 150 hydrophilic-type surfactant (for example 150 Rhodasurf 860P) ethoxylated tristyrylphenol phosphate 50 antifoam 5 propylene glycol 30 Aerosil 200 20 Attagel 50 40 water (qs 1 kg) 555 EXAMPLE SC3 (in g/kg) This example is more specifically adapted to the treatment of seeds.","active substances 50 hydrophilic-type surfactant (for example 5 Rhodasurf 860P) ethoxylated tristyrylphenol phosphate 15 antifoam 1 propylene glycol 30 colorant 20 Rhodopol G 1.5 Proxel GXL 1.5 water (qs 1 kg) 876 These formulations will preferably be produced using the following technique: The selected surfactants (hydrophilic-type surfactant+lipophilic-type surfactant+ethoxylated tristyrylphenol phosphate) are mixed into the required amount of water using a turbine mixer; following homogenization, the other constituents of the formula apart from the active substances are then mixed in.","Then the active substances are added and, optionally, the thickener of mineral origin (Aerosil 200 and Attagel 50) in order to obtain a mixture with a viscous consistency.","The resulting mixture is then ground using a high-speed turbo mixer mill and then a ball mill so as to give a D50 of the order of from 1 to 3 μm and a D90 of between 3 and 8 μm.","Where no thickener of mineral origin is used, the thickener of natural origin (Rhodopol G) is added next and the mixture is stirred until an appropriate viscosity is obtained.","The wettable (or sprayable) powders are commonly prepared such that they contain from 20% to 95% of active substances, and they commonly contain, in addition to the solid vehicle, from 0% to 30% of a wetting agent, from 3% to 20% of a dispersant, and, when necessary, from 0.1% to 10% of one or more stabilizers and/or other additives, such as penetrants, adhesives, or anticaking agents, colorants, etc.","In order to obtain the sprayable or wettable powders, the active substances are intimately mixed in appropriate mixers with the additional substances and are ground using mills or other appropriate grinders.","Sprayable powders are obtained whose wettability and suspension characteristics are advantageous; they can be suspended with water at any desired concentration, and these suspensions can be used very advantageously in particular for application, for example, to the leaves of plants or to seeds.","By way of example, here are various compositions of wettable powders (or sprayable powders): EXAMPLE WP 1 active substances 50% ethoxylated fatty alcohol (wetting 2.5% agent) ethoxylated phenylethylphenol 5% (dispersant) chalk (inert vehicle) 42.5% EXAMPLE WP 2 active substances 10% synthetic C13 oxo alcohol of 0.75% branched type, ethoxylated with 8 to 10 ethylene oxide units (wetting agent) neutral calcium lignosulphonate 12% (dispersant) calcium carbonate (inert filler) qs 100% EXAMPLE WP 3 This wettable powder contains the same ingredients as in the preceding example, in the following proportions: active substances 75% wetting agent 1.50% dispersant 8% calcium carbonate (inert filler) qs 100% EXAMPLE WP 4 active substances 90% ethoxylated fatty alcohol (wetting 4% agent) ethoxylated phenylethylphenol 6% (dispersant) EXAMPLE WP 5 active substances 50% mixture of anionic and nonionic 2.5% surfactants (wetting agent) sodium lignosulphonate (dispersant) 5% kaolinic clay (inert vehicle) 42.5% The aqueous emulsions and dispersions, for example the compositions obtained by diluting a wettable powder of the invention with water, are included within the general scope of the present invention.","The emulsions may be of the water-in-oil or oil-in-water type and they may have a thick consistency like that of a mayonnaise sauce.","The fungicidal compositions of the invention may be formulated in the form of water-dispersible granules which are likewise included within the scope of the invention.","These dispersible granules, with an apparent density which is generally between approximately 0.3 and 0.6, have a particle size which is generally between approximately 150 and 2 000 and preferably between 300 and 1 500 microns.","The active substance content of these granules is generally between approximately 1% and 90%, and preferably between 25% and 90%.","The remainder of the granules is composed essentially of a solid filler and, optionally, of surfactant adjuvants which impart water-dispersibility properties to the granules.","These granules may be essentially of two distinct types depending on whether the filler used is soluble or insoluble in water.","When the filler is water-soluble, it may be mineral or, preferably, organic.","Excellent results have been obtained with urea.","In the case where the filler is insoluble it is preferably mineral, such as for example kaolin or bentonite.","In that case it is advantageously accompanied by surfactants (in a proportion of from 2 to 20% by weight of the granules) of which more than half is composed, for example, of at least one, essentially anionic dispersant, such as an alkali metal or alkaline earth metal polynaphthalenesulphonate or an alkali metal or alkaline earth metal lignosulphonate, the remainder consisting of nonionic or anionic wetting agents such as an alkali or alkaline earth metal alkylnaphthalenesulphonate.","Furthermore, although not indispensable, it is possible to add other adjuvants such as antifoams.","The granules may be prepared by mixing the required ingredients then granulating the mixture by a number of techniques which are known per se (granulator, fluid bed, atomizer, extrusion, etc.).","The final procedure is normally a crushing operation followed by screening to the particle size selected within the abovementioned limits.","It is also possible to use granules obtained as before then impregnated with a composition comprising the active substances.","The granules are preferably obtained by extrusion, operating as indicated in the examples below.","EXAMPLE DG1 Dispersible Granules 90% by weight of active substances and 10% of urea pellets are mixed in a mixer.","The mixture is subsequently ground in a toothed roll crusher.","A powder is obtained which is wetted with approximately 8% by weight of water.","The moist powder is extruded in a perforated-roll extruder.","This gives granules which are dried and then crushed and screened, so as to retain only those granules with a size of between 150 and 2 000 microns.","EXAMPLE DG2 Dispersible Granules The following constituents are mixed in a mixer: active substances 75% wetting agent (sodium alkylnaphthalene 2% sulphonate) dispersant (sodium polynaphthalenesulphonate) 8% water-insoluble inert filler (kaolin) 15% This mixture is granulated in a fluid bed in the presence of water and then dried, crushed and screened to give granules with a size of between 0.15 and 0.80 mm.","These granules may be used alone or in solution or dispersion in water in such a way as to give the desired dose.","They may also be used for preparing compositions with other active substances, especially fungicides, the latter being in the form of wettable powders or granules or aqueous suspensions.","The fungicidal compositions of the invention commonly contain from 0.5 to 95% of the association of compound (I) and compound (II).","The composition in question may be a concentrated composition, i.e.","the commercial product combining compound (I) and compound (II).","It may also be a dilute composition which is ready to be applied to the crops to be treated.","In the latter case, dilution with water can be carried out either starting from a concentrated commercial composition containing compound (I) and compound (II) (this mixture is called a ready mix) or by means of mixing at the time of use (in this case, the mixture is referred to as a tank mix) two concentrated commercial compositions each containing compound (I) and compound (II).","The invention lastly provides a method of curatively or preventively combating phytopathogenic fungi in crops, characterized in that an effective, nonphytotoxic amount of a fungicidal composition of the invention is applied to the plants to be treated.","The phytopathogenic fungi in crops that can be combated by this method are, in particular those: from the group of the oomycetes: of the genus Phytophthora such as Phytophthora infestans (solanum late blight, particularly potato or tomato blight), of the family of the Peronosporaceae, especially Plasmopara viticola (grapevine downy mildew), Plasmopara halstedii (sunflower downy mildew), Pseudoperonospora sp.","(in particular, cucurbit downy mildew and hop downy mildew), Bremia lactucae (lettuce downy mildew), Peronospora tabacinae (tobacco downy mildew), Peronospora parasitica (cabbage downy mildew), Peronospora viciae (pea downy mildew), and Peronospora destructor (onion downy mildew); from the group of the adelomycetes: of the genus Alternaria, for example Alternaria solani (solanium early blight, and especially early blight in tomatoes and potatoes), of the genus Guignardia, especially Guignardia bidwelli (grapevine black rot), of the genus Oidium, for example grapevine powdery mildew (Uncinula necator), powdery mildew of leguminous crops, for example Erysiphe polygoni (crucifer powdery mildew), Leveillula taurica, Erysiphe cichoracearum, Sphaerotheca fuligena (powdery mildew in cucurbits, composites and tomatoes), Erysiphe communis (powdery mildew in beet and cabbage), Erysiphe pisi (powder mildew in peas and alfalfa), Erysiphe polyphaga (powdery mildew in beans and cucumber), Erysiphe umbelliferarum (powdery mildew in umbellifers, especially carrots), Sphaerotheca humuli (hop powdery mildew); from the group of soil fungi: of the genus Pythium sp., of the genus Aphanomyces sp., especially Aphanomyces euteiches (white root rot in peas), Aphanomyces cochlioides (root rot in beet).","The term “is/are applied to the plants to be treated” is intended to signify, in the sense of the present text, that the fungicidal compositions that are subjects of the invention may be applied by means of various treatment techniques such as: the spraying of a liquid comprising one of the said compositions onto the aerial parts of the said plants, dusting, incorporation of granules or powders into the soil, irrigation around the said plants, and, in the case of trees, injection or painting on, the dressing or coating of seeds of the said plants by means of a slurry comprising one of the said compositions.","The spraying of a liquid onto the aerial parts of the crops to be treated is the preferred treatment method.","The examples which follow are given purely by way of illustration of the invention, and do not impose any limitation whatsoever thereon.","Although the invention has been described in terms of numerous preferred variants, the person skilled in the art will appreciate that many modifications, substitutions, omissions and changes can be made without departing from the spirit of this invention.","It is thus clearly understood that the scope of the present invention is limited solely by the scope of the claims which follow, and by their equivalents.","EXAMPLE Test of a Composition Against Late Blight in Potatoes (Phytophthora infestans: Preventive Action) The following active substances are used for this test: compound (Ia) in the form of a suspension concentrate (10 SC) compound (II): propamocarb hydrochloride (Previcur-N®, in the form of a soluble concentrate (66.5 SL).","Potato plants (three weeks old) are treated by automatic spraying (Mardrive) at a rate of 200 l/ha.","Six identical tests are carried out.","Compounds (Ia) and (II) are mixed (tank mix) in different proportions which in the results table are expressed in g/ha.","All of the plants are inoculated 24 hours after treatment with a suspension of sporangia of Phytophthora infestans, containing 20 000 spores per ml.","The inoculated plants are covered for 2 days and then placed in a controlled atmosphere at 20° C. until the results are read off.","The degree of infestation is evaluated by rating on a scale ranging from 1 to 10, the value 0 denoting the absence of disease and the value 100 denoting complete infestation (100%).","For each treatment, the percentage disease control is calculated relative to untreated plants, and the theoretical results are calculated using the Colby formula described above.","The difference between the experimental results and the calculated results allows the degree of synergism between the two compounds to be determined.","The results are shown in the table below: Test on Compound (Ia) and Compound (II) Concentration Synergism (g/ha) Rating Efficacy % (Colby) Compound 5 4.3 48 — (Ia) 25 3.3 60 — Compound 400 8.3 0 — (II) 600 6.8 18 — (Ia) + (II) 1/80 5 + 400 2.8 66 18 (Ia) + (II) 1/120 5 + 600 2.2 74 17 (Ia) + (II) 1/16 25 + 400 1.5 82 22 (Ia) + (II) 1/24 25 + 600 1.0 88 21 Untreated control: rating 8.3 The compositions of the invention comprising compound (Ia) and compound (II) exhibit a high level of synergism irrespective of the doses of each of the active substances and irrespective of the proportions of compound (Ia) to compound (II)."]],"string":"[\n [\n \"Novel fungicide compositions based on pyridylmethylbenzamide and propamocarb derivative\",\n \"1) Fungicidal compositions comprising: a) at least one pyridylmethylbenzamide derivative of formula (I): in which the various radicals are as defined in the description, and b) at least one compound (II), which is propamocarb.\",\n \"2) Method of curatively or preventively combating phytopathogenic fungi in crops, characterized in that an effective, nonphytotoxic amount of one of these fungicidal compositions is applied to the aerial parts of the plants.\"\n ],\n [\n \"1.Fungicidal compositions comprising: a) at least one pyridylmethylbenzamide derivative of formula (I) in which: R1 is selected from a hydrogen atom, an optionally substituted alkyl radical and an optionally substituted acyl radical; R2 is selected from a hydrogen atom and an optionally substituted alkyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a hydroxyl radical, a cyano radical, a nitro radical, an —SF5 radical, a trialkylsilyl radical, an optionally substituted amino radical, an acyl radical, and a group E, OE or SE in which E is selected from an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl and heterocyclyl radical, each of which can be optionally substituted; c represents 0, 1, 2, 3 or 4; q represents 0, 1, 2, 3 or 4; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field; and b) at least one compound (II), which is propamocarb.\",\n \"2.Fungicidal compositions according to claim 1, characterized in that the compound (I) possesses one of the following characteristics, taken individually or in combination: R1 and R2, which are identical or different, are selected independently from a hydrogen atom and an optionally substituted alkyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a hydroxyl radical, a nitro radical, an optionally substituted amino radical, an acyl radical, and a group E, OE or SE in which E is selected from an alkyl, cycloalkyl, phenyl and heterocyclyl radical, each of which can be optionally substituted; c represents 0, 1, 2 or 3; q represents 0, 1, 2 or 3; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.\",\n \"3.Fungicidal compositions according to claim 1, characterized in that the compound (I) possesses one of the following characteristics, taken individually or in combination: R1 and R2, which are identical or different, are selected independently from a hydrogen atom and a methyl or ethyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a nitro radical, an optionally substituted amino radical and an alkyl, cycloalkyl, phenyl or heterocyclyl radical, each of which can be optionally substituted; c represents 1 or 2; q represents 1 or 2; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.\",\n \"4.Fungicidal compositions according to claim 1, characterized in that the compound (I) possesses one of the following characteristics: R1 and R2 each represent a hydrogen atom; R3 and R4, which are identical or different, are selected independently from a halogen atom, a nitro radical, an alkyl radical and a trifluoromethyl radical; c and q represent, independently of one another, 2; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.\",\n \"5.Fungicidal compositions according to claim 1, characterized in that the compound of formula (I) is selected from: 2,6-dichloro-N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}benzamide, N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}-2-fluoro-6-nitrobenzamide, and N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}-2-methyl-6-nitrobenzamide, and their possible tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.\",\n \"6.Fungicidal compositions according to claim 1, characterized in that the compound (D is selected from 2,6-dichloro-N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}benzamide, N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}-2-fluoro-6-nitrobenzamide, and N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl} -2-methyl-6-nitrobenzamide and the compound (II) is propamocarb.\",\n \"7.Fungicidal compositions according to claim 6, characterized in that the compound (I) is 2,6-dichloro-N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}benzamide and in that the compound (II) is propamocarb.\",\n \"8.Fungicidal compositions according to claim 1, characterized in that they comprise at least one compound of formula (I) and at least one compound (II), the compound (I)/compound (II) ratio being between 1/500 and 1/1.9.Fungicidal compositions according to claim 8, characterized in that the compound (I)/compound (II) ratio is between 1/200 and 1/5.10.Fungicidal compositions according to claim 9, characterized in that the compound (I)/compound (II) ratio is between 1/150 and 1/10.11.Fungicidal compositions according to claim 1, characterized in that the compound (I)/compound (II) ratio is chosen so as to produce a synergistic effect.\",\n \"12.Synergistic fungicidal compositions according to claim 11, characterized in that the compound (I)/compound (II) ratio is between 1/200 and 1/5.13.Synergistic fungicidal compositions according to claim 12, characterized in that the compound (I)/compound (II) ratio is between 1/140 and 1/10.14.Fungicidal compositions according to claim 1, characterized in that they comprise, in addition to compounds (I) and (II), an agriculturally suitable inert vehicle and optionally an agriculturally suitable surfactant.\",\n \"15.Fungicidal compositions according to claim 1, characterized in that they contain from 0.5 to 99% of the combination of compound (I) and compound (II).\",\n \"16.Method of curatively or preventively combating phytopathogenic fungi in crops, characterized in that an effective (agronomically effective), nonphytotoxic amount of a fungicidal composition according to claim 1 is applied to the soil in which the plants are growing or are liable to grow, to the leaves and/or the fruits of the plants or to the seeds of the plants.\",\n \"17.Method according to claim 16, characterized in that the fungicidal composition is applied by spraying a liquid onto the aerial parts of the crops to be treated.\",\n \"18.Method according to claim 16, characterized in that the amount of fungicidal composition corresponds to a dose of compound (I) and of compound (II) between approximately 1 g/ha and approximately 1 000 g/ha.\",\n \"19.Method according to claim 16, characterized in that the treated crop is tomato, potato or grapevine.\",\n \"20.Method according to claim 19, characterized in that the phytopathogenic fungus treated is downy mildew in tomatoes, potatoes and grapevines.\",\n \"21.Product comprising a compound of formula (I) and a compound of formula (II) as a combined preparation for simultaneous, separate or sequential use in the control of phytopathogenic fungi in crops in one locus.\",\n \"22.Method according to claim 17, characterized in that the amount of fungicidal composition corresponds to a dose of compound (I) and of compound (II) between approximately 1 g/ha and approximately 1 000 g/ha.\"\n ],\n [\n \"The present invention relates to novel fungicidal compositions comprising at least one pyridylmethylbenzamide derivative and propamocarb or one of its salts and being intended in particular for crop protection.\",\n \"It likewise relates to a method of protecting crops against fungal disease by applying these compositions.\",\n \"European patent application EP-A-1 056 723 in particular discloses compounds of the pyridylmethylbenzamide type which have a fungicidal action and allow prevention of the growth and development of phytopathogenic fungi which attack, or are likely to attack, crops.\",\n \"Propamocarb, moreover, is a widely known fungicidal active substance which is sold in particular under the name Previcur®.\",\n \"Propamocarb, in the form of the salt with hydrochloric acid, is described for example in patents DE 1567169 and DE 1643040 and also in “The Pesticide Manual”, a World Compendium, 11th edition, C. D. S. Tomlin, British Crop Protection Council, pages 1015-17, No.\",\n \"599.However, it is always desirable to improve the products which can be used by the farmer to combat fungal diseases in crops, and especially to combat downy mildews.\",\n \"It is also always desirable to reduce the amounts of chemicals dispersed into the environment in order to combat fungal infestations in crops, particularly by reducing the rates at which the products are applied.\",\n \"It is always desirable, finally, to increase the number of antifungal products available to the farmer, allowing him or her to select the product most suitable for the particular application.\",\n \"One aim of the invention is therefore to provide a novel fungicidal composition which is of use in solving the problems set out above.\",\n \"Another aim of the invention is to provide a novel fungicidal composition which is of use in the preventive and curative treatment of fungal diseases in crops such as, for example, solanums, cereals and grapevines.\",\n \"Another aim of the invention is to provide a novel fungicidal composition exhibiting improved efficacy against downy mildew, powdery mildew, rust and botrytis in cereals, solanums and grapevines.\",\n \"Another aim of the invention is to provide a novel fungicidal composition exhibiting improved efficacy against downy mildew and/or powdery mildew and/or botrytis in grapevines.\",\n \"It has now been found that these aims can be achieved in whole or in part by virtue of the fungicidal compositions of the present invention.\",\n \"The present invention therefore provides firstly fungicidal compositions comprising: a) at least one pyridylmethylbenzamide derivative of formula (I) in which: R1 is selected from a hydrogen atom, an optionally substituted alkyl radical and an optionally substituted acyl radical; R2 is selected from a hydrogen atom and an optionally substituted alkyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a hydroxyl radical, a cyano radical, a nitro radical, an —SF5 radical, a trialkylsilyl radical, an optionally substituted amino radical, an acyl radical, and a group E, OE or SE in which E is selected from an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl and heterocyclyl radical, each of which can be optionally substituted; c represents 0, 1, 2, 3 or 4; q represents 0, 1, 2, 3 or 4; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field; and b) at least one compound (II), which is propamocarb.\",\n \"In the definitions of the compounds of formula (I) set out above, the various radicals and chemical terms employed, unless specified otherwise, have the following meanings: “alkyl” or “alkyl-” denotes a saturated linear or branched hydrocarbon radical containing from 1 to 6 carbon atoms; “alkenyl” denotes a linear or branched hydrocarbon radical containing from 1 to 6 carbon atoms and an unsaturation in the form of a double bond; “alkynyl” denotes a linear or branched hydrocarbon radical containing from 1 to 6 carbon atoms and an unsaturation in the form of a triple bond; “alkoxy” denotes an alkyl-oxy radical; “acyl” denotes the formyl radical or an alkoxycarbonyl radical; “cycloalkyl” denotes a saturated cyclic hydrocarbon radical containing from 3 to 8 carbon atoms; “aryl” denotes a phenyl or naphthyl radical; “heterocyclyl” denotes an unsaturated or fully or partly saturated cyclic radical containing from 3 to 8 atoms selected from carbon, nitrogen, sulphur and oxygen nonlimiting examples including pyridyl, pyridinyl, quinolyl, furyl, thienyl, pyrrolyl and oxazolinyl; the term “optionally substituted” signifies that the radicals thus qualified may be substituted by one or more radicals selected from chlorine, bromine, fluorine, iodine, alkyl, alkoxy, hydroxyl, nitro, amino; cyano and acyl.\",\n \"The compounds of formula (I) are described for example in patent application EP-A-1 056 723 and preference among them will be given to the compounds possessing one of the following characteristics, taken individually or in combination: R1 and R2, which are identical or different, are selected independently from a hydrogen atom and an optionally substituted alkyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a hydroxyl radical, a nitro radical, an optionally substituted amino radical, an acyl radical, and a group E, OE or SE in which E is selected from an alkyl, cycloalkyl, phenyl and heterocyclyl radical, each of which can be optionally substituted; c represents 0, 1, 2 or 3; q represents 0, 1, 2 or 3; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.\",\n \"Among the compounds of formula (I) further preference will be given to the compounds possessing the following characteristics, taken individually or in combination: R1 and R2, which are identical or different, are selected independently from a hydrogen atom and a methyl or ethyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a nitro radical, an optionally substituted amino radical and an alkyl, cycloalkyl, phenyl or heterocyclyl radical, each of which can be optionally substituted; c represents 1 or 2; q represents 1 or 2; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.\",\n \"More particularly, the compounds of formula (I) possess the following characteristics: R1 and R2 each represent a hydrogen atom; R3 and R4, which are identical or different, are selected independently from a halogen atom, a nitro radical, an alkyl radical and a trifluoromethyl radical; c and q represent, independently of one another, 2; and their possible tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.\",\n \"By way of example, the following compounds of formula (I) are very particularly preferred within the context of the present invention: Compound (Ia): 2,6-dichloro-N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}benzamide; Compound (Ib): N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}-2-fluoro-6-nitrobenzamide; Compound (Ic): N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}-2-methyl-6-nitrobenzamide; and their possible tautomers and addition salts with an acid or a base that are acceptable in the agricultural field.\",\n \"The compound (II) defined above is probamocarb, namely propyl 3-(dimethylamino)propylcarbamate, which is described for example in “The Pesticide Manual”, 11th edition, C D S Tomlin, British Crop Protection Council, pages 1015-1017, No.\",\n \"599.Advantageously, the propamocarb used in the compositions of the present invention will be in the form of the hydrochloride, i.e.\",\n \"the addition salt of propamocarb with hydrochloric acid, known commercially under the name Previcur®.\",\n \"The propamocarb which is useful for the compositions of the present invention can also be used in the form of the salt with fosetyl, as described in patent application EP-A-1 056 755 or else in patent application WO-A-98/44801.Advantageously, the compositions of the present invention comprise compound (Ia) or compound (Ib) or compound (Ic) with compound (II).\",\n \"The preferred compositions of the present invention comprise compound (Ia) with compound (II).\",\n \"The present invention accordingly provides fungicidal compositions comprising at least one pyridylmethylbenzamide derivative of formula (I) as defined above and at least one compound (II) as defined above, the compound (I)/compound (II) ratio being between 1/500 and 1/1, preferably between 1/200 and 1/5, more preferably between 1/150 and 1/10.The compound (I)/compound (II) ratio is defined as being the ratio by weight of these two compounds.\",\n \"The same is true of any ratio of two chemical compounds mentioned subsequently in the present text, unless a different definition of this ratio is expressly indicated.\",\n \"It will be appreciated that the said fungicidal compositions may comprise a single compound (I) or more than one such compound and/or a single compound (II) or more than one such compound, and also one or more other fungicidal, herbicidal, insecticidal and/or plant growth regulator compounds, depending on the use for which the said compositions are intended.\",\n \"Accordingly, the fungicidal compositions of the present invention may further comprise, for example, one or more other active fungicidal substances selected from acibenzolar-S-methyl, azoxystrobin, benalaxyl, benomyl, blasticidin-S, bromuconazole, captafol, captan, carbendazim, carboxin, carpropamid, chlorothalonil, fungicidal compositions based on copper, copper derivatives such as copper hydroxide and copper oxychloride, cyazofamid, cymoxanil, cyproconazole, cyprodinyl, dichloran, diclocymet, diethofencarb, difenoconazole, diflumetorim, dimethomorph, diniconazole, discostrobin, dodemorph, dodine, edifenphos, epoxyconazole, ethaboxam, ethirimol, famoxadone, fenamidone, fenarimol, fenbuconazole, fenhexamide, fenpiclonil, fenpropidine, fenpropimorph, ferimzone, fluazinam, fludioxonil, flumetover, fluquinconazole, flusilazole, flusulfamide, flutolanil, flutriafol, folpel, furalaxyl, furametpyr, guazatine, hexaconazole, hymexazol, imizalil, iprobenphos, iprodione, isoprothiolane, kasugamycin, kresoxim-methyl, mancozeb, maneb, mefenoxam, mepanipyrim, metalaxyl and its enantiomeric forms such as metalaxyl-M, metconazole, metiram-zinc, metominostrobin, oxadixyl, pefurazoate, penconazole, pencycuron, phosphorous acid and its derivatives such as fosetyl-Al, phthalide, picoxystrobin, probenazole, prochloraz, procymidone, propiconazole, pyraclostrobin, pyrimethanil, pyroquilon, quinoxyfen, silthiofam, simeconazole, spiroxamine, tebuconazole, tetraconazole, thiabendazole, thifluzamide, thiophanate, for example thiophanate-methyl, thiram, triadimefon, triadimenol, tricyclazole, tridemorph, trifloxystrobin, triticonazole, valinamide derivatives such as, for example, iprovalicarb, vinclozolin, zineb and zoxamide.\",\n \"The invention further provides a method of curatively or preventively combating phytopathogenic fungi in crops, characterized in that an (agronomically) effective and nonphytotoxic amount of a fungicidal composition of the invention is applied to the soil in which the plants are growing or are liable to grow, to the leaves and/or the fruits of the plants or to the seeds of the plants.\",\n \"The compositions of the invention are advantageous for combating fungal diseases in numerous crops such as, for example, cereals, vegetables, solanums, market-garden crops, grapevines, fruits in general, and especially for combating downy mildew, damping off, septoria disease and Pythium sp.\",\n \"in these crops.\",\n \"The compositions of the invention may also be used for combating other phytopathogenic diseases in crops that are well known to the person skilled in the art to whom the compounds of formula (I) and the compounds (II) are available.\",\n \"These compounds generally improve markedly the respective isolated actions of compound (I) and of compound (II) for a certain number of fungi which are particularly harmful in crops, particularly for solanums (tomatoes, potatoes, etc.\",\n \"), more particularly for downy mildew in potatoes and downy mildew in tomatoes, while preserving an absence of phytotoxicity with regard to these crops.\",\n \"There is therefore an improvement in the spectrum of activity and a possibility of reducing the respective dose of each active substance used, the latter quality being particularly important for ecological reasons which are easy to comprehend.\",\n \"In the compositions of the invention, the compound (I)/compound (II) ratio is advantageously selected so as to produce a synergistic effect.\",\n \"The term “synergistic effect” refers in particular to the effect defined by S. R. Colby in an article titled “Calculating synergistic and antagonistic responses of herbicide combinations”, Weeds, (1967), 15, pages 20-22.The latter article makes use of the formula: E = X + Y - X Y 100 in which E represents the expected percentage inhibition of the disease for the combination of the two fungicides at defined doses (for example equal respectively to x and y), X is the observed percentage inhibition of the disease by compound (I) at a defined dose (equal to x), Y is the observed percentage inhibition of the disease by compound (II) at a defined dose (equal to y).\",\n \"When the observed percentage inhibition of the combination is greater than E, there is a synergistic effect.\",\n \"A synergistic effect is also understood to be that defined by applying the method of Tammes, “Isoboles, a graphic representation of synergism in pesticides”, Netherlands Journal of Plant Pathology, 70, (1964), pages 73-80.The abovementioned ranges for the compound (I)/compound (II) ratio are by no means limitative of the scope of the invention but rather are cited by way of indication, it being well within the abilities of the person skilled in the art to carry out complementary tests in order to find other values for the dosage ratio of these two compounds at which a synergistic effect is observed.\",\n \"Accordingly, the compositions of the invention comprising compound (I) and compound (II) allow quite remarkable synergistic properties to be observed.\",\n \"According to one variant of the compositions of the invention, the compound (I)/compound (II) ratio is advantageously between 1/200 and 1/5, preferably between 1/140 and 1/10.The invention therefore also comprises methods of treating plants to counter phytopathogenic diseases, characterized in that a composition comprising at least one compound of formula (I) with at least one compound (II) is applied.\",\n \"It is also possible to apply a composition comprising both active substances or else two compositions each comprising one of the two active substances, either simultaneously or in succession so as to have the conjoint effect.\",\n \"These compositions embrace not only compositions which are ready to be applied to the crop to be treated by means of an appropriate device, such as a sprayer, but also commercial concentrated compositions which have to be diluted before application to the crop.\",\n \"The present invention provides a method of combating a wide variety of phytopathogenic diseases in crops, particularly for combating septoria disease and downy mildew.\",\n \"These diseases can be combated by direct foliar application.\",\n \"The present invention therefore provides a method of curatively or preventively combating phytopathogenic diseases in crops which comprises treating the said crop (for example by application or by administration) with an effective, nonphytotoxic amount of a combination as defined above.\",\n \"By treatment of the crop is meant application or administration of a fungicidal composition as described above to the aerial parts of the crops or to the soil in which they are growing, which are infested or liable to be infested by a phytopathogenic disease, such as downy mildew or septoria disease, for example.\",\n \"By treatment of the crop is also meant the treatment of reproduction products of the crop, such as seeds or tubers for example.\",\n \"The compositions described below are generally used for application to growing plants or to loci on which crops are grown, or for the dressing or coating of seeds.\",\n \"Among the means which are suitable for applying the compositions of the invention mention may be made of the use of powders, foliar sprays, granules, mists or foams, or else means in the form of suspensions of finely divided or encapsulated compositions; for the treatment of soils or roots with liquid imbibitions, powders, granules, fumes or foams; for application to the seeds of plants, the use, as seed dressing or seed coating agents, of powders or of liquid slurries.\",\n \"The compositions of the invention are, appropriately, applied to the vegetation and in particular to the leaves infested with the phytopathogenic fungi.\",\n \"Another method of applying the compounds or compositions of the invention is to add a formulation comprising the active substances to the irrigation water.\",\n \"This irrigation may be an irrigation using sprinklers.\",\n \"The formulations which are suitable for applying the compositions of the invention comprise formulations suitable for use in the form, for example, of sprays, powders, granules, mists, foams, emulsions or others.\",\n \"In practice, for combating phytopathogenic diseases in crops, one method, for example, consists in applying an effective amount of a composition of the invention to the plants or to the medium in which they are growing.\",\n \"For a method of this kind, the active substances are generally applied to the locus itself in which the infestation is to be controlled, the said application taking place at an effective dose of between approximately 1 g and approximately 1 000 g of active substances in total per hectare of locus treated.\",\n \"Under ideal conditions, depending on the nature of the phytopathogenic fungus to be treated, a lower dose may provide adequate protection.\",\n \"Conversely, under poor climatic conditions, resistance or other factors may require higher doses of active substance.\",\n \"The effective working doses of the combinations employed in the invention may vary within wide proportions, according in particular to the nature of the phytopathogenic fungi to be eliminated or the degree of infestation, for example, of the plants by these fungi.\",\n \"The optimum dose commonly depends on a number of factors, for example on the type of phytopathogenic fungus to be treated, on the type or level of development of the infested plant, on the density of vegetation, or else on the method of application.\",\n \"More preferably, an effective dose of active substances (I) and (II) is between approximately 5 g/ha and approximately 700 g/ha.\",\n \"For their use in practice, the compositions of the invention can be used alone and may also advantageously be used in formulations comprising one or other of the active substances or else both together, in association or combination with one or more other, compatible components which are, for example, solid or liquid fillers or diluents, adjuvants, surfactants, or equivalents which are appropriate for the desired use and acceptable for agricultural uses.\",\n \"The formulations may be of any type which is known in the sector and suitable for application in all types of plantings or crops.\",\n \"These formulations, which can be prepared in any manner known in this sector, also form part of the invention.\",\n \"The formulations may also comprise other types of ingredients such as protective colloids, adhesives, thickeners, thixotropic agents, penetrants, oils for spraying, stabilizers, preservatives (especially antimould agents), sequestrants, or others, and other active ingredients which are known to possess pesticidal properties (especially fungicidal, insecticidal, acaricidal or nematicidal properties) or to possess plant growth regulator properties.\",\n \"More generally, the compounds used in the invention may be combined with any solid or liquid additives which correspond to the normal formulation techniques.\",\n \"In general, the formulations of the invention commonly contain from approximately 0.05% to approximately 99% (by weight) of one or more compositions of the invention, from approximately 1% to approximately 95% of one or more solid or liquid fillers, and optionally from approximately 0.1% to approximately 50% of one or more other, compatible compounds, such as surfactants or others.\",\n \"In the present specification, the term “filler” signifies an organic or inorganic, natural or synthetic component with which the active components are combined in order to facilitate their application to, for example, the plants, seeds, or soil.\",\n \"Consequently, this filler is generally inert and it must be acceptable (for example, acceptable for agronomic use, especially for treating plants).\",\n \"The filler can be solid, examples being clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers (for example ammonium salts), natural soil minerals, such as kaolins, clays, talc, lime, quartz, attapulgite, montmorillonite, bentonite or diatomaceous earths, or synthetic minerals, such as silica, alumina, or silicates, especially aluminium silicates or magnesium silicates.\",\n \"Solid fillers which are suitable for granules are as follows: natural rocks, crushed or broken, such as calcite, marble, pumice, sepiolite and dolomite; synthetic granules of organic or inorganic flours; granules of organic material such as sawdust, coconut shell, corn ear or corn husk or tobacco stem; kieselguhr, tricalcium phosphate, powdered cork, or adsorbent carbon black; water-soluble polymers, resins, waxes; or solid fertilizers.\",\n \"If desired, such compositions may comprise one or more compatible agents such as wetting agents, dispersants, emulsifiers or colorants which, when solid, may also serve as diluents.\",\n \"The fillers may also be liquid, examples including the following: water, alcohols, especially butanol or glycol, and their ethers or esters, especially methyl glycol acetate; ketones, especially acetone, cyclohexanone, methyl ethyl ketone, methyl isobutyl ketone or isophorone; petroleum fractions such as paraffinic or aromatic hydrocarbons, especially xylenes or alkylnaphthalenes; mineral oils or plant oils; chlorinated aliphatic hydrocarbons, especially trichloroethane or methylene chloride; chlorinated aromatic hydrocarbons, especially chlorobenzenes; water-soluble or highly polar solvents such as dimethylformamide, dimethyl sulphoxide, N,N-dimethylacetamide and N-methylpyrrolidone; N-octylpyrrolidone, liquefied gases; or others, either separately or in a mixture.\",\n \"The surfactant may be an emulsifier, a dispersant or a wetting agent, of ionic or nonionic type, or a mixture of these surfactants.\",\n \"Among them use is made, for example, of salts of polyacrylic acids, salts of lignosulphonic acids, salts of phenolsulphonic or naphthalenesulphonic acids, polycondensates of ethylene oxide with fatty alcohols or fatty acids or fatty esters or fatty amines, substituted phenols (especially alkylphenols or arylphenols), ester salts of sulphosuccinic acid, taurine derivatives (especially alkyl taurates), phosphoric esters of alcohols or of polycondensates of ethylene oxide with phenols, esters of fatty acids with polyols, or sulphate, sulphonate or phosphate functional derivatives of the above-described compounds.\",\n \"The presence of at least one surfactant is generally essential when the active substances and/or the inert filler are only sparingly soluble or insoluble in water and when the filler of the said composition to be applied is water.\",\n \"The formulations of the invention may further comprise other additives such as adhesives or colorants.\",\n \"Adhesives such as carboxymethylcellulose, or synthetic or natural polymers in the form of powders, granules or matrices, such as gum arabic, latex, polyvinylpyrrolidone, polyvinyl alcohol or polyvinyl acetate, natural phospholipids, such as cephalins or lecithins, or synthetic phospholipids can be used in the formulations.\",\n \"It is possible to use colorants such as inorganic pigments, such as the following, for example: iron oxides, titanium oxides, or Prussian blue; organic dyes, such as those of the alizarin, azo or metal phthalocyanine type; or trace elements such as salts of iron, manganese, boron, copper, cobalt, molybdenum or zinc.\",\n \"The formulations comprising the compositions of the invention that are employed for combating phytopathogenic fungi in crops may also comprise stabilizers, other fungicidal agents, insecticides, acaricides, nematicides, anthelmintics or anticoccidials, bactericides, attractants or repellents or pheromones for arthropods or vertebrates, deodorizers, flavourings or colorants.\",\n \"These substances may be selected purposely for improving the intensity, persistence, safety or spectrum of action on the phytopathogenic fungi in the crops or for rendering the composition capable of accomplishing other useful functions for the treated surfaces.\",\n \"For their use in agriculture, the compositions of the invention are accordingly formulated in various liquid or solid forms.\",\n \"As solid formulations, mention may be made of powders for dusting (with an active substance content which can be up to 100%) and granules, especially those obtained by extrusion, spraying, compacting, impregnation of a granulated support, or granulation from a powder (the active substance content of these granules being between 0.5 and 80% in the latter cases).\",\n \"The fungicidal compositions of the invention may also be used in the form of powders for dusting; it is also possible to use formulations comprising 50 g of active substances and 950 g of talc; it is also possible to use formulations containing 20 g of active substances, 10 g of finely divided silica and 970 g of talc; these constituents are mixed and ground and the mixture is applied by dusting.\",\n \"As formulations which are liquid or which are intended to constitute liquid compositions at the time of application, mention may be made of solutions, especially water-soluble concentrates, emulsifiable concentrates, emulsions, concentrated suspensions and wettable powders (or sprayable powder).\",\n \"The concentrated suspensions which can be applied by spraying are prepared so as to obtain a stable fluid product which does not settle and which leads to effective bioavailability of the active substances.\",\n \"These suspensions commonly contain from 5% to 75% of active substances, preferably from 10% to 25%, from 0.5 to 75% of surfactants, preferably from 5% to 50%, from 0 to 10% of appropriate additives, such as thickeners which are mineral or organic in origin, antifoams, corrosion inhibitors, adhesives, preservatives, such as Proxel GXL® for example, antifreezes and, as vehicle, water or an organic liquid in which the active substances are sparingly soluble or insoluble: certain organic solids or mineral salts may be dissolved in the vehicle in order to help prevent sedimentation or as antifreezes for the water.\",\n \"In certain cases, and especially for formulations intended for treating seeds, one or more colorants may be added.\",\n \"For foliar application, the choice of surfactants is essential in order to ensure effective bioavailability of the active substances; accordingly, use will be made preferably of a combination of a hydrophilic-type surfactant (HLB>10) and of a lipophilic-type surfactant (HLB<5).\",\n \"Such combinations of surfactants are described for example in the as-yet unpublished French patent application No.\",\n \"00 04015.By way of example, here are 3 possible formulations of the suspension concentrate type, adapted for different crops: EXAMPLE SC 1 (in g/kg) This example is more suitable for monocotyledonous crops (cereals, rice, etc.)\",\n \"active substances 150 hydrophilic-type surfactant (for example 300 Rhodasurf 860P) lipophilic-type surfactant (for example 150 Plurafac LF 700) ethoxylated tristyrylphenol phosphate 50 antifoam 5 propylene glycol 30 Aerosil 200 20 Attagel 50 40 water (qs 1 kg) 255 EXAMPLE SC2 (in g/kg) This example is more suitable for dicotyledonous crops (grapevines, fruit trees, etc.)\",\n \"active substances 150 hydrophilic-type surfactant (for example 150 Rhodasurf 860P) ethoxylated tristyrylphenol phosphate 50 antifoam 5 propylene glycol 30 Aerosil 200 20 Attagel 50 40 water (qs 1 kg) 555 EXAMPLE SC3 (in g/kg) This example is more specifically adapted to the treatment of seeds.\",\n \"active substances 50 hydrophilic-type surfactant (for example 5 Rhodasurf 860P) ethoxylated tristyrylphenol phosphate 15 antifoam 1 propylene glycol 30 colorant 20 Rhodopol G 1.5 Proxel GXL 1.5 water (qs 1 kg) 876 These formulations will preferably be produced using the following technique: The selected surfactants (hydrophilic-type surfactant+lipophilic-type surfactant+ethoxylated tristyrylphenol phosphate) are mixed into the required amount of water using a turbine mixer; following homogenization, the other constituents of the formula apart from the active substances are then mixed in.\",\n \"Then the active substances are added and, optionally, the thickener of mineral origin (Aerosil 200 and Attagel 50) in order to obtain a mixture with a viscous consistency.\",\n \"The resulting mixture is then ground using a high-speed turbo mixer mill and then a ball mill so as to give a D50 of the order of from 1 to 3 μm and a D90 of between 3 and 8 μm.\",\n \"Where no thickener of mineral origin is used, the thickener of natural origin (Rhodopol G) is added next and the mixture is stirred until an appropriate viscosity is obtained.\",\n \"The wettable (or sprayable) powders are commonly prepared such that they contain from 20% to 95% of active substances, and they commonly contain, in addition to the solid vehicle, from 0% to 30% of a wetting agent, from 3% to 20% of a dispersant, and, when necessary, from 0.1% to 10% of one or more stabilizers and/or other additives, such as penetrants, adhesives, or anticaking agents, colorants, etc.\",\n \"In order to obtain the sprayable or wettable powders, the active substances are intimately mixed in appropriate mixers with the additional substances and are ground using mills or other appropriate grinders.\",\n \"Sprayable powders are obtained whose wettability and suspension characteristics are advantageous; they can be suspended with water at any desired concentration, and these suspensions can be used very advantageously in particular for application, for example, to the leaves of plants or to seeds.\",\n \"By way of example, here are various compositions of wettable powders (or sprayable powders): EXAMPLE WP 1 active substances 50% ethoxylated fatty alcohol (wetting 2.5% agent) ethoxylated phenylethylphenol 5% (dispersant) chalk (inert vehicle) 42.5% EXAMPLE WP 2 active substances 10% synthetic C13 oxo alcohol of 0.75% branched type, ethoxylated with 8 to 10 ethylene oxide units (wetting agent) neutral calcium lignosulphonate 12% (dispersant) calcium carbonate (inert filler) qs 100% EXAMPLE WP 3 This wettable powder contains the same ingredients as in the preceding example, in the following proportions: active substances 75% wetting agent 1.50% dispersant 8% calcium carbonate (inert filler) qs 100% EXAMPLE WP 4 active substances 90% ethoxylated fatty alcohol (wetting 4% agent) ethoxylated phenylethylphenol 6% (dispersant) EXAMPLE WP 5 active substances 50% mixture of anionic and nonionic 2.5% surfactants (wetting agent) sodium lignosulphonate (dispersant) 5% kaolinic clay (inert vehicle) 42.5% The aqueous emulsions and dispersions, for example the compositions obtained by diluting a wettable powder of the invention with water, are included within the general scope of the present invention.\",\n \"The emulsions may be of the water-in-oil or oil-in-water type and they may have a thick consistency like that of a mayonnaise sauce.\",\n \"The fungicidal compositions of the invention may be formulated in the form of water-dispersible granules which are likewise included within the scope of the invention.\",\n \"These dispersible granules, with an apparent density which is generally between approximately 0.3 and 0.6, have a particle size which is generally between approximately 150 and 2 000 and preferably between 300 and 1 500 microns.\",\n \"The active substance content of these granules is generally between approximately 1% and 90%, and preferably between 25% and 90%.\",\n \"The remainder of the granules is composed essentially of a solid filler and, optionally, of surfactant adjuvants which impart water-dispersibility properties to the granules.\",\n \"These granules may be essentially of two distinct types depending on whether the filler used is soluble or insoluble in water.\",\n \"When the filler is water-soluble, it may be mineral or, preferably, organic.\",\n \"Excellent results have been obtained with urea.\",\n \"In the case where the filler is insoluble it is preferably mineral, such as for example kaolin or bentonite.\",\n \"In that case it is advantageously accompanied by surfactants (in a proportion of from 2 to 20% by weight of the granules) of which more than half is composed, for example, of at least one, essentially anionic dispersant, such as an alkali metal or alkaline earth metal polynaphthalenesulphonate or an alkali metal or alkaline earth metal lignosulphonate, the remainder consisting of nonionic or anionic wetting agents such as an alkali or alkaline earth metal alkylnaphthalenesulphonate.\",\n \"Furthermore, although not indispensable, it is possible to add other adjuvants such as antifoams.\",\n \"The granules may be prepared by mixing the required ingredients then granulating the mixture by a number of techniques which are known per se (granulator, fluid bed, atomizer, extrusion, etc.).\",\n \"The final procedure is normally a crushing operation followed by screening to the particle size selected within the abovementioned limits.\",\n \"It is also possible to use granules obtained as before then impregnated with a composition comprising the active substances.\",\n \"The granules are preferably obtained by extrusion, operating as indicated in the examples below.\",\n \"EXAMPLE DG1 Dispersible Granules 90% by weight of active substances and 10% of urea pellets are mixed in a mixer.\",\n \"The mixture is subsequently ground in a toothed roll crusher.\",\n \"A powder is obtained which is wetted with approximately 8% by weight of water.\",\n \"The moist powder is extruded in a perforated-roll extruder.\",\n \"This gives granules which are dried and then crushed and screened, so as to retain only those granules with a size of between 150 and 2 000 microns.\",\n \"EXAMPLE DG2 Dispersible Granules The following constituents are mixed in a mixer: active substances 75% wetting agent (sodium alkylnaphthalene 2% sulphonate) dispersant (sodium polynaphthalenesulphonate) 8% water-insoluble inert filler (kaolin) 15% This mixture is granulated in a fluid bed in the presence of water and then dried, crushed and screened to give granules with a size of between 0.15 and 0.80 mm.\",\n \"These granules may be used alone or in solution or dispersion in water in such a way as to give the desired dose.\",\n \"They may also be used for preparing compositions with other active substances, especially fungicides, the latter being in the form of wettable powders or granules or aqueous suspensions.\",\n \"The fungicidal compositions of the invention commonly contain from 0.5 to 95% of the association of compound (I) and compound (II).\",\n \"The composition in question may be a concentrated composition, i.e.\",\n \"the commercial product combining compound (I) and compound (II).\",\n \"It may also be a dilute composition which is ready to be applied to the crops to be treated.\",\n \"In the latter case, dilution with water can be carried out either starting from a concentrated commercial composition containing compound (I) and compound (II) (this mixture is called a ready mix) or by means of mixing at the time of use (in this case, the mixture is referred to as a tank mix) two concentrated commercial compositions each containing compound (I) and compound (II).\",\n \"The invention lastly provides a method of curatively or preventively combating phytopathogenic fungi in crops, characterized in that an effective, nonphytotoxic amount of a fungicidal composition of the invention is applied to the plants to be treated.\",\n \"The phytopathogenic fungi in crops that can be combated by this method are, in particular those: from the group of the oomycetes: of the genus Phytophthora such as Phytophthora infestans (solanum late blight, particularly potato or tomato blight), of the family of the Peronosporaceae, especially Plasmopara viticola (grapevine downy mildew), Plasmopara halstedii (sunflower downy mildew), Pseudoperonospora sp.\",\n \"(in particular, cucurbit downy mildew and hop downy mildew), Bremia lactucae (lettuce downy mildew), Peronospora tabacinae (tobacco downy mildew), Peronospora parasitica (cabbage downy mildew), Peronospora viciae (pea downy mildew), and Peronospora destructor (onion downy mildew); from the group of the adelomycetes: of the genus Alternaria, for example Alternaria solani (solanium early blight, and especially early blight in tomatoes and potatoes), of the genus Guignardia, especially Guignardia bidwelli (grapevine black rot), of the genus Oidium, for example grapevine powdery mildew (Uncinula necator), powdery mildew of leguminous crops, for example Erysiphe polygoni (crucifer powdery mildew), Leveillula taurica, Erysiphe cichoracearum, Sphaerotheca fuligena (powdery mildew in cucurbits, composites and tomatoes), Erysiphe communis (powdery mildew in beet and cabbage), Erysiphe pisi (powder mildew in peas and alfalfa), Erysiphe polyphaga (powdery mildew in beans and cucumber), Erysiphe umbelliferarum (powdery mildew in umbellifers, especially carrots), Sphaerotheca humuli (hop powdery mildew); from the group of soil fungi: of the genus Pythium sp., of the genus Aphanomyces sp., especially Aphanomyces euteiches (white root rot in peas), Aphanomyces cochlioides (root rot in beet).\",\n \"The term “is/are applied to the plants to be treated” is intended to signify, in the sense of the present text, that the fungicidal compositions that are subjects of the invention may be applied by means of various treatment techniques such as: the spraying of a liquid comprising one of the said compositions onto the aerial parts of the said plants, dusting, incorporation of granules or powders into the soil, irrigation around the said plants, and, in the case of trees, injection or painting on, the dressing or coating of seeds of the said plants by means of a slurry comprising one of the said compositions.\",\n \"The spraying of a liquid onto the aerial parts of the crops to be treated is the preferred treatment method.\",\n \"The examples which follow are given purely by way of illustration of the invention, and do not impose any limitation whatsoever thereon.\",\n \"Although the invention has been described in terms of numerous preferred variants, the person skilled in the art will appreciate that many modifications, substitutions, omissions and changes can be made without departing from the spirit of this invention.\",\n \"It is thus clearly understood that the scope of the present invention is limited solely by the scope of the claims which follow, and by their equivalents.\",\n \"EXAMPLE Test of a Composition Against Late Blight in Potatoes (Phytophthora infestans: Preventive Action) The following active substances are used for this test: compound (Ia) in the form of a suspension concentrate (10 SC) compound (II): propamocarb hydrochloride (Previcur-N®, in the form of a soluble concentrate (66.5 SL).\",\n \"Potato plants (three weeks old) are treated by automatic spraying (Mardrive) at a rate of 200 l/ha.\",\n \"Six identical tests are carried out.\",\n \"Compounds (Ia) and (II) are mixed (tank mix) in different proportions which in the results table are expressed in g/ha.\",\n \"All of the plants are inoculated 24 hours after treatment with a suspension of sporangia of Phytophthora infestans, containing 20 000 spores per ml.\",\n \"The inoculated plants are covered for 2 days and then placed in a controlled atmosphere at 20° C. until the results are read off.\",\n \"The degree of infestation is evaluated by rating on a scale ranging from 1 to 10, the value 0 denoting the absence of disease and the value 100 denoting complete infestation (100%).\",\n \"For each treatment, the percentage disease control is calculated relative to untreated plants, and the theoretical results are calculated using the Colby formula described above.\",\n \"The difference between the experimental results and the calculated results allows the degree of synergism between the two compounds to be determined.\",\n \"The results are shown in the table below: Test on Compound (Ia) and Compound (II) Concentration Synergism (g/ha) Rating Efficacy % (Colby) Compound 5 4.3 48 — (Ia) 25 3.3 60 — Compound 400 8.3 0 — (II) 600 6.8 18 — (Ia) + (II) 1/80 5 + 400 2.8 66 18 (Ia) + (II) 1/120 5 + 600 2.2 74 17 (Ia) + (II) 1/16 25 + 400 1.5 82 22 (Ia) + (II) 1/24 25 + 600 1.0 88 21 Untreated control: rating 8.3 The compositions of the invention comprising compound (Ia) and compound (II) exhibit a high level of synergism irrespective of the doses of each of the active substances and irrespective of the proportions of compound (Ia) to compound (II).\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471125"}}},{"rowIdx":576,"cells":{"text":{"kind":"list like","value":[["Automated immunohistochemical and in situ hybridization assay formulations","The present invention provides reagents for use in an automated environment for cell conditioning of biological samples wherein the cells or tissues are predisposed for access by reagent molecules for histochemical and cytochemical staining procedures.","The reagents comprise components optimized to faciliate molecular access to cells and cell constituents within the biological sample.","The present invention also provides reagents for use in an automated environment for removing or etching embedding media by exposing a biological sample to be stained in histochemical or cytochemical procedures without the dependence on organic solvents.","The reagents comprise components optimized to facilitate removal or etching of the embedding media from the biological sample."],["1.An aqueous composition of matter, comprising citrate buffer, ethylene glycol, sodium metabisulfite and a surfactant.","2.A composition according to claim 1, wherein the composition is buffered from approximately pH 4 to approximately pH 9.3.A composition according to claim 1, wherein composition is buffered at approximately pH 6.4.A composition according to claim 1, wherein the composition comprises less than or equal to approximately 100 mM citrate buffer.","5.A composition according to claim 1, wherein the composition comprises approximately 10 mM citrate buffer.","6.A composition according to claim 1, wherein the composition comprises less than or equal to approximately 10% ethylene glycol.","7.A composition according to claim 1, wherein the composition comprises approximately 5% ethylene glycol.","8.A composition according to claim 1, wherein the composition comprises less than or equal to approximately 10 mM sodium metabisulfite.","9.A composition according to claim 1, wherein the composition comprises 1 mM sodium metabisulfite.","10.A composition according to claim 1, wherein the composition comprises less than or equal to approximately 10% SDS.","11.A composition according to claim 1, wherein the composition comprises approximately 0.3% SDS.","12.A composition according to claim 1, wherein the composition comprises approximately 10 mM citrate buffer at approximately pH 6, approximately 5% ethylene glycol, approximately 1 mM sodium metabisulfite and approximately 0.3% SDS.","13.An aqueous composition of matter comprising a buffer, a glycol, sodium metabisulfite and a surfactant.","14.A composition according to claim 13, wherein the buffer is a citrate buffer.","15.A composition according to claim 14, wherein the buffer is at approximately pH 6.16.A composition according to claim 13, wherein the glycol is ethylene glycol.","17.A composition according to claim 13, wherein the concentration of the buffer is less than or equal to approximately 100 mM, the concentration of the glycol is less than or equal to approximately 10%, the concentration of the sodium metabisulfite is less than or equal to approximately 10 mM and the concentration of the SDS is less than or equal to approximately 10%.","18.A composition according to claim 17, wherein the buffer is citrate at a concentration of approximately 10 mM and the pH of the composition is approximately 6.19.A composition according to claim 18, wherein the glycol is ethylene glycol and the concentration of ethylene glycol is approximately 5%, wherein the concentration of sodium metabisulfite is approximately 1 mM and wherein the concentration of SDS is approximately 0.3%.","20.An aqueous composition of matter comprising approximately 10 mM citrate buffer at approximately pH 6, approximately 5% ethylene glycol, approximately 1 mM sodium metabisulfite and approximately 0.3% surfactant.","21.An aqueous composition of matter according to claim 20, wherein the surfactant is SDS."],["<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to solutions methods, and instruments for conditioning cells or tissues so as to increase the accessibility of various molecules to their respective targets and generally to improve tissue and cell readability of biological samples on automated instruments prior to immunohistochemical (IHC), in situ hybridization (ISH) or other histochemical or cytochemical manipulations.","2.Summary of the Related Art All patents, patent applications and non-patent articles or references mentioned herein are hereby incorporated by reference to the extent that they are not contradictory.","The diagnosis of disease based on the interpretation of tissue or cell samples taken from a diseased organism has expanded dramatically over the past few years.","In addition to traditional histological staining techniques and immunohistochemical assays, ill situ techniques such as in situ hybridization and in situ polymerase chain reaction are now used to help diagnose disease states in humans.","Thus, there are a variety of techniques that can assess not only cell morphology, but also the presence of specific macromolecules within cells and tissues.","For example, the diagnosis of breast, ovarian and other carcinomas may be facilitated by the use of techniques designed to identify the presence or absence of the c-erb2/HER-2/neu protooncogene or the protein(s) expressed therefrom.","The c-erb2/HER-2/neu protooncogene is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases.","Amplification and overexpression of the c-erb2/HER-2/neu protooncogene is found in about 30% of breast carcinomas, about 20% of ovarian carcinomas and others.","(Andrechek, E. R. et al., Proc.","Natl.","Acad.","Sci.","USA , vol.","97, no.","7, pp.","3444-3449 (2000); Doherty, J. K. et al., Proc.","Natl.","Acad.","Sci.","USA , vol.","96, pp.","10869-10874 (1999); Oh, J. J. et al., Nucleic Acids Res ., vol.","27, no.","20, pp.","4008-4017 (1999); Klapper, L. N. et al., Proc.","Natl.","Acad.","Sci.","USA , vol.","96, pp.","4995-5000 (1999); and references found in each of the aforementioned articles.)","Each of these techniques requires that sample cells or tissues undergo preparatory procedures that may include fixing the sample with chemicals such as an aldehyde (such as formaldehyde, glutaraldehyde), formalin substitutes, alcohol (such as ethanol, methanol, isopropanol) or embedding the sample in inert materials such as paraffin, celloidin, agars, polymers, resins, cryogenic media or a variety of plastic embedding media (such as epoxy resins and acrylics).","Other sample tissue or cell preparations require physical manipulation such as freezing (frozen tissue section) or aspiration through a fine needle (fine needle aspiration (FNA)).","Regardless of the tissue or cell sample or its method of preparation or preservation, the goal of the technologist is to obtain accurate, readable and reproducible results that permit the accurate interpretation of the data.","One way to provide accurate, readable and reproducible data is to prepare the tissue or cells in a fashion that optimizes the results of the test regardless of the technique employed.","In the case of immunohistochemistry and in situ techniques this means increasing the amount of signal obtained from the specific probe (e.g.","antibody, DNA, RNA, etc.).","In the case of histochemical staining it may mean increasing the intensity of the stain or increasing staining contrast.","Without preservation, tissue samples rapidly deteriorate such that their use in diagnostics is compromised shortly after removal from their host.","In 1893, Ferdinand Blum discovered that formaldehyde could be used to preserve or fix tissue so that it could be used in histochemical procedures.","The exact mechanisms by which formaldehyde acts in fixing tissues are not fully established, but they involve cross-linking of reactive sites within the same protein and between different proteins via methylene bridges (Fox et al., J. Histochem.","Cytochem.","33: 845-853 (1985)).","Recent evidence suggests that calcium ions also play a role (Morgan et al., J.","Path.","174: 301-307 (1994)).","These links cause changes in the quaternary and tertiary structures of proteins, but the primary and secondary structures appear to be preserved (Mason et al., J. Histochem.","Cytochem.","39: 225-229 (1991)).","The extent to which the cross-linking reaction occurs depends on conditions such as the concentration of formalin, pH, temperature and length of fixation (Fox et al., J. Histochem.","Cytochem.","33: 845-853 (1985)).","Some antigens, such as gastrin, somatostatin and α-1-antitrypsin, may be detected after formalin fixation, but for many antigens, such as intermediate filaments and leukocyte markers, immunodetection after formalin treatment is lost or markedly reduced (McNicol & Richmond, Histopathology 32: 97-103 (1998)).","Loss of antigen immunoreactivity is most noticeable at antigen epitopes that are discontinuous, i.e.","amino acid sequences where the formation of the epitope depends on the confluence of portions of the protein sequence that are not contiguous.","Antigen retrieval refers to the attempt to “undo” the structural changes that treatment of tissue with a cross-linking agent induces in the antigens resident within that tissue.","Although there are several theories that attempt to describe the mechanism of antigen retrieval (e.g., loosening or breaking of crosslinkages formed by formalin fixation), it is clear that modification of protein structure by formalin is reversible under conditions such as high-temperature heating.","It is also clear that several factors affect antigen retrieval: heating, pH, molarity and metal ions in solution (Shi et al., J. Histochem.","Cytochem.","45: 327-343 (1997)).","Microwave heating appears to be the most important factor for retrieval of antigens masked by formalin fixation.","Microwave heating (100°±5° C.) generally yields better results in antigen retrieval immunohistochemistry (AR-IHC).","Different heating methods have been described for antigen retrieval in IHC such as autoclaving (Pons et al, Appl.","Immunohistochem.","3: 265-267 (1995); Bankfalvi et al., J.","Path.","174: 223-228 (1994)); pressure cooking (Miller & Estran, Appl.","Immunohistochem.","3: 190-193 (1995); Norton et al., J.","Path.","173: 371-379 (1994)); water bath (Kawai et al., Path.","Int.","44: 759-764 (1994)); microwaving plus plastic pressure cooking (U.S. Pat.","No.","5,244,787; Pertschuk et al., J.","Cell Biochem.","19(suppl.","): 134-137 (1994)); and steam heating (Pasha et al., Lab.","Invest.","72: 167A (1995); Taylor et al., CAP Today 9: 16-22 (1995)).","Although some antigens yield satisfactory results when microwave heating is performed in distilled water, many antigens require the use of buffers during the heating process.","Some antigens have particular pH requirements such that adequate results will only be achieved in a narrow pH range.","Presently, most antigen retrieval solutions are used at a pH of approximately 6-8, but there is some indication that slightly more basic solutions may provide marginally superior results (Shi, et al., J. Histochem.","Cytochem.","45: 327-343 (1997)).","Although the chemical components of the antigen retrieval solution, including metal ions, may play a role as possible co-factors in the microwave heating procedure, thus far, no single chemical has been identified that is both essential and best for antigen retrieval.","Many solutions and methods are used routinely for staining enhancements.","These may include but are not limited to distilled water, EDTA, urea, Tris, glycine, saline and citrate buffer.","Solutions containing a variety of detergents (ionic or non-ionic surfactants) may also facilitate staining enhancement under a wide range of temperatures (from ambient to in excess of 100° C.).","In addition to cell surface molecules that may be present on the exterior portion of the cell, other molecules of interest in IHC, ISH and other histochemical and cytochemical manipulations are located within the cell, often on the nuclear envelope.","Some of these molecules undergo molecular transformation when exposed to a fixative (coagulative or precipitive) such as formalin.","Thus with respect to these molecules it is desirable to not only overcome the effects of fixation but also to increase the permeability of the cell in order to facilitate the interaction of organic and inorganic compounds with the cell.","Other tissue samples may not have been subjected to cross-linking agents prior to testing, but improved results with respect to these tissues is also important.","There are a variety of non-formalin methods for preserving and preparing cytological and histological samples.","Examples of these methods include, but are not limited to: a) hematology smears, cytospins™, ThiinPreps™, touch preps, cell lines, Ficoll separations, etc.","are routinely preserved in many ways which includes but are not limited to, air-drying, alcoholic fixation, spray fixatives and storage mediums such as sucrose/glycerin; b) tissues and cells (either fixed or unfixed) may be frozen and subsequently subjected to various stabilizing techniques which include, but are not limited to, preservation, fixation and desiccation; c) tissues and cells may be stabilized in a number of non-cross-linking aldehyde fixatives, non-aldehyde containing fixatives, alcoholic fixatives, oxidizing agents, heavy metal fixatives, organic acids and transport media.","One way to improve testing results is to increase the signal obtained from a given sample.","In a general sense, increased signal can be obtained by increasing the accessibility of a given molecule for its target.","As in the case for antigens found within the cell, targets within the cell can be made more accessible by increasing the permeability of the cell thereby permitting a greater number of molecules entry into the cell, thereby increasing the probability that the molecule will “find” its target.","Such increased permeability is especially important for techniques such as ISH, in situ PCR, IHC, histochemistry and cytochemistry.","Tissues and cells are also embedded in a variety of inert media (paraffin, celloidin, OCT™, agar, plastics or acrylics etc.)","to help preserve them for future analysis.","Many of these inert materials are hydrophobic and the reagents used for histological and cytological applications are predominantly hydrophilic; therefore, the inert medium may need to be removed from the biological sample prior to testing.","For example, paraffin embedded tissues sections are prepared for subsequent testing by removal of the paraffin from the tissue section by passing the slide through various organic solvents such as toluene, xylene, limonene or other suitable solvents.","These organic solvents are very volatile causing a variety of problems including requiring special processing (e.g., deparaffinization is performed in ventilated hoods) and requires special waste disposal.","The use of these organic solvents increases the cost of analysis and exposure risk associated with each tissue sample tested and has serious negative effects for the environment.","Presently, there is no available technique for removing inert media from sample tissue by directly heating the slide in an automated fashion.","Neither is it currently possible to remove inert media from sample tissue while conditioning the sample tissue or cell in a one-step automated staining process.","The methods of the present invention permit a) automated removal of embedding media without the use of organic solvents, thus exposing the cells for staining and thereby reducing time, cost and safety hazards, b) automated cell conditioning without automated removal of embedding media from the sample cell or tissue, c) a multi-step automated process that exposes the cells, performs cell conditioning and increases permeability of the cytological or histological specimens, thereby increasing sample readability and improving interpretation of test data.","The methods of the present invention can be used for improving the stainability and readability of most histological and cytological samples used in conjunction with cytological and histological staining techniques."],["<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to solutions, methods and instruments for cell or tissue conditioning, which improves the accessibility of molecules in biological samples during histochemical or cytochemical assays.","The conditioning may be performed at any point in a histochemical or cytochemical protocol.","The present invention further relates to an automated method for exposing biological samples for use in histological or cytological testing procedures by removing the embedding media without the use of organic solvents.","The present invention also relates to an automated method for the simultaneous exposing and cell conditioning of biological samples for histochemical and cytochemical applications."],["REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of U.S. application Ser.","No.","09/800,689, filed Mar.","7, 2001, which is a continuation-in-part of U.S. application Ser.","No.","09/721,096, filed Nov. 22, 2000, with Atty.","Docket No.","98,541-C. U.S. application Ser.","No.","09/721,096 claimed the benefit of priority from International Application No.","PCT/US99/20353, filed Sep. 3, 1999, which was published on Mar.","16, 2000 as WO 00/14507.International Application No.","PCT/US99/20353, in turn, claimed the benefit of priority from U.S.","Provisional Patent Application No.","60/099,018, filed Sep. 3, 1998; U.S. patent application Ser.","No.","09/259,240, filed Feb. 26, 1999; and International Application No.","PCT/US99/04181, filed Feb. 26, 1999, which was published on Sep. 2, 1999 as WO 99/44030.U.S.","patent application Ser.","No.","09/721,096, filed Nov. 22, 2000; International Patent App.","No.","PCT/US99/20353, filed Sep. 3, 1999, and published as WO 00/14507 on Mar.","16, 2000; U.S.","Provisional App.","No.","60/099,018, filed Sep. 3, 1998; U.S. patent application Ser.","No.","09/259,240, filed Feb. 26, 1999 and International Patent App.","No.","PCT/US99/04181, filed Feb. 26, 1999, and published as WO 99/44030 on Sep. 2, 1999, are all hereby incorporated by reference.","BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to solutions methods, and instruments for conditioning cells or tissues so as to increase the accessibility of various molecules to their respective targets and generally to improve tissue and cell readability of biological samples on automated instruments prior to immunohistochemical (IHC), in situ hybridization (ISH) or other histochemical or cytochemical manipulations.","2.Summary of the Related Art All patents, patent applications and non-patent articles or references mentioned herein are hereby incorporated by reference to the extent that they are not contradictory.","The diagnosis of disease based on the interpretation of tissue or cell samples taken from a diseased organism has expanded dramatically over the past few years.","In addition to traditional histological staining techniques and immunohistochemical assays, ill situ techniques such as in situ hybridization and in situ polymerase chain reaction are now used to help diagnose disease states in humans.","Thus, there are a variety of techniques that can assess not only cell morphology, but also the presence of specific macromolecules within cells and tissues.","For example, the diagnosis of breast, ovarian and other carcinomas may be facilitated by the use of techniques designed to identify the presence or absence of the c-erb2/HER-2/neu protooncogene or the protein(s) expressed therefrom.","The c-erb2/HER-2/neu protooncogene is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases.","Amplification and overexpression of the c-erb2/HER-2/neu protooncogene is found in about 30% of breast carcinomas, about 20% of ovarian carcinomas and others.","(Andrechek, E. R. et al., Proc.","Natl.","Acad.","Sci.","USA, vol.","97, no.","7, pp.","3444-3449 (2000); Doherty, J. K. et al., Proc.","Natl.","Acad.","Sci.","USA, vol.","96, pp.","10869-10874 (1999); Oh, J. J. et al., Nucleic Acids Res., vol.","27, no.","20, pp.","4008-4017 (1999); Klapper, L. N. et al., Proc.","Natl.","Acad.","Sci.","USA, vol.","96, pp.","4995-5000 (1999); and references found in each of the aforementioned articles.)","Each of these techniques requires that sample cells or tissues undergo preparatory procedures that may include fixing the sample with chemicals such as an aldehyde (such as formaldehyde, glutaraldehyde), formalin substitutes, alcohol (such as ethanol, methanol, isopropanol) or embedding the sample in inert materials such as paraffin, celloidin, agars, polymers, resins, cryogenic media or a variety of plastic embedding media (such as epoxy resins and acrylics).","Other sample tissue or cell preparations require physical manipulation such as freezing (frozen tissue section) or aspiration through a fine needle (fine needle aspiration (FNA)).","Regardless of the tissue or cell sample or its method of preparation or preservation, the goal of the technologist is to obtain accurate, readable and reproducible results that permit the accurate interpretation of the data.","One way to provide accurate, readable and reproducible data is to prepare the tissue or cells in a fashion that optimizes the results of the test regardless of the technique employed.","In the case of immunohistochemistry and in situ techniques this means increasing the amount of signal obtained from the specific probe (e.g.","antibody, DNA, RNA, etc.).","In the case of histochemical staining it may mean increasing the intensity of the stain or increasing staining contrast.","Without preservation, tissue samples rapidly deteriorate such that their use in diagnostics is compromised shortly after removal from their host.","In 1893, Ferdinand Blum discovered that formaldehyde could be used to preserve or fix tissue so that it could be used in histochemical procedures.","The exact mechanisms by which formaldehyde acts in fixing tissues are not fully established, but they involve cross-linking of reactive sites within the same protein and between different proteins via methylene bridges (Fox et al., J. Histochem.","Cytochem.","33: 845-853 (1985)).","Recent evidence suggests that calcium ions also play a role (Morgan et al., J.","Path.","174: 301-307 (1994)).","These links cause changes in the quaternary and tertiary structures of proteins, but the primary and secondary structures appear to be preserved (Mason et al., J. Histochem.","Cytochem.","39: 225-229 (1991)).","The extent to which the cross-linking reaction occurs depends on conditions such as the concentration of formalin, pH, temperature and length of fixation (Fox et al., J. Histochem.","Cytochem.","33: 845-853 (1985)).","Some antigens, such as gastrin, somatostatin and α-1-antitrypsin, may be detected after formalin fixation, but for many antigens, such as intermediate filaments and leukocyte markers, immunodetection after formalin treatment is lost or markedly reduced (McNicol & Richmond, Histopathology 32: 97-103 (1998)).","Loss of antigen immunoreactivity is most noticeable at antigen epitopes that are discontinuous, i.e.","amino acid sequences where the formation of the epitope depends on the confluence of portions of the protein sequence that are not contiguous.","Antigen retrieval refers to the attempt to “undo” the structural changes that treatment of tissue with a cross-linking agent induces in the antigens resident within that tissue.","Although there are several theories that attempt to describe the mechanism of antigen retrieval (e.g., loosening or breaking of crosslinkages formed by formalin fixation), it is clear that modification of protein structure by formalin is reversible under conditions such as high-temperature heating.","It is also clear that several factors affect antigen retrieval: heating, pH, molarity and metal ions in solution (Shi et al., J. Histochem.","Cytochem.","45: 327-343 (1997)).","Microwave heating appears to be the most important factor for retrieval of antigens masked by formalin fixation.","Microwave heating (100°±5° C.) generally yields better results in antigen retrieval immunohistochemistry (AR-IHC).","Different heating methods have been described for antigen retrieval in IHC such as autoclaving (Pons et al, Appl.","Immunohistochem.","3: 265-267 (1995); Bankfalvi et al., J.","Path.","174: 223-228 (1994)); pressure cooking (Miller & Estran, Appl.","Immunohistochem.","3: 190-193 (1995); Norton et al., J.","Path.","173: 371-379 (1994)); water bath (Kawai et al., Path.","Int.","44: 759-764 (1994)); microwaving plus plastic pressure cooking (U.S. Pat.","No.","5,244,787; Pertschuk et al., J.","Cell Biochem.","19(suppl.","): 134-137 (1994)); and steam heating (Pasha et al., Lab.","Invest.","72: 167A (1995); Taylor et al., CAP Today 9: 16-22 (1995)).","Although some antigens yield satisfactory results when microwave heating is performed in distilled water, many antigens require the use of buffers during the heating process.","Some antigens have particular pH requirements such that adequate results will only be achieved in a narrow pH range.","Presently, most antigen retrieval solutions are used at a pH of approximately 6-8, but there is some indication that slightly more basic solutions may provide marginally superior results (Shi, et al., J. Histochem.","Cytochem.","45: 327-343 (1997)).","Although the chemical components of the antigen retrieval solution, including metal ions, may play a role as possible co-factors in the microwave heating procedure, thus far, no single chemical has been identified that is both essential and best for antigen retrieval.","Many solutions and methods are used routinely for staining enhancements.","These may include but are not limited to distilled water, EDTA, urea, Tris, glycine, saline and citrate buffer.","Solutions containing a variety of detergents (ionic or non-ionic surfactants) may also facilitate staining enhancement under a wide range of temperatures (from ambient to in excess of 100° C.).","In addition to cell surface molecules that may be present on the exterior portion of the cell, other molecules of interest in IHC, ISH and other histochemical and cytochemical manipulations are located within the cell, often on the nuclear envelope.","Some of these molecules undergo molecular transformation when exposed to a fixative (coagulative or precipitive) such as formalin.","Thus with respect to these molecules it is desirable to not only overcome the effects of fixation but also to increase the permeability of the cell in order to facilitate the interaction of organic and inorganic compounds with the cell.","Other tissue samples may not have been subjected to cross-linking agents prior to testing, but improved results with respect to these tissues is also important.","There are a variety of non-formalin methods for preserving and preparing cytological and histological samples.","Examples of these methods include, but are not limited to: a) hematology smears, cytospins™, ThiinPreps™, touch preps, cell lines, Ficoll separations, etc.","are routinely preserved in many ways which includes but are not limited to, air-drying, alcoholic fixation, spray fixatives and storage mediums such as sucrose/glycerin; b) tissues and cells (either fixed or unfixed) may be frozen and subsequently subjected to various stabilizing techniques which include, but are not limited to, preservation, fixation and desiccation; c) tissues and cells may be stabilized in a number of non-cross-linking aldehyde fixatives, non-aldehyde containing fixatives, alcoholic fixatives, oxidizing agents, heavy metal fixatives, organic acids and transport media.","One way to improve testing results is to increase the signal obtained from a given sample.","In a general sense, increased signal can be obtained by increasing the accessibility of a given molecule for its target.","As in the case for antigens found within the cell, targets within the cell can be made more accessible by increasing the permeability of the cell thereby permitting a greater number of molecules entry into the cell, thereby increasing the probability that the molecule will “find” its target.","Such increased permeability is especially important for techniques such as ISH, in situ PCR, IHC, histochemistry and cytochemistry.","Tissues and cells are also embedded in a variety of inert media (paraffin, celloidin, OCT™, agar, plastics or acrylics etc.)","to help preserve them for future analysis.","Many of these inert materials are hydrophobic and the reagents used for histological and cytological applications are predominantly hydrophilic; therefore, the inert medium may need to be removed from the biological sample prior to testing.","For example, paraffin embedded tissues sections are prepared for subsequent testing by removal of the paraffin from the tissue section by passing the slide through various organic solvents such as toluene, xylene, limonene or other suitable solvents.","These organic solvents are very volatile causing a variety of problems including requiring special processing (e.g., deparaffinization is performed in ventilated hoods) and requires special waste disposal.","The use of these organic solvents increases the cost of analysis and exposure risk associated with each tissue sample tested and has serious negative effects for the environment.","Presently, there is no available technique for removing inert media from sample tissue by directly heating the slide in an automated fashion.","Neither is it currently possible to remove inert media from sample tissue while conditioning the sample tissue or cell in a one-step automated staining process.","The methods of the present invention permit a) automated removal of embedding media without the use of organic solvents, thus exposing the cells for staining and thereby reducing time, cost and safety hazards, b) automated cell conditioning without automated removal of embedding media from the sample cell or tissue, c) a multi-step automated process that exposes the cells, performs cell conditioning and increases permeability of the cytological or histological specimens, thereby increasing sample readability and improving interpretation of test data.","The methods of the present invention can be used for improving the stainability and readability of most histological and cytological samples used in conjunction with cytological and histological staining techniques.","SUMMARY OF THE INVENTION The present invention relates to solutions, methods and instruments for cell or tissue conditioning, which improves the accessibility of molecules in biological samples during histochemical or cytochemical assays.","The conditioning may be performed at any point in a histochemical or cytochemical protocol.","The present invention further relates to an automated method for exposing biological samples for use in histological or cytological testing procedures by removing the embedding media without the use of organic solvents.","The present invention also relates to an automated method for the simultaneous exposing and cell conditioning of biological samples for histochemical and cytochemical applications.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 is a perspective view of an instrument in which the formulations of the present invention may be used shown with the slide hood open and the carousel door removed.","FIG.","2 is a perspective view of an instrument in which the formulations of the present invention may be used shown in conjunction with a computer and other instruments with which it operates.","FIG.","3 is an exploded view of an instrument in which the formulations of the present invention may be used.","FIG.","4 is a perspective view of an instrument in which the formulations of the present invention may be used shown with a reagent dispenser.","FIG.","5 is a block diagram of the heating system of an instrument in which the formulations of the present invention may be used.","FIG.","6 is a flow chart for one embodiment for removing the embedding media from a slide.","FIGS.","7a-b are flow charts for one embodiment for cell conditioning.","DETAILED DESCRIPTION OF THE INVENTION The present invention provides reagents, formulations, aqueous compositions of matter and the like for use in an automated environment for cell conditioning of biological samples wherein the cells or tissues are predisposed for access by reagent molecules for histochemical and cytochemical staining procedures.","The reagents comprise components optimized to facilitate molecular access to cells and cell constituents within the biological sample.","The present invention also provides reagents for use in an automated environment for removing or etching embedding media by exposing a biological sample to be stained in histochemical or cytochemical procedures without the dependence on organic solvents.","The reagents comprise components optimized to facilitate removal or etching of the embedding media from the biological sample.","One embodiment in which the formulations of the present invention may be useful relates to the exposing of biological samples by removal of the inert materials in which biological samples have been embedded for preservation and support.","In a preferred embodiment, paraffin or other inert materials are removed from biological samples by heating one side of the biological sample.","This may be accomplished by contact heating of the microscope slide on which the embedded biological samples have been placed.","Other inert materials that can be removed from embedded biological samples include, but are not limited to, agars and cryogenic media.","This process of removal of inert embedding media or etching of embedding media is referred to herein as exposing.","In a preferred method in which the formulations of the present invention may be useful, the paraffin-embedded biological sample laying on the glass slide is first heated by a heating element.","The heating element exposes heat on one side of the biological sample (such as the thermal platforms disclosed in U.S. patent application Ser.","No.","09/259,240, herein incorporated by reference) within an automated staining instrument (U.S. patent application Ser.","No.","08/995,052 filed on Dec. 19, 1997 and U.S. provisional patent application Ser.","No.","60/076,198 filed on Feb. 27, 1998, both of which are herein incorporated by reference) such that the sample slide is dried and the paraffin is melted.","Heating Elements As discussed in U.S. patent application Ser.","No.","09/259,240 (which is incorporated by reference) and referring now in detail to the drawings wherein like parts are designated by like reference numerals throughout, there is illustrated in FIG.","1 a perspective view of the molecular pathology apparatus in which formulations according to the present invention are useful, such apparatus is designated generally by reference numeral 10.Apparatus 10 is designed to automatically stain or otherwise treat tissue mounted on microscope slides with nucleic acid probes, antibodies, and/or reagents associated therewith in the desired sequence, time and temperature.","Tissue sections so stained or treated are then to be viewed under a microscope by a medical practitioner who reads the slide for purposes of patient diagnosis, prognosis, or treatment selection.","In a preferred embodiment of an apparatus in which the formulations of the present invention are useful, apparatus 10 functions as one component or module of a system 12 (FIG.","2), which also comprises a host computer 14 preferably a personal computer, monitor 16, keyboard 18, mouse 20, bulk fluid containers 22, waste container 23 and related equipment.","Additional staining modules or other instruments may be added to system 12 to form a network with computer 14 functioning as a server.","Alternatively, some or all of these separate components could be incorporated into apparatus 10 making it a stand-alone instrument.","The preferred configuration of apparatus 10 as well as system 12 is generally as described in U.S. patent application Ser.","No.","08/995,052 filed Dec. 19, 1997 as well as in the Ventana NexES User's Guide available from Ventana Medical Systems, Inc. (Tuscon, Ariz.), both incorporated herein by reference, except with respect to the novel heating system, slide support, bulk fluids module, volume adjust, and slide wipe as disclosed below.","For purposes of clarity, detailed descriptions of those components found in both the present invention and the incorporated references are omitted.","In brief, apparatus 10 is a microprocessor controlled staining instrument that automatically applies chemical and biological reagents to tissue mounted on standard glass microscope slides.","A carousel supporting radially positioned glass slides is revolved by a stepper motor to place each slide under one of a series of reagent dispensers.","Apparatus 10 controls dispensing, washing, mixing, and heating to optimize reaction kinetics.","The computer controlled automation permits use of apparatus 10 in a walk-away manner, i.e.","with little manual labor.","More particularly, apparatus 10 comprises a housing formed of a lower section 30 removably mounted or hinged to an upper section 32.A slide carousel 34 is mounted within lower section 30 for rotation about axis A-A.","As set forth in greater detail below, a plurality of thermal platforms 50 are mounted radially about the perimeter of carousel 34 upon which standard glass slides with tissue samples may be placed.","Carousel 34 is preferably constructed of stainless steel.","It is a key feature of the present invention that the temperature of each slide may be individually controlled by means of various sensors and microprocessors as described herein.","Also housed within apparatus 10 (FIG.","3) are wash dispense nozzles 36, Coverslip™ dispense nozzle 37, fluid knife 38, wash volume adjust nozzle 39, bar code reader mirror 40, and air vortex mixers 42 the details of which are discussed hereinafter.","Rotatably mounted atop upper section 32 is a reagent carousel 28.Dispensers 26 are removably mounted to reagent tray 29 (FIG.","4) which, in turn, is adapted to engage carousel 28.Reagents may include any chemical or biological material conventionally applied to slides including nucleic acid probes or primers, polymerase, primary and secondary antibodies, digestion enzymes, pre-fixatives, post-fixatives, blocking agents, readout chemistry, and counterstains.","Reagent dispensers 26 are preferably bar code labeled 29 for identification by the computer.","For each slide, a single reagent is applied and then incubated for a precise period of time in a temperature-controlled environment.","Mixing of the reagents is accomplished by compressed air jets 42 aimed along the edge of the slide thus causing rotation of the reagent.","After the appropriate incubation, the reagent is washed off the slide using nozzles 36.Then the remaining wash buffer volume is adjusted using the volume adjust nozzle 39.Coverslip™ solution, to inhibit evaporation, is then applied to the slide via nozzle 37.Air knife 38 divides the pool of Coverslip™ followed by the application of the next reagent.","These steps are repeated as the carousels turn until the protocol is completed.","In addition to host computer 14, apparatus 10 preferably includes its own microprocessor 44 to which information from host computer 14 is downloaded.","In particular, the computer downloads to microprocessor 44 both the sequence of steps in a run program and the sensor monitoring and control logic called the “run rules” as well as the temperature parameters of the protocol.","Model No.","DS2251T 128K from Dallas Semiconductor, Dallas Tex.","is an example of a microprocessor that can perform this function.","Turning now to FIG.","5 there is shown a block diagram of the slide heating system 48.The system generally comprises about twenty thermal platforms 50, radially mounted to carousel 34, for heating the slides and monitoring the temperature thereof, and control electronics printed circuit board 52 also mounted to the slide carousel for monitoring the sensors and controlling the heaters.","Control electronics 52 are mounted under the rotating slide carousel 34.Information and power are transferred from the fixed instrument platform to the rotating carousel via a slip ring assembly 56.This information includes the temperature parameters needed for heating the slides (upper and lower) communicated from microprocessor 44 (after having been downloaded from computer 14) to control electronics 52 as described below.","If, during a run, the slide temperature is determined to be below the programmed lower limit, the thermal platform heats the slide.","Likewise, if the slide temperature is found to be above the upper limit, heating is stopped.","A power supply of sufficient capacity to provide about eight watts per heater is provided to meet the requisite rate of temperature rise (a/k/a “ramp up”).","Similarly, in an alternate embodiment, the cooling of the slides may be likewise controlled, as described subsequently.","In one alternate embodiment, cooling platforms are mounted below the slide.","The cooling platforms may comprise Peltier-type thermal transducers.","In an alternative embodiment, a cooling device such as a fan (not shown) may optionally be provided if rapid cooling of the slides is required for particular applications.","The cooling device will modify the ambient air for all of the platforms, necessitating the heaters corresponding to the slides which should not be cooled to compensate for the drop in ambient air temperature.","The slide heating system described herein uses conduction heating and heats slides individually.","The system provides more accurate on-slide temperature and allows for temperature settings on a slide by slide basis.","Heating of the Slide Typically, the biological sample is placed on a top surface of a slide (such as a glass slide).","The slide is then placed on top of the thermal platform 50, so that the bottom surface of the slide is in contact with the thermal platform.","As discussed previously, the thermal platform 50, via conduction, heats the bottom portion of the slide.","After the heating of the biological sample, the inert material may be removed from the slide by a fluid (as a gas or liquid).","For example, the inert material may be rinsed with de-ionized water and a surfactant.","Surfactants useful in the invention include, but are not limited to, Chemphos TC-227S (Chemron Corp.), Colawet DOSS70 (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70E (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70PG (Colonial Chemical, Inc., South Pittsburg, Tenn.), ESI-TERGE™ 320 (CCP/Atofina, Kansas City, Mo.","), Geropon T-77 (Rhodia, Inc.), Geropon AS-200 (Rhodia, Inc.), Hostapon KCG (Clarient Corp.), Monafax 057 (Uniquema/ICI Surfactants), and the like.","Preferably, the surfactant is Chemphos TC-227S or Hostapon KCG.","More preferably, the surfactant is sodium dodecylsulfate.","The current method for deparaffinization is markedly different from what was performed in the prior art.","Prior art methods include: (1) using organic solvents to dissolve the paraffin; or (2) manually using heat and dissolving agents to dissolve the paraffin.","In contrast, in one aspect of the current invention, the deparaffinization does not involve a chemical reaction that dissolves the paraffin.","In particular, the fluid which is placed on the sample does not solvate the paraffin.","Instead, the current method involve the melting of the paraffin from the tissue and the washing of it away with fluids.","In one embodiment, the fluid which is placed on top of the embedded sample is not miscible or capable of being mixed with the paraffin.","One example of this is water, which is not miscible with paraffin.","In addition, water has a higher density than liquified paraffin.","The paraffin, when melted, floats to the top of the fluid so that the top of the slide may be rinsed, rinsing off of the melted paraffin.","In another method of the present invention, a paraffin embedded biological sample is placed on a glass microscope slide and the microscope slide is placed on a heating element.","A reagent is placed on the biological sample slide, the biological sample slide is then exposed to elevated temperatures that will permit the melting of the inert material, and after which the inert material may be removed from the slide by a fluid (as a gas or liquid).","In a preferred embodiment of the present invention, reagents are used in conjunction with heating the embedded biological samples.","Suitable reagents may include, but are not limited to, de-ionized water, citrate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 6-10), phosphate buffer (pH 6.0-8.0), SSC buffer, APK Wash™, acidic buffers or solutions (pH 1-6.9), basic buffers or solutions (pH 7.1-14), mineral oil, Norpar, canola oil, and PAG oil.","Each of these reagents may also contain ionic or non-ionic surfactants such as Triton X-100, Tween, Brij, saponin and sodium dodecylsulfate.","Other surfactants useful in the invention include, but are not limited to, Chemphos TC-227S (Chemron Corp.), Colawet DOSS70 (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70E (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70PG (Colonial Chemical, Inc., South Pittsburg, Tenn.), ESI-TERGE™ 320 (CCP/Atofina, Kansas City, Mo.","), Geropon T-77 (Rhodia, Inc.), Geropon AS-200 (Rhodia, Inc.), Hostapon KCG (Clarient Corp.), Monafax 057 (Uniquema/ICI Surfactants), and the like.","Preferably, the surfactant is Chemphos TC-227S or Hostapon KCG.","More preferably, the surfactant is sodium dodecylsulfate.","In a method of the present invention, the temperature of the heating element is raised to a temperature in excess of the melting point of the inert material.","For example, the melting point of pure paraffin is listed as 50-57° C. in the Merck index.","Thus, in the method of the present invention, the temperature is in excess of the melting point of the paraffin in which the biological sample is embedded.","In a preferred method of the present invention, the temperature is raised in excess of 50° C. to about 130° C. In a method of the present invention, the duration of time required to melt the inert material will vary according to the temperature used and the embedding material.","Typically, in an automated system, a processor, such as a microprocessor, is used in conjunction with a memory.","The amount of time and the temperature required to melt the paraffin is contained within a table contained in the memory.","The paraffin embedded biological sample is subjected to elevated temperatures ranging from 5 minutes to 60 minutes.","The heating element used in the method of the present invention requires that sufficient contact be maintained between the surface on which the biological sample is placed and the heating element.","Referring to FIG.","6, there is shown a flow chart for one embodiment for removing the embedding media from a slide.","As shown at block 60, the surface of the slide is agitated.","In one embodiment, the vortex air mixers 42 are used to stir the biological sample on the surface of the slide.","As shown at block 62, the temperature for the slide is raised to a predetermined temperature.","In one embodiment, the temperature of the slides is raised by heating using the thermal platforms 50.As one example for in situ hybridization (ISH), the initial heating step heats the slide to 65° C. for 16 minutes and then to 75° C. for 2 minutes.","In an immunohistochemical (IHC) example, initial heating step raises the temperature of the thermal platforms to 75° C. for approximately 4 minutes.","This initial heating step, while not necessary to remove the embedding medium, is performed in order to (1) remove any moisture which is between the biological sample and the surface of the slide; and (2) begin melting the embedding media (the melting point for paraffin is 50-57° C., as discussed previously).","The embedding media is both on the outside of the sample (i.e., encasing the sample) and also infused in the tissue.","When performing this initial heating, the embedding media may pool away from the tissue.","Specifically, because the slide is at a slide angle (not completely horizontal), the embedding media may slowly pool at a point on the slide which is lower than where the sample sits.","In an alternate embodiment, the slide may be rinsed with a fluid (either liquid or gas) after the initial heating step to remove the embedding media that has pooled.","For example, fluids such as de-ionized water may be used to rinse the embedding media from the slide.","As shown at block 64, an aqueous fluid is applied to the slide.","Any aqueous fluid is acceptable such that the fluid covers the entire biological sample.","As discussed previously, examples of aqueous fluid include de-ionized water, citrate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 6-10), phosphate buffer (pH 6.0-8.0), SSC buffer, APK Wash™, acidic buffers or solutions (pH 1-6.9), basic buffers or solutions (pH 7.1-14), mineral oil, Norpar, canola oil, and PAG oil.","Moreover, the aqueous fluid may also contain ionic or non-ionic surfactants such as Triton X-100, Tween, Brij, saponin and sodium dodecylsulfate.","The surfactants lower the surface tension of the aqueous fluid, allowing the aqueous fluid to spread better over the surface of the slide.","In one embodiment, the aqueous fluid comprises de-ionized water with about 0.1% Triton X-100.Other surfactants useful in the invention include, but are not limited to, Chemphos TC-227S (Chemron Corp.), Colawet DOSS70 (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70E (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70PG (Colonial Chemical, Inc., South Pittsburg, Tenn.), ESI-TERGE™ 320 (CCP/Atofina, Kansas City, Mo.","), Geropon T-77 (Rhodia, Inc.), Geropon AS-200 (Rhodia, Inc.), Hostapon KCG (Clarient Corp.), Monafax 057 (Uniquema/ICI Surfactants), and the like.","Preferably, the surfactant is Chemphos TC-227S or Hostapon KCG.","More preferably, the surfactant is sodium dodecylsulfate.","An additional ingredient may be added, acting as an anti-microbial agent, so that the fluid prior to application on the slide does not contain microbes.","In one embodiment, the fluid includes a water content, by weight, of 99% or greater (ie., the fluid is composed of between 99%-100% water).","The use of water as a fluid to remove the embedding material is unlike what is conventionally used to remove the embedding material, such as organic solvents.","Water is totally immiscible with paraffin, a typical embedding material.","In contrast, organic solvents, such as toluene, xylene, limonene, are miscible with paraffin and therefore considered suitable for deparaffinization.","Further, the aqueous fluid should be applied in sufficient amounts and at sufficient times (accounting for evaporation of the aqueous fluid due to heating) such that the embedding media may float to the surface of the aqueous fluid and such that the biological sample on the slide will not dry out.","In one embodiment, the aqueous fluid is applied sequentially, with a first application of approximately 1 mL of aqueous fluid on the biological sample, and with a second application two minutes later of aqueous fluid.","The second application may be approximately 0.5 mL to 1 mL of aqueous fluid.","The fluid may be applied to the slide by using a nozzle which is position directly above the slide.","In this manner, the amount of fluid dropped onto the slide may be controlled.","Moreover, because the fluid embedding material (such as paraffin) may have a low surface tension, applying a stream of fluid onto the slide may not leave a sufficient amount of fluid on the top of the slide.","Thus, using the nozzle to drop the fluid onto the embedded sample is preferred as it allows more of the fluid to remain on the upper surface of the slide.","An additional consideration is the maintenance of the temperature of the biological sample above the melting point of the embedding media.","In the paraffin example, the melting point is approximately 50-57° C. In a preferred embodiment, the sample is heated to 75° C. However, since the aqueous fluid is not heated, the application of the aqueous fluid to the slide lowers the temperature of the biological sample temporarily to approximately 60-65° C., which is still above the melting point of paraffin.","Thus, the choice of temperature to heat the sample should be high enough so that the addition of fluids to the slide does not lower the temperature of the slide below the melting point of the embedding media.","Alternatively, the fluid applied to the slide may be heated prior to application so as not to reduce the temperature of the sample on the slide.","At block 66, an evaporation inhibitor is applied.","In a preferred embodiment, LIQUID COVERSLIP™ (LCS™) is applied.","Thereafter, the system waits for a predetermined amount of time as the slide is heated, as shown at block 68.In in situ hybridization (ISH), one example of the time for heating the slide is 6 minutes at 75° C. In an immunohistochemical (IHC) example, the incubation period is 8 minutes at 75° C. In one embodiment, the temperature of the slides remains constant during the initial heating until step 76, as discussed subsequently.","Thus, the heaters for the slides are turned on during the initial step of heating and remain on at the same temperature until after the embedding media is rinsed from the slide.","Alternatively, the setpoint temperature of the heaters may be adjusted from the time of initial heating until after the embedding media is rinsed from the slide.","During the heating, the embedding media floats to the top of the aqueous fluid.","In a preferred embodiment, the fluid which is applied to the slide has density which is greater than the embedding media.","In the specific example, the fluid is mostly composed of water (e.g., 99% or greater) and the embedding media is paraffin.","Paraffin, as an oil based product, is less dense than water.","Thus, the paraffin rises to the top of the water after being melted.","The slide is then rinsed, as shown at block 70, carrying away the embedding media in the aqueous fluid.","In a preferred embodiment, the same type of aqueous fluid used to cover the slide is also used to rinse the slide.","The rinse also leaves an amount of aqueous fluid on the biological sample.","In a preferred embodiment, approximately 0.25 mL is left on the slide.","Thereafter, aqueous fluid is further applied to the slide, as shown at block 72.In an alternate embodiment, the rinsing of the slide may also leave a sufficient amount of aqueous fluid on the slide such that additional aqueous fluid need not be applied.","At block 74, evaporation inhibitor is applied, which in a preferred embodiment is Liquid Coverslip™ (LCS™).","Thereafter, the temperature of the slide is reduced, as shown at block 76.In a preferred embodiment, this is done by reducing or eliminating the heat applied by the thermal platform 50.Moreover, in a preferred embodiment, the temperature of the slide is reduced to a predetermined temperature of 42° C. This predetermined temperature is chosen such that all slides, when processing, are at a known temperature (as opposed to being at ambient temperature, which may fluctuate).","Thereafter, the apparatus waits a predetermined amount of time, as shown at block 78.Another embodiment of the present invention relates to the exposing of biological samples without removal of the inert materials in which biological samples have been embedded for preservation and support.","In a preferred embodiment of the present invention, biological samples are readied for testing by contact heating of the microscope slide on which the embedded biological samples have been placed.","Other inert materials that are not removed from embedded biological samples include but are not limited to plastic or celloidin embedding media and/or other polymers and resins.","In a preferred method of the present invention, the embedded biological sample laying on the glass slide is first heated by the heating element.","The heating element exposes heat on one side of the biological sample, such as by using the thermal platforms 50 disclosed in U.S. patent application Ser.","No.","09/259,240 within an automated staining instrument (U.S. patent application Ser.","Nos.","08/995,052 and 60/076,198) such that the sample slide is dried.","In another method of the present invention, an embedded biological sample is placed on a glass microscope slide and the microscope slide is heated on one side (e.g., by placing the slide on a thermal platform).","A reagent is then placed on the biological sample slide and the biological sample slide, with the reagent, is then heated to a specified temperature (ranging from ambient to greater than 100° C.) and for a specified amount of time (ranging from 2 minutes to 12 hours).","This will cause etching of the surface of the inert embedding material, and after which the etching reagent may be removed from the slide by a fluid (as a gas or liquid).","As discussed previously, the amount of time and the specified temperature may be stored in memory.","In the preferred method of the present invention, reagents are used in conjunction with or without heating the embedded biological samples.","Suitable reagents may include, but are not limited to, de-ionized water, citrate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 6-10), phosphate buffer (pH 6.0-8.0), SSC buffer, APK Wash™, acidic buffers or solutions (pH 1-6.9), basic buffers or solutions (pH7.1-14) mineral oil, Norpar, canola oil, and PAG oil.","Each of these reagents may also contain ionic or non-ionic surfactants, which include, but are not limited to, Chemphos TC-227S (Chemron Corp.), Colawet DOSS70 (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70E (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70PG (Colonial Chemical, Inc., South Pittsburg, Tenn.), ESI-TERGE™ 320 (CCP/Atofina, Kansas City, Mo.","), Geropon T-77 (Rhodia, Inc.), Geropon AS-200 (Rhodia, Inc.), Hostapon KCG (Clarient Corp.), Monafax 057 (Uniquema/ICI Surfactants), and the like.","Preferably, the surfactant is Chemphos TC-227S or Hostapon KCG.","More preferably, the surfactant is sodium dodecylsulfate.","In the method of the present invention, the temperature of the heating element is set to an appropriate level for the drying or the etching of the embedded biological sample.","For example, etching may be carried out with a basic solution of methanol sodium hydroxide (sodium methoxide) at temperatures ranging from ambient to 37° C. In the method of the present invention, the duration of time required to etch the inert material will vary according to the temperature used and the embedding material (plastic or celloidin embedding media and/or other polymers and resins, etc.).","In a preferred method of the present invention the embedded biological sample is subjected to appropriate temperatures ranging from 2 minutes to 12 hours.","The heating element used in the method of the present invention requires that sufficient contact be maintained between the surface on which the biological sample is placed and the heating element.","A preferred embodiment of the present invention also comprises an automated method of cell conditioning, either concurrent with, subsequent to or independent of removal or etching of the inert embedding material from the biological sample.","Heating the biological sample in an appropriate (organic or inorganic) reagent has been found to improve the accessibility of the reagent to the target molecule in the cell (protein, nucleic acid, carbohydrate, lipid, pigment or other small molecule, etc.).","This process of improving accessibility of the reagent (organic or inorganic) to the molecular target is referred to herein as cell conditioning.","In one method of the present invention, cell conditioning is accomplished while the biological sample is being exposed as described above.","In this method of the present invention, a biological sample is placed on a glass microscope slide and the microscope slide is heated on one side (e.g., by placing the slide on a thermal platform) within an automated staining instrument (U.S. patent application Ser.","Nos.","08/995,052 and 60/076,198).","A reagent is placed on the biological sample and the temperature of the heating element may or may not be increased.","The biological sample is exposed to the appropriate temperature for an appropriate duration of time that will permit the melting or etching of the inert material and permit cell conditioning of the biological sample to be subsequently stained using histological or cytological techniques.","Referring to FIGS.","7a-b, there are shown flow charts of one embodiment for exposing a biological sample to permit cell conditioning.","As shown at block 80, it is determined whether cell conditioning is applied.","A variety of cell conditioners may be applied depending on the processing necessary.","As shown at block 82, the slide is rinsed with aqueous fluid.","Any aqueous fluid is acceptable such that the fluid covers the entire biological sample.","In a preferred embodiment, de-ionized water with Triton X-100™ is used.","Thereafter, an evaporation inhibitor is applied to the slide, as shown at block 84.In a preferred embodiment, Liquid Coverslip™ is applied.","Thereafter, the temperature of the slide is modified to a predetermined value, as shown at block 86.In a preferred embodiment, heaters heat the slide to 42° C. This initial heating is performed in order to ensure that all of the slides begin the cell conditioning at a predetermined temperature; otherwise, the temperature of the slide may be unknown if the temperature of the slides is determined by the ambient temperature.","Evaporation inhibitor is then applied, as shown at block 88.Thereafter, the temperature of the slides is raised to a predetermined temperature, as shown at block 90.This is the temperature at which the cell conditioning is performed.","In a preferred embodiment, the temperature of the heaters is set to 100° C. Thereafter, cell conditioner is applied, as shown at block 92 and evaporation inhibitor is applied, as shown at block 94.A multitude of cell conditioners may be applied, as discussed herein.","How lightly/heavily a sample is fixed determines the amount of cell conditioning necessary.","If a sample is lightly fixed, a mild cell conditioning is recommended.","Likewise, if the sample is moderately or heavily (extended) fixed, a moderate or a heavy cell conditioning, respectively, is recommended.","Improper cell conditioning may have adverse consequences on the processing of the sample.","In a preferred embodiment, cell conditioning mild/medium/heavy time is 30/60/90 minutes respectively.","Moreover, the cell conditioner is applied at predetermined increments within the processing in order to properly condition the cell and to avoid drying out the sample.","As shown in FIG.","7a, the number of times to iterate through the application of cell conditioner and LCS™ is determined, as shown at block 96.This variable is called “loop_counter.” Loop_counter is set to 0, as shown at block 98 in FIG.","7b.","A loop is entered (as shown at block 100) and the system waits a predetermined amount of time (as shown at block 102).","In a preferred embodiment, the predetermined amount of time is 6 minutes.","Cell conditioner is applied (as shown at block 104) and evaporation inhibitor is applied (as shown at block 106).","The loop_counter is then incremented, as shown at block 108.In a preferred embodiment, the number of times the loop is executed is 5/10/15 for mild/medium/heavy cell conditioning.","Thereafter, the temperature of the slide is reduced, as shown at block 110.In a preferred embodiment, the heater temperature is reduced to 42° C. Alternatively, the heater may be turned off and the slide temperature may revert to the ambient temperature.","Thereafter, the slide is rinsed, as shown at block 112.The reagents used for cell conditioning can be the same as those for exposing the embedded biological sample.","For example, for DNA targets, a cell conditioning solution may be a solution of EDTA; a common temperature setting may be 95° C. for a duration ranging from 2-90 minutes.","For protein targets, a cell conditioning solution may be a solution of boric acid buffer; a common temperature setting may be in excess of 100° C. for a duration ranging from 2-90 minutes.","For RNA targets, a cell conditioning solution may be a solution of SSC; a common temperature setting may be 75° C. for a duration ranging from 2-90 minutes.","For histochemical reactions, such as a Hematoxylin and Eosin (H&E) stain, a cell conditioning solution may be treated de-ionized water; a common temperature may range from 60-S0° C. for a duration of 2-90 minutes.","A partial list of possible reagents appears in Analytical Morphology, Gu, ed., Eaton Publishing Co. (1997) at pp.","1-40.The solutions should generally be of known molarity, pH, and composition.","Sodium dodecyl sulfate (SDS) and/or ethylene glycol is preferably added to the conditioning solution.","In addition, metal ions or other materials may be added to these reagents to increase effectiveness of the cell conditioning.","In another method of the present invention, cell conditioning is accomplished subsequent to the biological sample being exposed as described above.","In this method of the present invention a biological sample is placed on a glass microscope slide and the microscope slide is heated on one side (e.g., by placing the slide on a thermal platform) within an automated staining instrument (U.S. patent application Ser.","Nos.","08/995,052 and 60/076,198).","In this method (one embodiment of which is shown in FIG.","6), the embedded biological sample laying on the glass slide is first heated by the heating element within an automated staining instrument such that the sample slide is dried and the embedding material is melted or etched and removed by the application of a fluid.","Subsequent to exposing the biological sample, an appropriate reagent is applied in order to permit cell conditioning of the biological sample to be subsequently stained using histological or cytological techniques.","The reagents used for cell conditioning can be the same as those for exposing the embedded biological sample.","For example, for DNA targets, a cell conditioning solution may be a solution of SSC; a common temperature setting may be 95° C. for a duration ranging from 2-90 minutes.","For protein targets, a cell conditioning solution may be a solution of phosphate buffer; a common temperature setting may be in excess of 100° C. for a duration ranging from 2-90 minutes.","For RNA targets, a cell conditioning solution may be a solution of SSC; a common temperature setting may be 75° C. for a duration ranging from 2-90 minutes.","For histochemical reactions, such as a Trichrome stain, a cell conditioning solution may be Bouins; a common temperature may range from 60-80° C. for a duration of 2-30 minutes.","In yet another method of the present invention, cell conditioning is accomplished without the biological sample being exposed.","In this method of the present invention, a biological sample is placed on a glass microscope slide and the microscope slide placed on a heating element within an automated staining instrument.","A reagent is placed on the biological sample and the temperature of the heating element may or may not be increased.","Cell conditioning of the biological sample may be performed prior to being stained using histological or cytological techniques.","The reagents used for cell conditioning can be the same as those for exposing the embedded biological sample.","For example, for DNA targets, a cell conditioning solution may be a solution of sodium citrate; a common temperature setting may be 90° C. for a duration ranging from 2-90 minutes.","For protein targets, a cell conditioning solution may be a solution of urea; a common temperature setting may be in excess of 100° C. for a duration ranging from 2-90 minutes.","For whole cells, a cell conditioning solution may be a solution of methanol; a common temperature setting may be ambient for a duration ranging from 4-10 minutes.","For histochemical reactions, such as an Acid Fast Bacilli (AFB) stain, a cell conditioning solution may be peanut oil; a common temperature may range from 60-70° C. for a duration of 30-60 minutes.","The present invention also comprises cell conditioning of cytological preps, such as fine needle aspirations (FNA) smears, touch preps, Ficoll, Cytospins®, Thins Preps®, cervical-vaginal pap smears, blood or body fluid films, etc., that are neither fixed with an aldehyde nor embedded in a matrix, such as paraffin.","Many are fixed in an alcohol, such as methanol or ethanol, others will be sprayed with hair spray or other aerosol fixative and dried, and still others will be placed in cytological fixatives, which may include carbowax and Saccomanno's (organic or inorganic) reagent among others.","The cells are either centrifuged or filtered to a slide or directly touched to a glass slide and smeared in some cases (PAP's) or applied directly against the slide (touch preps).","The term “Biological sample” includes, but is not limited to, any collection of cells (either loose or in tissue) that can be mounted on a standard glass microscope slide including, without limitation, sections of organs, tumors sections, bodily fluids, smears, frozen sections, blood, cytology preps, microorganisms, tissue arrays and cell lines.","The term “Stain” includes, but is not limited to, any biological or chemical entity which, when applied to targeted molecules in biological sample, renders the molecules detectable under microscopic examination.","Stains include without limitation detectable nucleic acid probes, antibodies, and other reagents which in combination or by themselves result in a colored end product (by bright field or fluorescence).","The terms “Reagent”, “Buffer”, “Additive”, “Component” and “Solution” as used herein for exposing or deparaffinizing (i.e., the process of deparaffinization) may comprise the following component or components, all of which are available from Sigma Chemical, unless otherwise noted: de-ionized water, de-ionized water with about 0.1% Triton X-100, 10 mM phosphate at around pH 6.1, 10 mM phosphate with about 0.1% Triton X-100 at around pH 6.1, 10 mM citrate at around pH 6, 10 mM citrate with about 0.1% Triton X-100, 2×SSC, 10 mM Tris[hydroxymethyl]aminomethane chloride (i.e., Tris-Cl) at around pH 8.2, 10 mM Tris-CJ with about 0.1% Triton X-100 at around pH 8.2.A person skilled in the art to which this invention pertains will recognize that the concentration or concentrations of the component or components listed above may be varied without altering the characteristics of the reagent, buffer, additive or solution for exposing or deparaffinizing.","The terms “Reagent”, “Buffer”, “Additive”, “Component”, “Solution” and “Cell Conditioner” as used herein for cell conditioning may comprise the following component or components, all of which are available from Sigma Chemical, unless otherwise noted: 5 mM citrate at around pH 6, 5 mM citrate with about 0.5% sodium dodecyl sulfate (SDS) at around pH 6, 10 mM citrate at around pH 6, 10 mM citrate with about 0.5% SDS at around pH 6, 20 mM citrate at around pH 6, 20 mM citrate with about 0.5% SDS at around pH 6, 50 mM citrate at around pH 6, 50 mM citrate with about 0.5% SDS at around pH 6, 1 mM ethylene diamine tetraacetic acid (EDTA) at around pH 8, 1 mM EDTA with about 0.075% SDS at around pH 8, 10 mM EDTA at around pH 8, 10 mM EDTA with about 0.075% SDS at around pH 8, 20 mM EDTA at around pH 8, 20 mM EDTA with about 0.075% SDS at around pH 8, 50 mM EDTA at around pH 8, 50 mM EDTA with about 0.075% SDS at around pH 8, 10 mM citrate with about 0.5% SDS and about 1% ethylene glycol at around pH 6, 10 mM citrate with about 0.5% SDS and about 5% ethylene glycol at around pH 6, 10 mM citrate with about 0.5% SDS and about 10% ethylene glycol at around pH 6, 1 mM EDTA with about 0.075% SDS and about 1% ethylene glycol at around pH 8, 1 mM EDTA with about 0.075% SDS and about 5% ethylene glycol at around pH 8, 1 mM EDTA with about 0.075% SDS and about 10% ethylene glycol at around pH 8, phosphate/citrate/EDTA at about pH 9, 10 mM citrate with about 10 mM urea at around pH 6, 10 mM citrate with about 1 mM urea at around pH 6.2, 10 mM sodium citrate with about 1.4 mM MgCl2 and about 0.1% SDS at around pH 7, 10 mM sodium citrate with about 1.4 mM MgCl2 and about 0.1% SDS at around pH 7.99, 10 mM Tris-Cl at around pH 8, 10 mM Tris-Cl with about 20% formamide at around pH 8, 10 mM citrate with about 5% dimethyl sulfoxide (DMSO) at around pH 6, 10 mM citrate with about 0.1% Triton X-100 and about 20% formamide at around pH 6, 10 mM phosphate with 5×SSC and about 2.5% chrondroitin A at around pH 7, 10 mM Tris-Cl with about 10 mM EDTA and about 0.1% Triton X-100 and about 20% formamide at around pH 8.2, 10 mM citrate with about 20% glycerol at around pH 6, 10 mM citrate with about 0.1% Triton X-100 and about 10 mM glycine at around pH 6, 1 mM EDTA with about 1 mM citrate and about 0.25% SDS at around pH 7.8, Norpar/mineral oil (high temperature coverslip), PAG-100 oil, 10 mM citrate with about 2% SDS at a pH of around 6 to around 6.2, 10 mM citrate with about 1% SDS at a pH of around 6 to around 6.2, 10 mM citrate with about 0.5% SDS at a pH of around 6 to around 6.2, 10 mM citrate with about 0.25% SDS at a pH of around 6 to around 6.2, 1 mM EDTA with about 2% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 1% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.5% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.25% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.1% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.075% SDS at a pH of around 7.5 to around 8, 0.5 mM EDTA with about 0.25% SDS at around pH 8, 10 mM EDTA with about 0.5% SDS at around pH 9.6.A person skilled in the art to which this invention pertains will recognize that the concentration or concentrations of the component or components listed above may be varied without altering the characteristics of the reagent, buffer, additive or solution for cell conditioning.","A preferred embodiment of the present invention is the basic formulation comprising 10 mM Tris base, 7.5 mM boric acid, 1 mM EDTA (disodium salt), 0.05% ProClin™ 300 (Supelco, Inc., Bellefonte, Pa.) at pH 8.5.Other embodiments contemplated include the basic formulation wherein the Tris base concentration ranges from about 5 mM to about 20 mM, wherein the boric acid concentration ranges from about 5 mM to about 40 mM, wherein the EDTA concentration ranges from about 0.5 mM to about 2 mM or wherein the pH ranges from around pH 7 to around pH 9.Temperature ranges contemplated are from around 95° C. to around 100° C. Without intending to be construed as a limitation, it should be noted that no significant difference is seen when the concentration of Tris base or EDTA varies over the range specified above, significantly better results were obtained when the concentration of boric acid was 10 mM as opposed to 20 mM or 40 mM, slightly better results were obtained when the concentration of boric acid was 10 mM as opposed to 5 mM, conditioning is optimal at a pH of around 8 to around 8.6.Comparing 10 mM Tris+20 mM boric acid+1 mM EDTA+0.5% Brij 35 with 10 mM Tris+20 mM boric acid+0.5% Brij 35, both reagents displayed strongest conditioning at 100° C. with significantly weaker conditioning at 95° C. Other embodiments contemplated include, but are not limited to, citrate buffer (a combination of sodium citrate trisodium salt dihydrate and citric acid monohydrate hydrate), 10 mM Tris+20 mM boric acid+1 mM EDTA, 10 mM Tris+20 mM boric acid, 10 mM Tris+1 mM EDTA, 10 mM Tris, 20 mM boric acid, 1 mM EDTA, 20 mM Tris+20 mM boric acid+1 mM EDTA, 5 mM Tris+20 mM boric acid+1 mM EDTA, 10 mM Tris+20 mM boric acid+2 mM EDTA, 10 mM Tris+20 mM boric acid+0.5 mM EDTA, 10 mM Tris+40 mM boric acid+1 mM EDTA, 10 mM Tris+10 mM boric acid+1 mM EDTA, 10 mM Tris+5 mM boric acid+1 mM EDTA, 10 mM Tris+7.5 mM boric acid+1 mM EDTA, 10 mM Tris+20 mM boric acid+1 mM EDTA+5% ethylene glycol, 10 mM Tris+20 mM boric acid+5% ethylene glycol, 10 mM Tris+20 mM boric acid+1 mM EDTA+0.1% SDS, 10 mM Tris+20 mM boric acid+0.1% SDS, 10 mM Tris+20 mM boric acid+1 mM EDTA+5% DMSO, 10 mM Tris+20 mM boric acid+5% DMSO, 10 mM Tris+20 mM boric acid+1 mM EDTA+10% DMSO, 10 mM Tris+20 mM boric acid+10% DMSO, 10 mM Tris+20 mM boric acid+1 mM EDTA+5% formamide, 10 mM Tris+20 mM boric acid+5% formamide, 10 mM Tris+20 mM boric acid+1 mM EDTA+10% formamide, 10 mM Tris+20 mM boric acid+10% formamide, 10 mM Tris+20 mM boric acid+1 mM EDTA+0.5% Brij 35, 10 mM Tris+20 mM boric acid+0.5% Brij 35, 10 mM Tris+20 mM boric acid+1 mM EDTA+0.1% Brij 35, 10 mM Tris+20 mM boric acid+0.1% Brij 35, 10 mM Tris+20 mM boric acid+1 mM EDTA+0.5% Triton X-100, 10 mM Tris+20 mM boric acid+0.5% Triton X-100.The term “10×SSC” refers to a 10 molar concentration of sodium chrloide/sodium citrate solution, comprising deionized water as needed to make a liter solution, 87.66 g NaCl, 44.12 g citric acid trisodium salt, dihydrate, adjusted to pH 7.0 with HCl or NaOH, as appropriate.","0.5 ml ProClin 300 is added as preservative.","For all phosphate buffers prepared at any concentration (X molar), prepare X molar solutions of (1) HPO4−2 using K2HPO4 or NaHPO4, and (2) H2PO4− using KH2PO4 or NaH2PO4.Another preferred embodiment of the cell conditioner (cell conditioning solution 2 or CC2) comprises about 10 mM citrate buffer at about pH 6, about 5% ethylene glycol, about 1 mM sodium metabisulfite (Morphosave™, Ventana Medical Systems, Inc., Tucson, Ariz.; U.S. Pat.","No.","5,432,056) and about 0.3% sodium dodecyl sulfate (SDS).","The concentration of citrate may range from about 5 mM to about 50 mM.","The pH of the buffer may range from about 4 to about S. The concentration of ethylene glycol may range from about 1% to about 10%.","The concentration of sodium metabisulfite may range from about 0.1 mM to about 10 mM, preferably from about 0.5 mM to about 1.5 mM.","The concentration of SDS may range from about 0.1% to about 1%, preferably from about 0.25% to about 0.5%.","The following examples are presented for illustrative purposes only and are not intended, nor should they be construed, as limiting the invention in any way.","Those skilled in the art will recognize that variations on the following can be made without exceeding the spirit or scope of the invention.","All patents, patent applications, and other publications are hereby incorporated by reference in their entirety.","EXAMPLES Example 1 Automated “Exposing” and “Cell Conditioning” with Biological Samples Stained with H&E Biological samples, including breast, stomach, brain, tonsil and kidney, that had been embedded in paraffin were exposed according to the following procedure: slides containing the above referenced biological sample were placed on an automated instrument (Ventana Medical Systems, Inc., Tucson, Ariz.) and subjected to the exposing protocol described below.","Generally, the slides containing paraffin embedded biological samples were dry heated to 65° C. for six (6) minutes then rinsed with any of the following: 1) 1× citrate buffer, 2) de-ionized water, 3) 10 mM phosphate buffer (pH=6.3), or 4) 10 mM Tris-HCl buffer (pH=7.4) each containing 0.1% Triton X-100.Exposing Protocol 1 1.Incubate for 2 minutes 2.Rinse slide 3.Adjust slide volume and apply LIQUID COVERSLIPLIQUID COVERSLIP™ 4.Incubate for 6 minutes 5.Rinse slide 6.Adjust slide volume and apply LCS™ 7.Increase temperature to 65.0° C. 8.Rinse slide 9.Adjust slide volume and apply LCS™ 10.Incubate for 4 minutes 11.Adjust slide volume and apply LCS™ 12.Incubate for 4 minutes 13.Adjust slide volume and apply LCS™ 14.Incubate for 4 minutes 15.Rinse slide 16.Decrease temperature to 42.0° C. 17.Adjust slide volume and apply LCS™ 18.Incubate for 4 minutes 19.Rinse slide 20.Decrease temperature to 42.0° C. 21.Adjust slide volume and apply LCS™ 22.Incubate for 4 minutes 23.Rinse slide After automated exposing, the biological sample was stained with hematoxylin and eosin by the following method.","Slides were placed in hematoxylin 1 (Richard Allen Scientific, Kalamazoo, Mich.) for 1.5 minutes and then rinsed with running de-ionized water for one minute.","Slides were then placed in acid alcohol clarifier (Richard Allen Scientific) for one minute and then rinsed with running de-ionized water for one minute.","Slides were then placed in diluting ammonia-bluing reagent for one minute (Richard Allen Scientific, Kalamazoo, Mich.) and then rinsed in running de-ionized water for one minute.","Slides were then rinsed in 95% ethanol, and then placed in 2.5% eosin Y (Richard Allen Scientific, Kalamazoo, Mich.) for 2.5 minutes.","The biological samples on the slides were dehydrated by exposing the biological sample to a 100% ethanol bath for one minute.","This process was repeated three times followed by exposure of the biological sample to a xylene bath for three minutes, twice.","After the dehydration step the biological sample was covered with a coverslip.","Control biological samples were deparaffinized by a traditional solvent-based deparaffinization technique.","Paraffin-embedded biological samples placed on microscope slides and preserved in paraffin were completely submersed in a xylene bath for five minutes.","Slides containing biological samples were placed in a second xylene bath for five minutes.","After removal from the second xylene bath, the slides were placed in a 100% ethanol bath for three minutes.","Slides were then placed in a second 100% ethanol bath for three minutes and then placed in a 90% ethanol solution for two minutes.","The slides were then placed in 80% ethanol for one minute followed by complete immersion in distilled water for one to three minutes.","After deparaffinization, the biological samples were stained with hematoxylin and eosin as described above.","The biological samples that were deparaffinized by the solvent technique and by the automated heating technique were compared after staining by hematoxylin and eosin.","Morphology on all sets of slide was acceptable and essentially equivalent The tonsil and brain biological samples that were exposed by the automated heating method showed more intensified hematoxylin staining than the biological samples deparaffinized by standard solvent techniques.","Example 2 Automated “Exposing” of Biological Samples with Simultaneous “Cell Conditioning” Biological samples of kidney and tonsil that had been formalin fixed and embedded in paraffin were exposed according to the protocol described in Example 1.After automated exposing, the biological sample was subjected to the automated DAB paraffin protocol used for immunohistochemical staining.","The protocol for DAB staining is described below: DAB Protocol 1.Incubate for 2 minutes 2.Rinse slide 3.Adjust slide volume and apply LCS™ 4.Rinse slide 5.Adjust slide volume and apply LCS™ 6.Rinse slide 7.Adjust slide volume and apply LCS™ 8.Apply one drop of inhibitor 9.Incubate for 4 minutes 10.Adjust slide volume and apply LCS™ 11.Apply one drop of primary antibody 12.Incubate for 32 minutes 13.Adjust slide volume and apply LCS™ 14.Apply one drop of Biotinylated Ig 15.Incubate for 8 minutes 16.Rinse slide 17.Adjust slide volume and apply LCS™ 18.Apply one drop of Avidin-HRPO 19.Incubate for 8 minutes 20.Rinse slide 21.Adjust slide volume and apply LCS™ 22.Apply one drop of DAB and one drop DAB H2O2 23.Incubate for 8 minutes 24.Rinse slide 25.Adjust slide volume and apply LIQUID COVERSLIP™ 26.Apply one drop of Copper 27.Incubate for 4 minutes 28.Rinse slide The primary antibody used for the kidney biological sample was Anti-CD15 (Ventana Medical Systems, Inc. Tucson, Ariz., Catalogue no.","250-2504).","The primary antibody used for the tonsil biological sample was Anti-CD45RO (Ventana Medical Systems, Inc. Tucson, Ariz., Catalogue no.","250-2563).","The DAB staining kit used was obtained from Ventana Medical Systems, Inc. Tucson, Ariz., Catalogue no.","250-001.Control biological samples were deparaffinized by a traditional solvent-based deparaffinization technique, as described in Example 1.After deparaffinization the biological samples were placed in a pressure cooker (Model #62104 Nordic Ware, Minneapolis, Minn.) containing 1.5 L 1× citrate buffer.","The pressure cooker was then sealed and placed in a microwave oven (Model #MQSO836E, Matsushita, Franklin Park, Ill.).","With the microwave oven set on “high,” the samples were subjected to microwave heating for approximately 30 minutes.","After microwaving the samples were then “cured” for 30 minutes in the pressure cooker with the lid securely fastened.","After curing the biological samples were placed in 1× citrate buffer for two minutes.","The biological samples were then removed from the citrate buffer and the end of the slides blotted to removed excess citrate buffer.","After blotting, the slides were placed on the automated instrument and immunohistochemically stained as described above.","The biological samples deparaffinized by the solvent technique and by the automated exposing and simultaneous cell conditioning technique were compared after immunohistochemical staining.","Morphology and staining on all sets of slides was acceptable and essentially equivalent.","Example 3 Two Step Automated “Exposing” and “Cell Conditioning” Biological samples of tonsil and breast that had been preserved in paraffin and treated with formaldehyde were treated by the following protocol: Exposing and Cell Conditioning Protocol 1.Incubate for 2 minutes 2.Increase thermofoil temperature to 65.0° C. 3.Incubate for 6 minutes 4.Rinse slide and apply LCS™ 5.Incubate for 6 minutes 6.Rinse slide and apply LCS™ 7.Increase thermofoil temperature to 100.0° C. 8.Adjust slide volume and apply LCS™ 9.Rinse slide 10.Adjust slide volume and apply LCS™ 11.Incubate for 4 minutes 12.Adjust slide volume and apply LCS™ 13.Incubate for 4 minutes 14.Adjust slide volume and apply LCS™ 15.Incubate for 4 minutes 16.Adjust slide volume and apply LCS™ 17.Incubate for 4 minutes 18.Adjust slide volume and apply LCS™ 19.Incubate for 4 minutes 20.Adjust slide volume and apply LCS™ 21.Incubate for 4 minutes 22.Adjust slide volume and apply LCS™ 23.Incubate for 4 minutes 24.Adjust slide volume and apply LCS™ 25.Incubate for 4 minutes 26.Adjust slide volume and apply LCS™ 27.Incubate for 4 minutes 28.Rinse slide 29.Decrease temperature to 42.0° C. 30.Adjust slide volume and apply LCS™ 31.Incubate for 4 minutes 32.Rinse slide 33.Decrease temperature to 20.0° C. 34.Adjust slide volume and apply LCS™ 35.Incubate for 4 minutes 36.Rinse slide The buffer used in the protocol was SSC buffer with either 20% formamide or 0.1% Triton.","After the biological sample was subjected to the above protocol, the DAB paraffin protocol used for immunohistochemical staining of Example 2 was applied.","Tonsil biological sample was treated with anti-Ki67 as a primary antibody.","Breast samples were treated with anti-estrogen receptor (6F11) or anti-progesterone receptor (1A6) as a primary antibody.","All primary antibodies are available through Ventana.","Control biological samples were deparaffinized by a traditional solvent-based de-paraffinization technique, as described in Example 1.After deparaffinization the biological samples were placed in a pressure cooker (Model #62104 Nordic Ware, Minneapolis, Minn.) containing 1.5 L 1× citrate buffer.","The pressure cooker was then sealed and placed in a microwave oven (Model #MQSO836E, Matsushita, Franklin Park, Ill.).","With the microwave oven set on “high”, the samples were subjected to microwave heating for approximately 30 minutes.","After microwaving the samples were then “cured” for 30 minutes in the pressure cooker with the lid securely fastened.","After curing the biological samples were placed in 1× citrate buffer for two minutes.","The biological samples were then removed from the citrate buffer and the end of the slides were blotted to removed excess citrate buffer.","After blotting the slide were placed on the automated instrument and immunohistochemically stained as described above.","The biological samples deparaffinized by solvent technique and by the automated heating technique were compared after immunohistochemical staining.","Morphology on all sets of slide was acceptable and essentially equivalent.","Staining with Ki67 on all sets of slides was equivalent.","ER (6F11) and PR (1A6) staining was slightly weaker with automated cell conditioning indicating the process does work but more development and optimization is required.","Example 4 Automated Cell Conditioning of Non Paraffin Embedded Cell Lines for in situ hybridization Thin Preps™ Hela (ATCC lot # 980427H), Caski (ATCC lot # 980416C) and Siha (ATCC lot # 980416S) cell lines stored in Cytyk preparation solution (lot # 01139Q) were deposited on microscope slides using the Cytyk 2000 instrument.","After deposition the slides were placed in alcohol to keep moist until use on the Discovery® In-Situ staining module (Ventana Medical Systems Inc., Tucson, Ariz.).","Slides were loaded into the instrument and wetted with 2×SSC made from 20×SSC (Ventana P/N 650-012).","Slides were run through a cell conditioning protocol currently referred to as Depar 30 where the slides are rinsed with 2×SSC and the temperature of the slides is increased to 95° C. for a period of approximately 30 minutes.","The slides are then cooled to 37° C. and rinsed with APK Wash® prior to the in-situ staining run.","Using the protocol Blue Swap ISH the cell lines were stained for HPV 16/18 (Enzo HPV 16/18 Bio Probe cat # 32874).","Prior to probe application the cell lines are enzymatically digested with Protease 2 (Ventana P/N 250-2019).","After the probe application the probe and biological sample are denatured simultaneously at 95° C. for 8 minutes.","The non-specifically bound probe is washed off with stringency washes of 2×SSC at 55° C. The probe is then detected with Streptavidin Alk Phos and NBT/BCIP.","The cell lines were dehydrated after staining with a one-minute exposure to 95% ethanol and a one-minute exposure to 100% ethanol repeated 2 times.","Following the ethanol the slides were exposed to xylene for 3 minutes twice.","After dehydration the slides were coverslipped.","The stained cell lines after conditioning showed acceptable morphology, weak staining and there was high background on these slides indicating a need for the process to be developed more.","Depar 30 Protocol Wet Load Slides 1.Skip Application & Incubate for 2 minutes 2.Rinse Slides (2×SSC Buffer) (Warm Slides to 65° C.) 3.Adjust Slide Volume, then apply LCS™ 4.Skip Application & Incubate 6 minutes 5.Rinse Slides (2×SSC Buffer) (Warm Slides to 95° C.) 6.Adjust Slide Volume, then Apply LCS™ 7.Rinse Slides 8.Adjust Slide Volume, then Apply LCS™ 9.Skip Application & Incubate for 4 minutes 10.Adjust Slide Volume, then Apply LCS™ 11.Skip Application & Incubate for 4 minutes 12.Adjust Slide Volume, then Apply LCS™ 13.Skip Application & Incubate for 4 minutes 14.Adjust Slide Volume, then Apply LCS™ 15.Skip Application & Incubate for 4 minutes 16.Adjust Slide Volume, then Apply LCS™ 17.Skip Application & Incubate for 4 minutes 18.Adjust Slide Volume, then Apply LCS™ 19.Skip Application & Incubate for 4 minutes 20.Adjust Slide Volume, then Apply LCS™ 21.Skip Application & Incubate for 4 minutes 22.Rinse Slides (2×SSC Buffer) (Warm Slides to 37° C.) 23.Adjust Slide Volume, then Apply LCS™ 24.Skip Application & Incubate for 4 minutes 25.Rinse Slides (APK Wash) 26.Adjust Slide Volume, then Apply LCS™ Example 5 Automated “Exposing” and “Cell Conditioning” for Single Copy DNA Detection Slides containing formalin fixed, paraffin embedded cell lines Caski (R96-1050A) and Siha (R96-96-C2), both generously provided by Dr. Raymond Tubbs, Cleveland Clinic Pathology Dept., Cleveland Ohio, were stained on Ventana target slides.","Slides were dry loaded onto the instrument and the slide temperature was increased to 65° C. The Depar 30 protocol was run wherein the slides are rinsed with 2×SSC Buffer while at 65° C. then the heat is increased to 95° C. for about 40 minutes.","The slides were then cooled to 37° C. and rinsed with APK wash. At this time the following in situ protocol was run: In situ Protocol: Tubbs 1 (Ventana APK Wash was used for all rinse steps) Protease Digestion: Protease 2, 4 minutes, 37° C. Inhibitor Step: Ventana Inhibitor from DAB kit 32 minutes 37° C. Probe: Enzo HPV Bio Probe 16/18 Control Probe: Enzo HPV Bio Probe 6/11 Denaturation: 95° C., 8 minutes Hybridization: 37° C., 64 minutes 2 Stringency Washes 2×SSC, 60° C., 8 minutes each 3rd Stringency Wash 2×SSC, 37° C., 4 minutes Probe Detection: Streptavidin HRPO (Dako GenPoint Cat.","#K0620) Amplification: Biotinyl Tyramide (Dako GenPoint Cat.","# K0620) Detection: Streptavidin HRPO (Dako GenPoint Cat.","#K0620) or Streptavidin Alk Phos (Vector Cat.","# SA5100) Chromogen DAB (Dako Gen Point Cat.","# K0620) or Ventana NBT/BCIP (Kit P/N250-060) or Ventana Naphthol/Fast Red (Kit P/N250-030) Example 6 Automated “Cell Conditioning” for Non-paraffin Embedded Samples The protocol for DAB staining as described in Example 2 was used in this Example.","The cell conditioning steps for these antibodies was done after using a Cytyk 2000® instrument to make ThinPreps® of cell lines.","The ThinPreps® were stained using antibodies to ER, PgR, Ki67, P53 on Ventana ES instruments, NexES instruments and a manual procedure (Cytyk, Inc.).","A duplicate group of slides have been stained on the NexES Insita module, allowing the cell conditioning steps to be performed by automation.","Although the example stated above is specific to the Cytyk® instrument and staining of the ThinPreps®, the experience is not limited to that mode of making cytological preps.","Example 7 “Cell conditioning” of Frozen Biological Sample Frozen tonsil blocks were prepared by cutting six sections from each block and placing the sample on microscope slides.","Four slides from each block were placed on the Discovery™ in situ module and put through protocol Depar 10.Slides are dry heated to 65° for 6 minutes then rinsed with 0.1M EDTA buffer pH 8.After rinsing, the slide is incubated at 65° for 20 minutes.","Slides were then cooled to 37° C. and rinsed with APK Wash. Two slides from each block were left untreated as controls.","Following the Depar 10 treatment two treated slides from each block and one untreated slide were stained for H & E as described in Example 1.Two treated slides from each block and one untreated from each block are stained for LCA.","Run outcomes: for both the H & E and antibody staining there was no staining difference between the treated and untreated slides.","Example 8 Automated “Exposing” and “Cell Conditioning” for Immunohistochemistry Various antibodies were assayed in the NexES Plus® (Ventana Medical Systems, Inc., Tuscon, Ariz.) automated environment according to the following protocol and flow chart: Protocol: (1) ***** Select EZ Prep ***** (2) ***** Start Timed Steps ***** (3) If deparaffinization is selected (4) Warm slide to 75° C. and incubate for 4 minutes (5) Adjust volume (6) Apply LCS™ (7) Incubate for 8 minutes (8) Rinse slide (9) Adjust volume (10) Apply LCS™ (11) Warm slide to 42° C. and incubate for 2 minutes (12) If cell conditioning is selected (13) If conditioner #1 is selected (14) Rinse slide (15) Adjust slide volume (16) Apply LCS™ (17) Warm slide to 42° C. and incubate for 2 minutes (18) Apply cell conditioning coverslip (19) Warm slide to 100° C. and incubate for 2 minutes (20) Apply cell conditioner #1 (21) Apply LCS™ (22) If standard is selected (23) Incubate for 6 minutes (24) Apply cell conditioner #1 (25) Apply LCS™ (26) Repeat (21)-(23) 9 times (27) Warm slide to 42° C. and incubate for 2 minutes (28) If mild is selected (29) Incubate for 6 minutes (30) Apply cell conditioner #1 (31) Apply LCS™ (32) Repeat (27)-(29) 4 times (33) Warm slide to 42° C. and incubate for 2 minutes (34) If extended is selected (35) Incubate for 6 minutes (36) Apply cell conditioner #1 (37) Apply LCS™ (38) Repeat (33)-(35) 14 times (39) Warm slide to 42° C. and incubate for 2 minutes (40) If conditioner #1 is not selected (41) If conditioner #2 is selected (42) Rinse slide (43) Adjust slide volume (44) Apply LCS™ (45) Warm slide to 42° C. and incubate for 2 minutes (46) Apply cell conditioning coverslip (47) Warm slide to 100° C. and incubate for 2 minutes (48) Apply cell conditioner #2 (49) Apply LCS™ (50) If standard is selected (51) Incubate for 6 minutes (52) Apply cell conditioner #1 (53) Apply LCS™ (54) Repeat (49)-(51) 9 times (55) Warm slide to 42° C. and incubate for 2 minutes (56) If mild is selected (57) Incubate for 6 minutes (58) Apply cell conditioner #1 (59) Apply LCS™ (60) Repeat (55)-(57) 4 times (61) Warm slide to 42° C. and incubate for 2 minutes (62) If extended is selected (63) Incubate for 6 minutes (64) Apply cell conditioner #1 (65) Apply LCS™ (66) Repeat (61)-(63) 14 times (67) Warm slide to 42° C. and incubate for 2 minutes (68) Rinse slide (69) Adjust slide volume (70) Apply LCS™ (71) Disable slide heater (72) ***** Select Reaction Buffer ***** Table I summarizes the results from automated immunohistochemistry assays performed on the NexES Plus (also known as the BENCHMARK™ IHC) using the automated deparaffinization and cell conditioning protocols described herein.","The slides from the automated immunohistochemistry assays were compared to slides prepared using manual deparaffinization/cell conditioning protocols on a NexES instrument.","Specific antibody slides were prepared in triplicate.","Negative control slides were prepared singly.","Slides from both assays were analyzed by persons skilled in the art and assigned ratings for specific staining intensity and non-specific background staining intensity.","The ratings for all of the slides from the NexES Plus fully automated assays exceeded the ratings for the manually deparaffinized and cell conditioned NexES slides in both specific staining intensity and non-specific background intensity.","Slides were analyzed and rated by a minimum of two independent persons skilled in the art and average staining intensities were determined.","The column marked “Qualified Tissue” in Table 1 represents the standard control tissue used to standardize the antibody used on it.","The standardized antibody is given a rating of 4.0.As can be seen in the column marked “Average Staining Intensity,” most of the automated deparaffinized/cell conditioned slides met or exceeded those results.","TABLE 1 Antibody and Cell Conditioning Results Incubation Average Qualified Time Negative Staining Negative Antibody Clone Tissue Pretreatment (minutes) Control Intensity Control anti-PR 1A6 Endo11299#2 60 min.","cell conditioning 32 NC Ig 4 0 anti-ER 6F11 UT31698V 60 min.","cell conditioning 32 NC Ig 4 0 anti-Pan Keratin AE1, AE3, PCK26 SK197A 4 min.","Protease 1 16 NC Ig 4 0 anti-C-erbB-2 CB11 TMC197G 60 min.","cell conditioning 32 NC Ig 4 0 anti-S100 polyclonal p19 None 16 Rbt NC 4 0 anti-CD20 L26 T1398H None 16 NC Ig 3.9 0 anti-Vimentin 3B4 245A 8 min.","Protease 2 16 NC Ig 4 0 anti-LCA RP2/18 T31099B None 16 NC Ig 4 0 anti-Melanosome HMB45 97-715C None 16 NC Ig 4 0 anti-CEA TF-3H8-1 CCA98157 None 16 NC Ig 4 0 anti-CD45RO A6 T9810163 None 16 NC Ig 3.6 0 anti-Chromogratin LK2H10 pN None 16 NC Ig 4 0 anti-EMA Mc5 BRCA98139BC None 16 NC Ig 3.75 0 anti-Kappa Polyclonal T4282 4 min.","Protease 1 16 Rbt NC 4 0 anti-Lambda Polyclonal 51099 4 min.","Protease 1 16 Rbt NC 4 0 anti-CD15 MMA K121698 None 16 NC Ig 4 0 anti-Desmin DE-R-11 SI173BC 16 min Protease 2 16 NC Ig 4 0 anti-Muscle Actin HUC1-1 DIA8699A None 16 NC Ig 4 0 anti-PSA Polyclonal 271A None 16 Rbt NC 4 0 anti-Ki67 MM1 P92-3622 60 min.","cell conditioning 16 NC Ig 3.9 0 anti-p53 bp-53-11 EV246B 60 min.","cell conditioning 16 NC Ig 4 0 anti-Keratin 5D3 L103CB 8 min Protease 1 16 NC Ig 4 0 anti-NSE BBS/NC/VI-H14 #2 None 16 NC Ig 3.9 0 anti-bcl-2 bcl-2/100/D5 Block6 60 min.","cell conditioning 32 NC Ig 4 0 anti-CD30 1G12 HodgkinsI912696C 60 min.","cell conditioning 16 NC Ig 3.75 0 anti-CD43 L60 T11698H None 16 NC Ig 3.75 0 anti-GFAP Polyclonal CBR9872E None 16 Rbt NC 4 0 anti-Synaptophysin 27G12 406A ND 16 ND 4 0 NOTE: In Table 1, ND stands for No Data.","The primary antibodies (available from Ventana Medical Systems, Inc., Tuscon, Ariz.) referred to in Table 1 (above) are described below, in alphabetical order: anti-bcl-2: Clone bcl-2/100/D5 is a monoclonal antibody used to detect bcl-2, which is an inhibitor of apoptosis.","This antibody may be an aid to distinguish between reactive and neoplastic follicular proliferation.","Control: Tonsil.","(VMSI # 760-2693) anti-C-erbB-2 (HER2/neu): Clone CB11 is a monoclonal antibody to c-erb-B2, which is localized on the cell membrane, and occasionally, in the cytoplasm of some neoplastic cells.","(VMSI # 760-2994) anti-CD15: Clone MMA is a monoclonal antibody used to aid in the identification of cells of the granulocytic lineage and/or Reed-Sternberg differentiation.","Control: Hodgkin's Lymphoma.","(VMSI # 760-2504) anti-CD20: Clone L26 is a monoclonal antibody used to aid in the identification of cells of the B lymphocytic lineage.","Contol: Tonsil.","(VMSI # 760-2531/760-2137) anti-CD43: Clone L60 is a monoclonal antibody that specifically binds to antigens located in the plasma membrane of normal granulocytes and T lymphocytes.","Control: Tonsil.","(VMSI # 760-2511) anti-CD45RA: Clone X148 is a monoclonal antibody that specifically binds to antigens located in the plasma membrane of normal B lymphocytes and a subset of T lymphocytes.","Control: Tonsil.","(VMSI # 76-2510) anti-CD45RO: Clone A6 is a monoclonal antibody that binds to the plasma membrane of cells of the T lineage and a subset of B-cells.","Control: Tonsil.","(VMSI # 760-2563) anti-CEA: Clone TF-3H8-1 specifically binds to antigens located in the plasma membrane and cytoplasmic regions of mucosal epithelial cells.","Control: Colon Carcinoma.","(VMSI # 760-2507/760-2141) anti-Chromogranin: Clone LK2H10 is a monoclonal antibody that binds the chromogranin protein located in the secretory granules of normal and neoplastic neuroendocrine cells.","Control: Pancreas.","(VMSI # 760-2519/760-2140) anti-Desmin: Clone DE-R-11 is a monoclonal antibody used to aid in the identification of cells of the myocytic lineage.","Desmin is an intermediate filament found in mature smooth, striated and cardiac muscle.","Control: Vas Deferens.","(VMSI # 760-2513) anti-EGFR: Clone 31G7 is directed against a transmembrane glycoprotein present on a variety of cells.","(VMSI # 760-2548) anti-EMA: Clone Mc5 is a monoclonal antibody used to aid in the identification of cells of epithelial lineage.","EMA is of value in distinguishing both large-cell anaplastic carcinoma from diffuse histiocytic lymphoma, and small-cell anaplastic carcinoma from well and poorly differentiated lymphocytic lymphomas.","Control: Carcinoma.","(VMSI # 760-2508) anti-ER: Clone 6F11 is a monoclonal antibody used to detect the presence of estrogen receptor.","(VMSI # 760-2596/760-2132) Clone CC4-5 is a monoclonal antibody also used to detect the presence of estrogen receptor.","(VMSI #760-2546/760-2138) anti-GFAP: GFAP polyclonal antibody is directed against glial fibrillary acidic protein present in the cytoplasm of most human astrocytes and ependymal cells.","This reagent may be used to aid in the identification of cells of the glial lineage.","Control: Brain.","(VMSI # 760-2516) anti-Kappa: Kappa light chains are expressed by cells of the B-cell lineage.","Light chain production by lymphoid cells is genetically restricted such that the immunoglobulin molecules produced by an individual cell will only contain a single light chain class.","This clonal restriction may be used to indicate the polyclonal or monoclonal nature of B-cell and plasma cell populations.","Control: Plasmacytoma.","(VMSI # 760-2514) anti-Keratin: Clone 5D3 is a monoclonal antibody raised against human epidermal keratins.","This antibody may be used to aid in the identification of cells of the epithelial lineage.","This antibody reacts with cytokeratins 8 and 18.Control: Carcinoma.","(VMSI # 760-2501) Clone AE1 is a monoclonal antibody raised against human epidermal keratins.","This antibody may be used to aid in the identification of cells of the ductal epithelial lineage.","AE1 reacts with cytokeratins 10, 13, 14, 15 and 19.Control: Liver.","(VMSI # 760-2521) anti-Pan Keratin: Pan Keratin is a cocktail of monoclonal antibody clones AE1/AE3/PCK26 used to aid in the identification of cells of the epithelial lineage.","This antibody cocktail reacts with all cytokeratins except 9, 11 and 12.Control: Skin.","(VMSI # 760-2595/760-2135) anti-Ki67: Clone MM1 is a monoclonal antibody that specifically binds to nuclear antigens associated with cell proliferation that are present throughout the active cell cycle (G1, S, G2 and M phases), but absent in resting (G0 phase) cells.","This reagent may be used to aid in the identification of proliferating cells.","Control: Tonsil.","(VMSI # 760-2520) anti-Lambda: Lambda light chains are expressed by cells of the B-cell lineage.","Light chain production by lymphoid cells is genetically restricted, such that the immunoglobulin molecules produced by an individual cell will only contain a single light chain class.","This clonal restriction may be an aid to indicate the polyclonal or monoclonal nature of B-cell and plasma cell populations.","Control: Plasmacytoma.","(VMSI # 760-2515) anti-LCA: Clone RP2/18 is a is a monoclonal antibody used to aid in the identification of cells of lymphocytic descent.","This antibody specifically binds to antigens located predominantly in the plasma membrane and cytoplasmic rim of lymphocytes with variable reactivity to monocytes/histocytes and polymorphs.","Control: Lymphoma.","(VMSI # 760-2505/760-2136) anti-Melanosome: Clone HMB45 monoclonal antibody is used to aid in the identification of cells of the melanocytic lineage.","It reacts with an antigen expressed in abnormal melanocytes and melanoma cells.","Control: Melanoma.","(VMSI # 760-2518M60-2139) anti-Muscle Actin: Clone HUC1-1 is a monoclonal antibody used to aid in the identification of cells of myocytic descent.","Muscle actin is expressed in cells of the striated, smooth and cardiac muscle lineage.","Control: Ileum.","(VMSI # 760-2502) anti-NSE: Clone BBS/NC/VI-H14 is a monoclonal antibody that reacts with an antigen that may be expressed in the cytoplasm of neurons, neuro-endocrine cells, endocrine tumors, carcinoids and Merkel cell tumors.","Control: Pancreas.","(VMSI # 76-2517) anti-p53: Clone Bp-53-11 is a monoclonal antibody directed against the p53 protein.","This reagent may be used to aid in the identification of abnormally proliferating cells in neoplastic cell populations.","Control: Carcinoma.","(VMSI # 760-2540) anti-PCNA: Clone PC10 is a monoclonal antibody that may be used to aid in the identification of proliferating cells in various cell populations.","The PCNA antigen is expressed in all proliferating cells in the G1, S, G2 and M phases.","Control: Tonsil.","(VMSI # 760-2503) anti-PSA: PSA polyclonal antibody reacts with the secretory protein expressed in the cytoplasm of prostate epithelial cells.","Control: Prostate.","(VMSI # 760-2506) anti-PSAP: Clone PASE/4LJ is a monoclonal antibody used to aid in the identification of cells of the prostate lineage.","PSAP specifically binds to antigens located in the cytoplasmic regions of the normal prostate epithelial cells.","Control: Prostate.","(VMSI # 760-2509) anti-PR: Clone 1A6 is a monoclonal antibody used to aid in the identification of the progesterone receptor in human tissue.","(VMSI # 760-2547/760-2133) anti-S100: S100 polyclonal antibody is used to aid in the identification of cells of normal and abnormal neuro-endocrine descent.","Control: Skin.","(VMSI # 760-2523/760-2133) anti-Vimentin: Clone 3B4 is used to aid in the identification of cells of mesenchymal origin.","Control: Vas Deferens.","(VMSI # 760-2512/760-2134) Negative Control: Polyclonal serum applied to negative tissue controls as part of quality control procedures for polyclonal antibodies.","(VMSI # 760-1023) Negative Control Ig: Clone MOPC-21 is applied to negative tissue controls as part of quality control procedures for monoclonal antibodies.","(VMSI # 760-2014) Example 9 Automated “Cell Conditioning” with or without “Exposing” for Immunohistochemistry (IHC), Immunocytochemistry (ICC) or In situ Hybribidization (ISH) The CC2 solution may be used on automated immunohistochemical, immunocytochemical or nz situ hybridization instruments (e.g., BenchMark™ ISH, Ventana Medical Systems, Inc., Tucson, Ariz.; BenchMark™ IHC, Ventana Medical Systems, Inc., Tucson, Ariz.; etc.)","with reagents designed to detect the c-erb2/HER-2/neu gene or protein(s) expressed therefrom (e.g., INFORM® HER-2/neu DNA test, Ventana Medical Systems, Inc., Tucson, Ariz.; PATHWAY™ HER-2/neu (CB11) protein test, Ventana Medical Systems, Inc., Tucson, Ariz.; etc.).","For an ISH assay, the CC2 solution may preferably be used in an automated pretreatment step following deparaffinization with the CC2 solution applied to the tissue at about 90° C. for about 10 minutes prior to application of the DNA probe.","The heat treatment in conjunction with the CC2 solution allows the genomic DNA to become better suited for binding with the c-erb2/HER-2/neu DNA probe.","For an IHC assay, the CC2 solution may preferably be used in an automated cell conditioning step following deparaffinization with the CC2 solution applied to the tissue at about 95° C. for about 30 minutes.","The high temperature in conjunction with the CC2 solution allows the c-erb2/HER-2/neu protein (antigen or epitope) to become better suited for binding with the anti-c-erb2/HER-2/neu antibody.","These are representative contexts in which the CC2 solution is preferably used.","The CC2 solution is also useful in many other assays, techniques, protocols or procedures designed to detect the presence or absence of targeted.","DNA or protein molecules under consideration.","From the foregoing detailed description, it will be appreciated that numerous changes and modifications can be made to the aspects of the invention without departure from the true spirit and scope of the invention.","This true spirit and scope of the invention is defined by the appended claims, to be interpreted in light of the foregoing specification."]],"string":"[\n [\n \"Automated immunohistochemical and in situ hybridization assay formulations\",\n \"The present invention provides reagents for use in an automated environment for cell conditioning of biological samples wherein the cells or tissues are predisposed for access by reagent molecules for histochemical and cytochemical staining procedures.\",\n \"The reagents comprise components optimized to faciliate molecular access to cells and cell constituents within the biological sample.\",\n \"The present invention also provides reagents for use in an automated environment for removing or etching embedding media by exposing a biological sample to be stained in histochemical or cytochemical procedures without the dependence on organic solvents.\",\n \"The reagents comprise components optimized to facilitate removal or etching of the embedding media from the biological sample.\"\n ],\n [\n \"1.An aqueous composition of matter, comprising citrate buffer, ethylene glycol, sodium metabisulfite and a surfactant.\",\n \"2.A composition according to claim 1, wherein the composition is buffered from approximately pH 4 to approximately pH 9.3.A composition according to claim 1, wherein composition is buffered at approximately pH 6.4.A composition according to claim 1, wherein the composition comprises less than or equal to approximately 100 mM citrate buffer.\",\n \"5.A composition according to claim 1, wherein the composition comprises approximately 10 mM citrate buffer.\",\n \"6.A composition according to claim 1, wherein the composition comprises less than or equal to approximately 10% ethylene glycol.\",\n \"7.A composition according to claim 1, wherein the composition comprises approximately 5% ethylene glycol.\",\n \"8.A composition according to claim 1, wherein the composition comprises less than or equal to approximately 10 mM sodium metabisulfite.\",\n \"9.A composition according to claim 1, wherein the composition comprises 1 mM sodium metabisulfite.\",\n \"10.A composition according to claim 1, wherein the composition comprises less than or equal to approximately 10% SDS.\",\n \"11.A composition according to claim 1, wherein the composition comprises approximately 0.3% SDS.\",\n \"12.A composition according to claim 1, wherein the composition comprises approximately 10 mM citrate buffer at approximately pH 6, approximately 5% ethylene glycol, approximately 1 mM sodium metabisulfite and approximately 0.3% SDS.\",\n \"13.An aqueous composition of matter comprising a buffer, a glycol, sodium metabisulfite and a surfactant.\",\n \"14.A composition according to claim 13, wherein the buffer is a citrate buffer.\",\n \"15.A composition according to claim 14, wherein the buffer is at approximately pH 6.16.A composition according to claim 13, wherein the glycol is ethylene glycol.\",\n \"17.A composition according to claim 13, wherein the concentration of the buffer is less than or equal to approximately 100 mM, the concentration of the glycol is less than or equal to approximately 10%, the concentration of the sodium metabisulfite is less than or equal to approximately 10 mM and the concentration of the SDS is less than or equal to approximately 10%.\",\n \"18.A composition according to claim 17, wherein the buffer is citrate at a concentration of approximately 10 mM and the pH of the composition is approximately 6.19.A composition according to claim 18, wherein the glycol is ethylene glycol and the concentration of ethylene glycol is approximately 5%, wherein the concentration of sodium metabisulfite is approximately 1 mM and wherein the concentration of SDS is approximately 0.3%.\",\n \"20.An aqueous composition of matter comprising approximately 10 mM citrate buffer at approximately pH 6, approximately 5% ethylene glycol, approximately 1 mM sodium metabisulfite and approximately 0.3% surfactant.\",\n \"21.An aqueous composition of matter according to claim 20, wherein the surfactant is SDS.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to solutions methods, and instruments for conditioning cells or tissues so as to increase the accessibility of various molecules to their respective targets and generally to improve tissue and cell readability of biological samples on automated instruments prior to immunohistochemical (IHC), in situ hybridization (ISH) or other histochemical or cytochemical manipulations.\",\n \"2.Summary of the Related Art All patents, patent applications and non-patent articles or references mentioned herein are hereby incorporated by reference to the extent that they are not contradictory.\",\n \"The diagnosis of disease based on the interpretation of tissue or cell samples taken from a diseased organism has expanded dramatically over the past few years.\",\n \"In addition to traditional histological staining techniques and immunohistochemical assays, ill situ techniques such as in situ hybridization and in situ polymerase chain reaction are now used to help diagnose disease states in humans.\",\n \"Thus, there are a variety of techniques that can assess not only cell morphology, but also the presence of specific macromolecules within cells and tissues.\",\n \"For example, the diagnosis of breast, ovarian and other carcinomas may be facilitated by the use of techniques designed to identify the presence or absence of the c-erb2/HER-2/neu protooncogene or the protein(s) expressed therefrom.\",\n \"The c-erb2/HER-2/neu protooncogene is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases.\",\n \"Amplification and overexpression of the c-erb2/HER-2/neu protooncogene is found in about 30% of breast carcinomas, about 20% of ovarian carcinomas and others.\",\n \"(Andrechek, E. R. et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA , vol.\",\n \"97, no.\",\n \"7, pp.\",\n \"3444-3449 (2000); Doherty, J. K. et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA , vol.\",\n \"96, pp.\",\n \"10869-10874 (1999); Oh, J. J. et al., Nucleic Acids Res ., vol.\",\n \"27, no.\",\n \"20, pp.\",\n \"4008-4017 (1999); Klapper, L. N. et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA , vol.\",\n \"96, pp.\",\n \"4995-5000 (1999); and references found in each of the aforementioned articles.)\",\n \"Each of these techniques requires that sample cells or tissues undergo preparatory procedures that may include fixing the sample with chemicals such as an aldehyde (such as formaldehyde, glutaraldehyde), formalin substitutes, alcohol (such as ethanol, methanol, isopropanol) or embedding the sample in inert materials such as paraffin, celloidin, agars, polymers, resins, cryogenic media or a variety of plastic embedding media (such as epoxy resins and acrylics).\",\n \"Other sample tissue or cell preparations require physical manipulation such as freezing (frozen tissue section) or aspiration through a fine needle (fine needle aspiration (FNA)).\",\n \"Regardless of the tissue or cell sample or its method of preparation or preservation, the goal of the technologist is to obtain accurate, readable and reproducible results that permit the accurate interpretation of the data.\",\n \"One way to provide accurate, readable and reproducible data is to prepare the tissue or cells in a fashion that optimizes the results of the test regardless of the technique employed.\",\n \"In the case of immunohistochemistry and in situ techniques this means increasing the amount of signal obtained from the specific probe (e.g.\",\n \"antibody, DNA, RNA, etc.).\",\n \"In the case of histochemical staining it may mean increasing the intensity of the stain or increasing staining contrast.\",\n \"Without preservation, tissue samples rapidly deteriorate such that their use in diagnostics is compromised shortly after removal from their host.\",\n \"In 1893, Ferdinand Blum discovered that formaldehyde could be used to preserve or fix tissue so that it could be used in histochemical procedures.\",\n \"The exact mechanisms by which formaldehyde acts in fixing tissues are not fully established, but they involve cross-linking of reactive sites within the same protein and between different proteins via methylene bridges (Fox et al., J. Histochem.\",\n \"Cytochem.\",\n \"33: 845-853 (1985)).\",\n \"Recent evidence suggests that calcium ions also play a role (Morgan et al., J.\",\n \"Path.\",\n \"174: 301-307 (1994)).\",\n \"These links cause changes in the quaternary and tertiary structures of proteins, but the primary and secondary structures appear to be preserved (Mason et al., J. Histochem.\",\n \"Cytochem.\",\n \"39: 225-229 (1991)).\",\n \"The extent to which the cross-linking reaction occurs depends on conditions such as the concentration of formalin, pH, temperature and length of fixation (Fox et al., J. Histochem.\",\n \"Cytochem.\",\n \"33: 845-853 (1985)).\",\n \"Some antigens, such as gastrin, somatostatin and α-1-antitrypsin, may be detected after formalin fixation, but for many antigens, such as intermediate filaments and leukocyte markers, immunodetection after formalin treatment is lost or markedly reduced (McNicol & Richmond, Histopathology 32: 97-103 (1998)).\",\n \"Loss of antigen immunoreactivity is most noticeable at antigen epitopes that are discontinuous, i.e.\",\n \"amino acid sequences where the formation of the epitope depends on the confluence of portions of the protein sequence that are not contiguous.\",\n \"Antigen retrieval refers to the attempt to “undo” the structural changes that treatment of tissue with a cross-linking agent induces in the antigens resident within that tissue.\",\n \"Although there are several theories that attempt to describe the mechanism of antigen retrieval (e.g., loosening or breaking of crosslinkages formed by formalin fixation), it is clear that modification of protein structure by formalin is reversible under conditions such as high-temperature heating.\",\n \"It is also clear that several factors affect antigen retrieval: heating, pH, molarity and metal ions in solution (Shi et al., J. Histochem.\",\n \"Cytochem.\",\n \"45: 327-343 (1997)).\",\n \"Microwave heating appears to be the most important factor for retrieval of antigens masked by formalin fixation.\",\n \"Microwave heating (100°±5° C.) generally yields better results in antigen retrieval immunohistochemistry (AR-IHC).\",\n \"Different heating methods have been described for antigen retrieval in IHC such as autoclaving (Pons et al, Appl.\",\n \"Immunohistochem.\",\n \"3: 265-267 (1995); Bankfalvi et al., J.\",\n \"Path.\",\n \"174: 223-228 (1994)); pressure cooking (Miller & Estran, Appl.\",\n \"Immunohistochem.\",\n \"3: 190-193 (1995); Norton et al., J.\",\n \"Path.\",\n \"173: 371-379 (1994)); water bath (Kawai et al., Path.\",\n \"Int.\",\n \"44: 759-764 (1994)); microwaving plus plastic pressure cooking (U.S. Pat.\",\n \"No.\",\n \"5,244,787; Pertschuk et al., J.\",\n \"Cell Biochem.\",\n \"19(suppl.\",\n \"): 134-137 (1994)); and steam heating (Pasha et al., Lab.\",\n \"Invest.\",\n \"72: 167A (1995); Taylor et al., CAP Today 9: 16-22 (1995)).\",\n \"Although some antigens yield satisfactory results when microwave heating is performed in distilled water, many antigens require the use of buffers during the heating process.\",\n \"Some antigens have particular pH requirements such that adequate results will only be achieved in a narrow pH range.\",\n \"Presently, most antigen retrieval solutions are used at a pH of approximately 6-8, but there is some indication that slightly more basic solutions may provide marginally superior results (Shi, et al., J. Histochem.\",\n \"Cytochem.\",\n \"45: 327-343 (1997)).\",\n \"Although the chemical components of the antigen retrieval solution, including metal ions, may play a role as possible co-factors in the microwave heating procedure, thus far, no single chemical has been identified that is both essential and best for antigen retrieval.\",\n \"Many solutions and methods are used routinely for staining enhancements.\",\n \"These may include but are not limited to distilled water, EDTA, urea, Tris, glycine, saline and citrate buffer.\",\n \"Solutions containing a variety of detergents (ionic or non-ionic surfactants) may also facilitate staining enhancement under a wide range of temperatures (from ambient to in excess of 100° C.).\",\n \"In addition to cell surface molecules that may be present on the exterior portion of the cell, other molecules of interest in IHC, ISH and other histochemical and cytochemical manipulations are located within the cell, often on the nuclear envelope.\",\n \"Some of these molecules undergo molecular transformation when exposed to a fixative (coagulative or precipitive) such as formalin.\",\n \"Thus with respect to these molecules it is desirable to not only overcome the effects of fixation but also to increase the permeability of the cell in order to facilitate the interaction of organic and inorganic compounds with the cell.\",\n \"Other tissue samples may not have been subjected to cross-linking agents prior to testing, but improved results with respect to these tissues is also important.\",\n \"There are a variety of non-formalin methods for preserving and preparing cytological and histological samples.\",\n \"Examples of these methods include, but are not limited to: a) hematology smears, cytospins™, ThiinPreps™, touch preps, cell lines, Ficoll separations, etc.\",\n \"are routinely preserved in many ways which includes but are not limited to, air-drying, alcoholic fixation, spray fixatives and storage mediums such as sucrose/glycerin; b) tissues and cells (either fixed or unfixed) may be frozen and subsequently subjected to various stabilizing techniques which include, but are not limited to, preservation, fixation and desiccation; c) tissues and cells may be stabilized in a number of non-cross-linking aldehyde fixatives, non-aldehyde containing fixatives, alcoholic fixatives, oxidizing agents, heavy metal fixatives, organic acids and transport media.\",\n \"One way to improve testing results is to increase the signal obtained from a given sample.\",\n \"In a general sense, increased signal can be obtained by increasing the accessibility of a given molecule for its target.\",\n \"As in the case for antigens found within the cell, targets within the cell can be made more accessible by increasing the permeability of the cell thereby permitting a greater number of molecules entry into the cell, thereby increasing the probability that the molecule will “find” its target.\",\n \"Such increased permeability is especially important for techniques such as ISH, in situ PCR, IHC, histochemistry and cytochemistry.\",\n \"Tissues and cells are also embedded in a variety of inert media (paraffin, celloidin, OCT™, agar, plastics or acrylics etc.)\",\n \"to help preserve them for future analysis.\",\n \"Many of these inert materials are hydrophobic and the reagents used for histological and cytological applications are predominantly hydrophilic; therefore, the inert medium may need to be removed from the biological sample prior to testing.\",\n \"For example, paraffin embedded tissues sections are prepared for subsequent testing by removal of the paraffin from the tissue section by passing the slide through various organic solvents such as toluene, xylene, limonene or other suitable solvents.\",\n \"These organic solvents are very volatile causing a variety of problems including requiring special processing (e.g., deparaffinization is performed in ventilated hoods) and requires special waste disposal.\",\n \"The use of these organic solvents increases the cost of analysis and exposure risk associated with each tissue sample tested and has serious negative effects for the environment.\",\n \"Presently, there is no available technique for removing inert media from sample tissue by directly heating the slide in an automated fashion.\",\n \"Neither is it currently possible to remove inert media from sample tissue while conditioning the sample tissue or cell in a one-step automated staining process.\",\n \"The methods of the present invention permit a) automated removal of embedding media without the use of organic solvents, thus exposing the cells for staining and thereby reducing time, cost and safety hazards, b) automated cell conditioning without automated removal of embedding media from the sample cell or tissue, c) a multi-step automated process that exposes the cells, performs cell conditioning and increases permeability of the cytological or histological specimens, thereby increasing sample readability and improving interpretation of test data.\",\n \"The methods of the present invention can be used for improving the stainability and readability of most histological and cytological samples used in conjunction with cytological and histological staining techniques.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to solutions, methods and instruments for cell or tissue conditioning, which improves the accessibility of molecules in biological samples during histochemical or cytochemical assays.\",\n \"The conditioning may be performed at any point in a histochemical or cytochemical protocol.\",\n \"The present invention further relates to an automated method for exposing biological samples for use in histological or cytological testing procedures by removing the embedding media without the use of organic solvents.\",\n \"The present invention also relates to an automated method for the simultaneous exposing and cell conditioning of biological samples for histochemical and cytochemical applications.\"\n ],\n [\n \"REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of U.S. application Ser.\",\n \"No.\",\n \"09/800,689, filed Mar.\",\n \"7, 2001, which is a continuation-in-part of U.S. application Ser.\",\n \"No.\",\n \"09/721,096, filed Nov. 22, 2000, with Atty.\",\n \"Docket No.\",\n \"98,541-C. U.S. application Ser.\",\n \"No.\",\n \"09/721,096 claimed the benefit of priority from International Application No.\",\n \"PCT/US99/20353, filed Sep. 3, 1999, which was published on Mar.\",\n \"16, 2000 as WO 00/14507.International Application No.\",\n \"PCT/US99/20353, in turn, claimed the benefit of priority from U.S.\",\n \"Provisional Patent Application No.\",\n \"60/099,018, filed Sep. 3, 1998; U.S. patent application Ser.\",\n \"No.\",\n \"09/259,240, filed Feb. 26, 1999; and International Application No.\",\n \"PCT/US99/04181, filed Feb. 26, 1999, which was published on Sep. 2, 1999 as WO 99/44030.U.S.\",\n \"patent application Ser.\",\n \"No.\",\n \"09/721,096, filed Nov. 22, 2000; International Patent App.\",\n \"No.\",\n \"PCT/US99/20353, filed Sep. 3, 1999, and published as WO 00/14507 on Mar.\",\n \"16, 2000; U.S.\",\n \"Provisional App.\",\n \"No.\",\n \"60/099,018, filed Sep. 3, 1998; U.S. patent application Ser.\",\n \"No.\",\n \"09/259,240, filed Feb. 26, 1999 and International Patent App.\",\n \"No.\",\n \"PCT/US99/04181, filed Feb. 26, 1999, and published as WO 99/44030 on Sep. 2, 1999, are all hereby incorporated by reference.\",\n \"BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to solutions methods, and instruments for conditioning cells or tissues so as to increase the accessibility of various molecules to their respective targets and generally to improve tissue and cell readability of biological samples on automated instruments prior to immunohistochemical (IHC), in situ hybridization (ISH) or other histochemical or cytochemical manipulations.\",\n \"2.Summary of the Related Art All patents, patent applications and non-patent articles or references mentioned herein are hereby incorporated by reference to the extent that they are not contradictory.\",\n \"The diagnosis of disease based on the interpretation of tissue or cell samples taken from a diseased organism has expanded dramatically over the past few years.\",\n \"In addition to traditional histological staining techniques and immunohistochemical assays, ill situ techniques such as in situ hybridization and in situ polymerase chain reaction are now used to help diagnose disease states in humans.\",\n \"Thus, there are a variety of techniques that can assess not only cell morphology, but also the presence of specific macromolecules within cells and tissues.\",\n \"For example, the diagnosis of breast, ovarian and other carcinomas may be facilitated by the use of techniques designed to identify the presence or absence of the c-erb2/HER-2/neu protooncogene or the protein(s) expressed therefrom.\",\n \"The c-erb2/HER-2/neu protooncogene is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases.\",\n \"Amplification and overexpression of the c-erb2/HER-2/neu protooncogene is found in about 30% of breast carcinomas, about 20% of ovarian carcinomas and others.\",\n \"(Andrechek, E. R. et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, vol.\",\n \"97, no.\",\n \"7, pp.\",\n \"3444-3449 (2000); Doherty, J. K. et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, vol.\",\n \"96, pp.\",\n \"10869-10874 (1999); Oh, J. J. et al., Nucleic Acids Res., vol.\",\n \"27, no.\",\n \"20, pp.\",\n \"4008-4017 (1999); Klapper, L. N. et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, vol.\",\n \"96, pp.\",\n \"4995-5000 (1999); and references found in each of the aforementioned articles.)\",\n \"Each of these techniques requires that sample cells or tissues undergo preparatory procedures that may include fixing the sample with chemicals such as an aldehyde (such as formaldehyde, glutaraldehyde), formalin substitutes, alcohol (such as ethanol, methanol, isopropanol) or embedding the sample in inert materials such as paraffin, celloidin, agars, polymers, resins, cryogenic media or a variety of plastic embedding media (such as epoxy resins and acrylics).\",\n \"Other sample tissue or cell preparations require physical manipulation such as freezing (frozen tissue section) or aspiration through a fine needle (fine needle aspiration (FNA)).\",\n \"Regardless of the tissue or cell sample or its method of preparation or preservation, the goal of the technologist is to obtain accurate, readable and reproducible results that permit the accurate interpretation of the data.\",\n \"One way to provide accurate, readable and reproducible data is to prepare the tissue or cells in a fashion that optimizes the results of the test regardless of the technique employed.\",\n \"In the case of immunohistochemistry and in situ techniques this means increasing the amount of signal obtained from the specific probe (e.g.\",\n \"antibody, DNA, RNA, etc.).\",\n \"In the case of histochemical staining it may mean increasing the intensity of the stain or increasing staining contrast.\",\n \"Without preservation, tissue samples rapidly deteriorate such that their use in diagnostics is compromised shortly after removal from their host.\",\n \"In 1893, Ferdinand Blum discovered that formaldehyde could be used to preserve or fix tissue so that it could be used in histochemical procedures.\",\n \"The exact mechanisms by which formaldehyde acts in fixing tissues are not fully established, but they involve cross-linking of reactive sites within the same protein and between different proteins via methylene bridges (Fox et al., J. Histochem.\",\n \"Cytochem.\",\n \"33: 845-853 (1985)).\",\n \"Recent evidence suggests that calcium ions also play a role (Morgan et al., J.\",\n \"Path.\",\n \"174: 301-307 (1994)).\",\n \"These links cause changes in the quaternary and tertiary structures of proteins, but the primary and secondary structures appear to be preserved (Mason et al., J. Histochem.\",\n \"Cytochem.\",\n \"39: 225-229 (1991)).\",\n \"The extent to which the cross-linking reaction occurs depends on conditions such as the concentration of formalin, pH, temperature and length of fixation (Fox et al., J. Histochem.\",\n \"Cytochem.\",\n \"33: 845-853 (1985)).\",\n \"Some antigens, such as gastrin, somatostatin and α-1-antitrypsin, may be detected after formalin fixation, but for many antigens, such as intermediate filaments and leukocyte markers, immunodetection after formalin treatment is lost or markedly reduced (McNicol & Richmond, Histopathology 32: 97-103 (1998)).\",\n \"Loss of antigen immunoreactivity is most noticeable at antigen epitopes that are discontinuous, i.e.\",\n \"amino acid sequences where the formation of the epitope depends on the confluence of portions of the protein sequence that are not contiguous.\",\n \"Antigen retrieval refers to the attempt to “undo” the structural changes that treatment of tissue with a cross-linking agent induces in the antigens resident within that tissue.\",\n \"Although there are several theories that attempt to describe the mechanism of antigen retrieval (e.g., loosening or breaking of crosslinkages formed by formalin fixation), it is clear that modification of protein structure by formalin is reversible under conditions such as high-temperature heating.\",\n \"It is also clear that several factors affect antigen retrieval: heating, pH, molarity and metal ions in solution (Shi et al., J. Histochem.\",\n \"Cytochem.\",\n \"45: 327-343 (1997)).\",\n \"Microwave heating appears to be the most important factor for retrieval of antigens masked by formalin fixation.\",\n \"Microwave heating (100°±5° C.) generally yields better results in antigen retrieval immunohistochemistry (AR-IHC).\",\n \"Different heating methods have been described for antigen retrieval in IHC such as autoclaving (Pons et al, Appl.\",\n \"Immunohistochem.\",\n \"3: 265-267 (1995); Bankfalvi et al., J.\",\n \"Path.\",\n \"174: 223-228 (1994)); pressure cooking (Miller & Estran, Appl.\",\n \"Immunohistochem.\",\n \"3: 190-193 (1995); Norton et al., J.\",\n \"Path.\",\n \"173: 371-379 (1994)); water bath (Kawai et al., Path.\",\n \"Int.\",\n \"44: 759-764 (1994)); microwaving plus plastic pressure cooking (U.S. Pat.\",\n \"No.\",\n \"5,244,787; Pertschuk et al., J.\",\n \"Cell Biochem.\",\n \"19(suppl.\",\n \"): 134-137 (1994)); and steam heating (Pasha et al., Lab.\",\n \"Invest.\",\n \"72: 167A (1995); Taylor et al., CAP Today 9: 16-22 (1995)).\",\n \"Although some antigens yield satisfactory results when microwave heating is performed in distilled water, many antigens require the use of buffers during the heating process.\",\n \"Some antigens have particular pH requirements such that adequate results will only be achieved in a narrow pH range.\",\n \"Presently, most antigen retrieval solutions are used at a pH of approximately 6-8, but there is some indication that slightly more basic solutions may provide marginally superior results (Shi, et al., J. Histochem.\",\n \"Cytochem.\",\n \"45: 327-343 (1997)).\",\n \"Although the chemical components of the antigen retrieval solution, including metal ions, may play a role as possible co-factors in the microwave heating procedure, thus far, no single chemical has been identified that is both essential and best for antigen retrieval.\",\n \"Many solutions and methods are used routinely for staining enhancements.\",\n \"These may include but are not limited to distilled water, EDTA, urea, Tris, glycine, saline and citrate buffer.\",\n \"Solutions containing a variety of detergents (ionic or non-ionic surfactants) may also facilitate staining enhancement under a wide range of temperatures (from ambient to in excess of 100° C.).\",\n \"In addition to cell surface molecules that may be present on the exterior portion of the cell, other molecules of interest in IHC, ISH and other histochemical and cytochemical manipulations are located within the cell, often on the nuclear envelope.\",\n \"Some of these molecules undergo molecular transformation when exposed to a fixative (coagulative or precipitive) such as formalin.\",\n \"Thus with respect to these molecules it is desirable to not only overcome the effects of fixation but also to increase the permeability of the cell in order to facilitate the interaction of organic and inorganic compounds with the cell.\",\n \"Other tissue samples may not have been subjected to cross-linking agents prior to testing, but improved results with respect to these tissues is also important.\",\n \"There are a variety of non-formalin methods for preserving and preparing cytological and histological samples.\",\n \"Examples of these methods include, but are not limited to: a) hematology smears, cytospins™, ThiinPreps™, touch preps, cell lines, Ficoll separations, etc.\",\n \"are routinely preserved in many ways which includes but are not limited to, air-drying, alcoholic fixation, spray fixatives and storage mediums such as sucrose/glycerin; b) tissues and cells (either fixed or unfixed) may be frozen and subsequently subjected to various stabilizing techniques which include, but are not limited to, preservation, fixation and desiccation; c) tissues and cells may be stabilized in a number of non-cross-linking aldehyde fixatives, non-aldehyde containing fixatives, alcoholic fixatives, oxidizing agents, heavy metal fixatives, organic acids and transport media.\",\n \"One way to improve testing results is to increase the signal obtained from a given sample.\",\n \"In a general sense, increased signal can be obtained by increasing the accessibility of a given molecule for its target.\",\n \"As in the case for antigens found within the cell, targets within the cell can be made more accessible by increasing the permeability of the cell thereby permitting a greater number of molecules entry into the cell, thereby increasing the probability that the molecule will “find” its target.\",\n \"Such increased permeability is especially important for techniques such as ISH, in situ PCR, IHC, histochemistry and cytochemistry.\",\n \"Tissues and cells are also embedded in a variety of inert media (paraffin, celloidin, OCT™, agar, plastics or acrylics etc.)\",\n \"to help preserve them for future analysis.\",\n \"Many of these inert materials are hydrophobic and the reagents used for histological and cytological applications are predominantly hydrophilic; therefore, the inert medium may need to be removed from the biological sample prior to testing.\",\n \"For example, paraffin embedded tissues sections are prepared for subsequent testing by removal of the paraffin from the tissue section by passing the slide through various organic solvents such as toluene, xylene, limonene or other suitable solvents.\",\n \"These organic solvents are very volatile causing a variety of problems including requiring special processing (e.g., deparaffinization is performed in ventilated hoods) and requires special waste disposal.\",\n \"The use of these organic solvents increases the cost of analysis and exposure risk associated with each tissue sample tested and has serious negative effects for the environment.\",\n \"Presently, there is no available technique for removing inert media from sample tissue by directly heating the slide in an automated fashion.\",\n \"Neither is it currently possible to remove inert media from sample tissue while conditioning the sample tissue or cell in a one-step automated staining process.\",\n \"The methods of the present invention permit a) automated removal of embedding media without the use of organic solvents, thus exposing the cells for staining and thereby reducing time, cost and safety hazards, b) automated cell conditioning without automated removal of embedding media from the sample cell or tissue, c) a multi-step automated process that exposes the cells, performs cell conditioning and increases permeability of the cytological or histological specimens, thereby increasing sample readability and improving interpretation of test data.\",\n \"The methods of the present invention can be used for improving the stainability and readability of most histological and cytological samples used in conjunction with cytological and histological staining techniques.\",\n \"SUMMARY OF THE INVENTION The present invention relates to solutions, methods and instruments for cell or tissue conditioning, which improves the accessibility of molecules in biological samples during histochemical or cytochemical assays.\",\n \"The conditioning may be performed at any point in a histochemical or cytochemical protocol.\",\n \"The present invention further relates to an automated method for exposing biological samples for use in histological or cytological testing procedures by removing the embedding media without the use of organic solvents.\",\n \"The present invention also relates to an automated method for the simultaneous exposing and cell conditioning of biological samples for histochemical and cytochemical applications.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is a perspective view of an instrument in which the formulations of the present invention may be used shown with the slide hood open and the carousel door removed.\",\n \"FIG.\",\n \"2 is a perspective view of an instrument in which the formulations of the present invention may be used shown in conjunction with a computer and other instruments with which it operates.\",\n \"FIG.\",\n \"3 is an exploded view of an instrument in which the formulations of the present invention may be used.\",\n \"FIG.\",\n \"4 is a perspective view of an instrument in which the formulations of the present invention may be used shown with a reagent dispenser.\",\n \"FIG.\",\n \"5 is a block diagram of the heating system of an instrument in which the formulations of the present invention may be used.\",\n \"FIG.\",\n \"6 is a flow chart for one embodiment for removing the embedding media from a slide.\",\n \"FIGS.\",\n \"7a-b are flow charts for one embodiment for cell conditioning.\",\n \"DETAILED DESCRIPTION OF THE INVENTION The present invention provides reagents, formulations, aqueous compositions of matter and the like for use in an automated environment for cell conditioning of biological samples wherein the cells or tissues are predisposed for access by reagent molecules for histochemical and cytochemical staining procedures.\",\n \"The reagents comprise components optimized to facilitate molecular access to cells and cell constituents within the biological sample.\",\n \"The present invention also provides reagents for use in an automated environment for removing or etching embedding media by exposing a biological sample to be stained in histochemical or cytochemical procedures without the dependence on organic solvents.\",\n \"The reagents comprise components optimized to facilitate removal or etching of the embedding media from the biological sample.\",\n \"One embodiment in which the formulations of the present invention may be useful relates to the exposing of biological samples by removal of the inert materials in which biological samples have been embedded for preservation and support.\",\n \"In a preferred embodiment, paraffin or other inert materials are removed from biological samples by heating one side of the biological sample.\",\n \"This may be accomplished by contact heating of the microscope slide on which the embedded biological samples have been placed.\",\n \"Other inert materials that can be removed from embedded biological samples include, but are not limited to, agars and cryogenic media.\",\n \"This process of removal of inert embedding media or etching of embedding media is referred to herein as exposing.\",\n \"In a preferred method in which the formulations of the present invention may be useful, the paraffin-embedded biological sample laying on the glass slide is first heated by a heating element.\",\n \"The heating element exposes heat on one side of the biological sample (such as the thermal platforms disclosed in U.S. patent application Ser.\",\n \"No.\",\n \"09/259,240, herein incorporated by reference) within an automated staining instrument (U.S. patent application Ser.\",\n \"No.\",\n \"08/995,052 filed on Dec. 19, 1997 and U.S. provisional patent application Ser.\",\n \"No.\",\n \"60/076,198 filed on Feb. 27, 1998, both of which are herein incorporated by reference) such that the sample slide is dried and the paraffin is melted.\",\n \"Heating Elements As discussed in U.S. patent application Ser.\",\n \"No.\",\n \"09/259,240 (which is incorporated by reference) and referring now in detail to the drawings wherein like parts are designated by like reference numerals throughout, there is illustrated in FIG.\",\n \"1 a perspective view of the molecular pathology apparatus in which formulations according to the present invention are useful, such apparatus is designated generally by reference numeral 10.Apparatus 10 is designed to automatically stain or otherwise treat tissue mounted on microscope slides with nucleic acid probes, antibodies, and/or reagents associated therewith in the desired sequence, time and temperature.\",\n \"Tissue sections so stained or treated are then to be viewed under a microscope by a medical practitioner who reads the slide for purposes of patient diagnosis, prognosis, or treatment selection.\",\n \"In a preferred embodiment of an apparatus in which the formulations of the present invention are useful, apparatus 10 functions as one component or module of a system 12 (FIG.\",\n \"2), which also comprises a host computer 14 preferably a personal computer, monitor 16, keyboard 18, mouse 20, bulk fluid containers 22, waste container 23 and related equipment.\",\n \"Additional staining modules or other instruments may be added to system 12 to form a network with computer 14 functioning as a server.\",\n \"Alternatively, some or all of these separate components could be incorporated into apparatus 10 making it a stand-alone instrument.\",\n \"The preferred configuration of apparatus 10 as well as system 12 is generally as described in U.S. patent application Ser.\",\n \"No.\",\n \"08/995,052 filed Dec. 19, 1997 as well as in the Ventana NexES User's Guide available from Ventana Medical Systems, Inc. (Tuscon, Ariz.), both incorporated herein by reference, except with respect to the novel heating system, slide support, bulk fluids module, volume adjust, and slide wipe as disclosed below.\",\n \"For purposes of clarity, detailed descriptions of those components found in both the present invention and the incorporated references are omitted.\",\n \"In brief, apparatus 10 is a microprocessor controlled staining instrument that automatically applies chemical and biological reagents to tissue mounted on standard glass microscope slides.\",\n \"A carousel supporting radially positioned glass slides is revolved by a stepper motor to place each slide under one of a series of reagent dispensers.\",\n \"Apparatus 10 controls dispensing, washing, mixing, and heating to optimize reaction kinetics.\",\n \"The computer controlled automation permits use of apparatus 10 in a walk-away manner, i.e.\",\n \"with little manual labor.\",\n \"More particularly, apparatus 10 comprises a housing formed of a lower section 30 removably mounted or hinged to an upper section 32.A slide carousel 34 is mounted within lower section 30 for rotation about axis A-A.\",\n \"As set forth in greater detail below, a plurality of thermal platforms 50 are mounted radially about the perimeter of carousel 34 upon which standard glass slides with tissue samples may be placed.\",\n \"Carousel 34 is preferably constructed of stainless steel.\",\n \"It is a key feature of the present invention that the temperature of each slide may be individually controlled by means of various sensors and microprocessors as described herein.\",\n \"Also housed within apparatus 10 (FIG.\",\n \"3) are wash dispense nozzles 36, Coverslip™ dispense nozzle 37, fluid knife 38, wash volume adjust nozzle 39, bar code reader mirror 40, and air vortex mixers 42 the details of which are discussed hereinafter.\",\n \"Rotatably mounted atop upper section 32 is a reagent carousel 28.Dispensers 26 are removably mounted to reagent tray 29 (FIG.\",\n \"4) which, in turn, is adapted to engage carousel 28.Reagents may include any chemical or biological material conventionally applied to slides including nucleic acid probes or primers, polymerase, primary and secondary antibodies, digestion enzymes, pre-fixatives, post-fixatives, blocking agents, readout chemistry, and counterstains.\",\n \"Reagent dispensers 26 are preferably bar code labeled 29 for identification by the computer.\",\n \"For each slide, a single reagent is applied and then incubated for a precise period of time in a temperature-controlled environment.\",\n \"Mixing of the reagents is accomplished by compressed air jets 42 aimed along the edge of the slide thus causing rotation of the reagent.\",\n \"After the appropriate incubation, the reagent is washed off the slide using nozzles 36.Then the remaining wash buffer volume is adjusted using the volume adjust nozzle 39.Coverslip™ solution, to inhibit evaporation, is then applied to the slide via nozzle 37.Air knife 38 divides the pool of Coverslip™ followed by the application of the next reagent.\",\n \"These steps are repeated as the carousels turn until the protocol is completed.\",\n \"In addition to host computer 14, apparatus 10 preferably includes its own microprocessor 44 to which information from host computer 14 is downloaded.\",\n \"In particular, the computer downloads to microprocessor 44 both the sequence of steps in a run program and the sensor monitoring and control logic called the “run rules” as well as the temperature parameters of the protocol.\",\n \"Model No.\",\n \"DS2251T 128K from Dallas Semiconductor, Dallas Tex.\",\n \"is an example of a microprocessor that can perform this function.\",\n \"Turning now to FIG.\",\n \"5 there is shown a block diagram of the slide heating system 48.The system generally comprises about twenty thermal platforms 50, radially mounted to carousel 34, for heating the slides and monitoring the temperature thereof, and control electronics printed circuit board 52 also mounted to the slide carousel for monitoring the sensors and controlling the heaters.\",\n \"Control electronics 52 are mounted under the rotating slide carousel 34.Information and power are transferred from the fixed instrument platform to the rotating carousel via a slip ring assembly 56.This information includes the temperature parameters needed for heating the slides (upper and lower) communicated from microprocessor 44 (after having been downloaded from computer 14) to control electronics 52 as described below.\",\n \"If, during a run, the slide temperature is determined to be below the programmed lower limit, the thermal platform heats the slide.\",\n \"Likewise, if the slide temperature is found to be above the upper limit, heating is stopped.\",\n \"A power supply of sufficient capacity to provide about eight watts per heater is provided to meet the requisite rate of temperature rise (a/k/a “ramp up”).\",\n \"Similarly, in an alternate embodiment, the cooling of the slides may be likewise controlled, as described subsequently.\",\n \"In one alternate embodiment, cooling platforms are mounted below the slide.\",\n \"The cooling platforms may comprise Peltier-type thermal transducers.\",\n \"In an alternative embodiment, a cooling device such as a fan (not shown) may optionally be provided if rapid cooling of the slides is required for particular applications.\",\n \"The cooling device will modify the ambient air for all of the platforms, necessitating the heaters corresponding to the slides which should not be cooled to compensate for the drop in ambient air temperature.\",\n \"The slide heating system described herein uses conduction heating and heats slides individually.\",\n \"The system provides more accurate on-slide temperature and allows for temperature settings on a slide by slide basis.\",\n \"Heating of the Slide Typically, the biological sample is placed on a top surface of a slide (such as a glass slide).\",\n \"The slide is then placed on top of the thermal platform 50, so that the bottom surface of the slide is in contact with the thermal platform.\",\n \"As discussed previously, the thermal platform 50, via conduction, heats the bottom portion of the slide.\",\n \"After the heating of the biological sample, the inert material may be removed from the slide by a fluid (as a gas or liquid).\",\n \"For example, the inert material may be rinsed with de-ionized water and a surfactant.\",\n \"Surfactants useful in the invention include, but are not limited to, Chemphos TC-227S (Chemron Corp.), Colawet DOSS70 (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70E (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70PG (Colonial Chemical, Inc., South Pittsburg, Tenn.), ESI-TERGE™ 320 (CCP/Atofina, Kansas City, Mo.\",\n \"), Geropon T-77 (Rhodia, Inc.), Geropon AS-200 (Rhodia, Inc.), Hostapon KCG (Clarient Corp.), Monafax 057 (Uniquema/ICI Surfactants), and the like.\",\n \"Preferably, the surfactant is Chemphos TC-227S or Hostapon KCG.\",\n \"More preferably, the surfactant is sodium dodecylsulfate.\",\n \"The current method for deparaffinization is markedly different from what was performed in the prior art.\",\n \"Prior art methods include: (1) using organic solvents to dissolve the paraffin; or (2) manually using heat and dissolving agents to dissolve the paraffin.\",\n \"In contrast, in one aspect of the current invention, the deparaffinization does not involve a chemical reaction that dissolves the paraffin.\",\n \"In particular, the fluid which is placed on the sample does not solvate the paraffin.\",\n \"Instead, the current method involve the melting of the paraffin from the tissue and the washing of it away with fluids.\",\n \"In one embodiment, the fluid which is placed on top of the embedded sample is not miscible or capable of being mixed with the paraffin.\",\n \"One example of this is water, which is not miscible with paraffin.\",\n \"In addition, water has a higher density than liquified paraffin.\",\n \"The paraffin, when melted, floats to the top of the fluid so that the top of the slide may be rinsed, rinsing off of the melted paraffin.\",\n \"In another method of the present invention, a paraffin embedded biological sample is placed on a glass microscope slide and the microscope slide is placed on a heating element.\",\n \"A reagent is placed on the biological sample slide, the biological sample slide is then exposed to elevated temperatures that will permit the melting of the inert material, and after which the inert material may be removed from the slide by a fluid (as a gas or liquid).\",\n \"In a preferred embodiment of the present invention, reagents are used in conjunction with heating the embedded biological samples.\",\n \"Suitable reagents may include, but are not limited to, de-ionized water, citrate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 6-10), phosphate buffer (pH 6.0-8.0), SSC buffer, APK Wash™, acidic buffers or solutions (pH 1-6.9), basic buffers or solutions (pH 7.1-14), mineral oil, Norpar, canola oil, and PAG oil.\",\n \"Each of these reagents may also contain ionic or non-ionic surfactants such as Triton X-100, Tween, Brij, saponin and sodium dodecylsulfate.\",\n \"Other surfactants useful in the invention include, but are not limited to, Chemphos TC-227S (Chemron Corp.), Colawet DOSS70 (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70E (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70PG (Colonial Chemical, Inc., South Pittsburg, Tenn.), ESI-TERGE™ 320 (CCP/Atofina, Kansas City, Mo.\",\n \"), Geropon T-77 (Rhodia, Inc.), Geropon AS-200 (Rhodia, Inc.), Hostapon KCG (Clarient Corp.), Monafax 057 (Uniquema/ICI Surfactants), and the like.\",\n \"Preferably, the surfactant is Chemphos TC-227S or Hostapon KCG.\",\n \"More preferably, the surfactant is sodium dodecylsulfate.\",\n \"In a method of the present invention, the temperature of the heating element is raised to a temperature in excess of the melting point of the inert material.\",\n \"For example, the melting point of pure paraffin is listed as 50-57° C. in the Merck index.\",\n \"Thus, in the method of the present invention, the temperature is in excess of the melting point of the paraffin in which the biological sample is embedded.\",\n \"In a preferred method of the present invention, the temperature is raised in excess of 50° C. to about 130° C. In a method of the present invention, the duration of time required to melt the inert material will vary according to the temperature used and the embedding material.\",\n \"Typically, in an automated system, a processor, such as a microprocessor, is used in conjunction with a memory.\",\n \"The amount of time and the temperature required to melt the paraffin is contained within a table contained in the memory.\",\n \"The paraffin embedded biological sample is subjected to elevated temperatures ranging from 5 minutes to 60 minutes.\",\n \"The heating element used in the method of the present invention requires that sufficient contact be maintained between the surface on which the biological sample is placed and the heating element.\",\n \"Referring to FIG.\",\n \"6, there is shown a flow chart for one embodiment for removing the embedding media from a slide.\",\n \"As shown at block 60, the surface of the slide is agitated.\",\n \"In one embodiment, the vortex air mixers 42 are used to stir the biological sample on the surface of the slide.\",\n \"As shown at block 62, the temperature for the slide is raised to a predetermined temperature.\",\n \"In one embodiment, the temperature of the slides is raised by heating using the thermal platforms 50.As one example for in situ hybridization (ISH), the initial heating step heats the slide to 65° C. for 16 minutes and then to 75° C. for 2 minutes.\",\n \"In an immunohistochemical (IHC) example, initial heating step raises the temperature of the thermal platforms to 75° C. for approximately 4 minutes.\",\n \"This initial heating step, while not necessary to remove the embedding medium, is performed in order to (1) remove any moisture which is between the biological sample and the surface of the slide; and (2) begin melting the embedding media (the melting point for paraffin is 50-57° C., as discussed previously).\",\n \"The embedding media is both on the outside of the sample (i.e., encasing the sample) and also infused in the tissue.\",\n \"When performing this initial heating, the embedding media may pool away from the tissue.\",\n \"Specifically, because the slide is at a slide angle (not completely horizontal), the embedding media may slowly pool at a point on the slide which is lower than where the sample sits.\",\n \"In an alternate embodiment, the slide may be rinsed with a fluid (either liquid or gas) after the initial heating step to remove the embedding media that has pooled.\",\n \"For example, fluids such as de-ionized water may be used to rinse the embedding media from the slide.\",\n \"As shown at block 64, an aqueous fluid is applied to the slide.\",\n \"Any aqueous fluid is acceptable such that the fluid covers the entire biological sample.\",\n \"As discussed previously, examples of aqueous fluid include de-ionized water, citrate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 6-10), phosphate buffer (pH 6.0-8.0), SSC buffer, APK Wash™, acidic buffers or solutions (pH 1-6.9), basic buffers or solutions (pH 7.1-14), mineral oil, Norpar, canola oil, and PAG oil.\",\n \"Moreover, the aqueous fluid may also contain ionic or non-ionic surfactants such as Triton X-100, Tween, Brij, saponin and sodium dodecylsulfate.\",\n \"The surfactants lower the surface tension of the aqueous fluid, allowing the aqueous fluid to spread better over the surface of the slide.\",\n \"In one embodiment, the aqueous fluid comprises de-ionized water with about 0.1% Triton X-100.Other surfactants useful in the invention include, but are not limited to, Chemphos TC-227S (Chemron Corp.), Colawet DOSS70 (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70E (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70PG (Colonial Chemical, Inc., South Pittsburg, Tenn.), ESI-TERGE™ 320 (CCP/Atofina, Kansas City, Mo.\",\n \"), Geropon T-77 (Rhodia, Inc.), Geropon AS-200 (Rhodia, Inc.), Hostapon KCG (Clarient Corp.), Monafax 057 (Uniquema/ICI Surfactants), and the like.\",\n \"Preferably, the surfactant is Chemphos TC-227S or Hostapon KCG.\",\n \"More preferably, the surfactant is sodium dodecylsulfate.\",\n \"An additional ingredient may be added, acting as an anti-microbial agent, so that the fluid prior to application on the slide does not contain microbes.\",\n \"In one embodiment, the fluid includes a water content, by weight, of 99% or greater (ie., the fluid is composed of between 99%-100% water).\",\n \"The use of water as a fluid to remove the embedding material is unlike what is conventionally used to remove the embedding material, such as organic solvents.\",\n \"Water is totally immiscible with paraffin, a typical embedding material.\",\n \"In contrast, organic solvents, such as toluene, xylene, limonene, are miscible with paraffin and therefore considered suitable for deparaffinization.\",\n \"Further, the aqueous fluid should be applied in sufficient amounts and at sufficient times (accounting for evaporation of the aqueous fluid due to heating) such that the embedding media may float to the surface of the aqueous fluid and such that the biological sample on the slide will not dry out.\",\n \"In one embodiment, the aqueous fluid is applied sequentially, with a first application of approximately 1 mL of aqueous fluid on the biological sample, and with a second application two minutes later of aqueous fluid.\",\n \"The second application may be approximately 0.5 mL to 1 mL of aqueous fluid.\",\n \"The fluid may be applied to the slide by using a nozzle which is position directly above the slide.\",\n \"In this manner, the amount of fluid dropped onto the slide may be controlled.\",\n \"Moreover, because the fluid embedding material (such as paraffin) may have a low surface tension, applying a stream of fluid onto the slide may not leave a sufficient amount of fluid on the top of the slide.\",\n \"Thus, using the nozzle to drop the fluid onto the embedded sample is preferred as it allows more of the fluid to remain on the upper surface of the slide.\",\n \"An additional consideration is the maintenance of the temperature of the biological sample above the melting point of the embedding media.\",\n \"In the paraffin example, the melting point is approximately 50-57° C. In a preferred embodiment, the sample is heated to 75° C. However, since the aqueous fluid is not heated, the application of the aqueous fluid to the slide lowers the temperature of the biological sample temporarily to approximately 60-65° C., which is still above the melting point of paraffin.\",\n \"Thus, the choice of temperature to heat the sample should be high enough so that the addition of fluids to the slide does not lower the temperature of the slide below the melting point of the embedding media.\",\n \"Alternatively, the fluid applied to the slide may be heated prior to application so as not to reduce the temperature of the sample on the slide.\",\n \"At block 66, an evaporation inhibitor is applied.\",\n \"In a preferred embodiment, LIQUID COVERSLIP™ (LCS™) is applied.\",\n \"Thereafter, the system waits for a predetermined amount of time as the slide is heated, as shown at block 68.In in situ hybridization (ISH), one example of the time for heating the slide is 6 minutes at 75° C. In an immunohistochemical (IHC) example, the incubation period is 8 minutes at 75° C. In one embodiment, the temperature of the slides remains constant during the initial heating until step 76, as discussed subsequently.\",\n \"Thus, the heaters for the slides are turned on during the initial step of heating and remain on at the same temperature until after the embedding media is rinsed from the slide.\",\n \"Alternatively, the setpoint temperature of the heaters may be adjusted from the time of initial heating until after the embedding media is rinsed from the slide.\",\n \"During the heating, the embedding media floats to the top of the aqueous fluid.\",\n \"In a preferred embodiment, the fluid which is applied to the slide has density which is greater than the embedding media.\",\n \"In the specific example, the fluid is mostly composed of water (e.g., 99% or greater) and the embedding media is paraffin.\",\n \"Paraffin, as an oil based product, is less dense than water.\",\n \"Thus, the paraffin rises to the top of the water after being melted.\",\n \"The slide is then rinsed, as shown at block 70, carrying away the embedding media in the aqueous fluid.\",\n \"In a preferred embodiment, the same type of aqueous fluid used to cover the slide is also used to rinse the slide.\",\n \"The rinse also leaves an amount of aqueous fluid on the biological sample.\",\n \"In a preferred embodiment, approximately 0.25 mL is left on the slide.\",\n \"Thereafter, aqueous fluid is further applied to the slide, as shown at block 72.In an alternate embodiment, the rinsing of the slide may also leave a sufficient amount of aqueous fluid on the slide such that additional aqueous fluid need not be applied.\",\n \"At block 74, evaporation inhibitor is applied, which in a preferred embodiment is Liquid Coverslip™ (LCS™).\",\n \"Thereafter, the temperature of the slide is reduced, as shown at block 76.In a preferred embodiment, this is done by reducing or eliminating the heat applied by the thermal platform 50.Moreover, in a preferred embodiment, the temperature of the slide is reduced to a predetermined temperature of 42° C. This predetermined temperature is chosen such that all slides, when processing, are at a known temperature (as opposed to being at ambient temperature, which may fluctuate).\",\n \"Thereafter, the apparatus waits a predetermined amount of time, as shown at block 78.Another embodiment of the present invention relates to the exposing of biological samples without removal of the inert materials in which biological samples have been embedded for preservation and support.\",\n \"In a preferred embodiment of the present invention, biological samples are readied for testing by contact heating of the microscope slide on which the embedded biological samples have been placed.\",\n \"Other inert materials that are not removed from embedded biological samples include but are not limited to plastic or celloidin embedding media and/or other polymers and resins.\",\n \"In a preferred method of the present invention, the embedded biological sample laying on the glass slide is first heated by the heating element.\",\n \"The heating element exposes heat on one side of the biological sample, such as by using the thermal platforms 50 disclosed in U.S. patent application Ser.\",\n \"No.\",\n \"09/259,240 within an automated staining instrument (U.S. patent application Ser.\",\n \"Nos.\",\n \"08/995,052 and 60/076,198) such that the sample slide is dried.\",\n \"In another method of the present invention, an embedded biological sample is placed on a glass microscope slide and the microscope slide is heated on one side (e.g., by placing the slide on a thermal platform).\",\n \"A reagent is then placed on the biological sample slide and the biological sample slide, with the reagent, is then heated to a specified temperature (ranging from ambient to greater than 100° C.) and for a specified amount of time (ranging from 2 minutes to 12 hours).\",\n \"This will cause etching of the surface of the inert embedding material, and after which the etching reagent may be removed from the slide by a fluid (as a gas or liquid).\",\n \"As discussed previously, the amount of time and the specified temperature may be stored in memory.\",\n \"In the preferred method of the present invention, reagents are used in conjunction with or without heating the embedded biological samples.\",\n \"Suitable reagents may include, but are not limited to, de-ionized water, citrate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 6-10), phosphate buffer (pH 6.0-8.0), SSC buffer, APK Wash™, acidic buffers or solutions (pH 1-6.9), basic buffers or solutions (pH7.1-14) mineral oil, Norpar, canola oil, and PAG oil.\",\n \"Each of these reagents may also contain ionic or non-ionic surfactants, which include, but are not limited to, Chemphos TC-227S (Chemron Corp.), Colawet DOSS70 (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70E (Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70PG (Colonial Chemical, Inc., South Pittsburg, Tenn.), ESI-TERGE™ 320 (CCP/Atofina, Kansas City, Mo.\",\n \"), Geropon T-77 (Rhodia, Inc.), Geropon AS-200 (Rhodia, Inc.), Hostapon KCG (Clarient Corp.), Monafax 057 (Uniquema/ICI Surfactants), and the like.\",\n \"Preferably, the surfactant is Chemphos TC-227S or Hostapon KCG.\",\n \"More preferably, the surfactant is sodium dodecylsulfate.\",\n \"In the method of the present invention, the temperature of the heating element is set to an appropriate level for the drying or the etching of the embedded biological sample.\",\n \"For example, etching may be carried out with a basic solution of methanol sodium hydroxide (sodium methoxide) at temperatures ranging from ambient to 37° C. In the method of the present invention, the duration of time required to etch the inert material will vary according to the temperature used and the embedding material (plastic or celloidin embedding media and/or other polymers and resins, etc.).\",\n \"In a preferred method of the present invention the embedded biological sample is subjected to appropriate temperatures ranging from 2 minutes to 12 hours.\",\n \"The heating element used in the method of the present invention requires that sufficient contact be maintained between the surface on which the biological sample is placed and the heating element.\",\n \"A preferred embodiment of the present invention also comprises an automated method of cell conditioning, either concurrent with, subsequent to or independent of removal or etching of the inert embedding material from the biological sample.\",\n \"Heating the biological sample in an appropriate (organic or inorganic) reagent has been found to improve the accessibility of the reagent to the target molecule in the cell (protein, nucleic acid, carbohydrate, lipid, pigment or other small molecule, etc.).\",\n \"This process of improving accessibility of the reagent (organic or inorganic) to the molecular target is referred to herein as cell conditioning.\",\n \"In one method of the present invention, cell conditioning is accomplished while the biological sample is being exposed as described above.\",\n \"In this method of the present invention, a biological sample is placed on a glass microscope slide and the microscope slide is heated on one side (e.g., by placing the slide on a thermal platform) within an automated staining instrument (U.S. patent application Ser.\",\n \"Nos.\",\n \"08/995,052 and 60/076,198).\",\n \"A reagent is placed on the biological sample and the temperature of the heating element may or may not be increased.\",\n \"The biological sample is exposed to the appropriate temperature for an appropriate duration of time that will permit the melting or etching of the inert material and permit cell conditioning of the biological sample to be subsequently stained using histological or cytological techniques.\",\n \"Referring to FIGS.\",\n \"7a-b, there are shown flow charts of one embodiment for exposing a biological sample to permit cell conditioning.\",\n \"As shown at block 80, it is determined whether cell conditioning is applied.\",\n \"A variety of cell conditioners may be applied depending on the processing necessary.\",\n \"As shown at block 82, the slide is rinsed with aqueous fluid.\",\n \"Any aqueous fluid is acceptable such that the fluid covers the entire biological sample.\",\n \"In a preferred embodiment, de-ionized water with Triton X-100™ is used.\",\n \"Thereafter, an evaporation inhibitor is applied to the slide, as shown at block 84.In a preferred embodiment, Liquid Coverslip™ is applied.\",\n \"Thereafter, the temperature of the slide is modified to a predetermined value, as shown at block 86.In a preferred embodiment, heaters heat the slide to 42° C. This initial heating is performed in order to ensure that all of the slides begin the cell conditioning at a predetermined temperature; otherwise, the temperature of the slide may be unknown if the temperature of the slides is determined by the ambient temperature.\",\n \"Evaporation inhibitor is then applied, as shown at block 88.Thereafter, the temperature of the slides is raised to a predetermined temperature, as shown at block 90.This is the temperature at which the cell conditioning is performed.\",\n \"In a preferred embodiment, the temperature of the heaters is set to 100° C. Thereafter, cell conditioner is applied, as shown at block 92 and evaporation inhibitor is applied, as shown at block 94.A multitude of cell conditioners may be applied, as discussed herein.\",\n \"How lightly/heavily a sample is fixed determines the amount of cell conditioning necessary.\",\n \"If a sample is lightly fixed, a mild cell conditioning is recommended.\",\n \"Likewise, if the sample is moderately or heavily (extended) fixed, a moderate or a heavy cell conditioning, respectively, is recommended.\",\n \"Improper cell conditioning may have adverse consequences on the processing of the sample.\",\n \"In a preferred embodiment, cell conditioning mild/medium/heavy time is 30/60/90 minutes respectively.\",\n \"Moreover, the cell conditioner is applied at predetermined increments within the processing in order to properly condition the cell and to avoid drying out the sample.\",\n \"As shown in FIG.\",\n \"7a, the number of times to iterate through the application of cell conditioner and LCS™ is determined, as shown at block 96.This variable is called “loop_counter.” Loop_counter is set to 0, as shown at block 98 in FIG.\",\n \"7b.\",\n \"A loop is entered (as shown at block 100) and the system waits a predetermined amount of time (as shown at block 102).\",\n \"In a preferred embodiment, the predetermined amount of time is 6 minutes.\",\n \"Cell conditioner is applied (as shown at block 104) and evaporation inhibitor is applied (as shown at block 106).\",\n \"The loop_counter is then incremented, as shown at block 108.In a preferred embodiment, the number of times the loop is executed is 5/10/15 for mild/medium/heavy cell conditioning.\",\n \"Thereafter, the temperature of the slide is reduced, as shown at block 110.In a preferred embodiment, the heater temperature is reduced to 42° C. Alternatively, the heater may be turned off and the slide temperature may revert to the ambient temperature.\",\n \"Thereafter, the slide is rinsed, as shown at block 112.The reagents used for cell conditioning can be the same as those for exposing the embedded biological sample.\",\n \"For example, for DNA targets, a cell conditioning solution may be a solution of EDTA; a common temperature setting may be 95° C. for a duration ranging from 2-90 minutes.\",\n \"For protein targets, a cell conditioning solution may be a solution of boric acid buffer; a common temperature setting may be in excess of 100° C. for a duration ranging from 2-90 minutes.\",\n \"For RNA targets, a cell conditioning solution may be a solution of SSC; a common temperature setting may be 75° C. for a duration ranging from 2-90 minutes.\",\n \"For histochemical reactions, such as a Hematoxylin and Eosin (H&E) stain, a cell conditioning solution may be treated de-ionized water; a common temperature may range from 60-S0° C. for a duration of 2-90 minutes.\",\n \"A partial list of possible reagents appears in Analytical Morphology, Gu, ed., Eaton Publishing Co. (1997) at pp.\",\n \"1-40.The solutions should generally be of known molarity, pH, and composition.\",\n \"Sodium dodecyl sulfate (SDS) and/or ethylene glycol is preferably added to the conditioning solution.\",\n \"In addition, metal ions or other materials may be added to these reagents to increase effectiveness of the cell conditioning.\",\n \"In another method of the present invention, cell conditioning is accomplished subsequent to the biological sample being exposed as described above.\",\n \"In this method of the present invention a biological sample is placed on a glass microscope slide and the microscope slide is heated on one side (e.g., by placing the slide on a thermal platform) within an automated staining instrument (U.S. patent application Ser.\",\n \"Nos.\",\n \"08/995,052 and 60/076,198).\",\n \"In this method (one embodiment of which is shown in FIG.\",\n \"6), the embedded biological sample laying on the glass slide is first heated by the heating element within an automated staining instrument such that the sample slide is dried and the embedding material is melted or etched and removed by the application of a fluid.\",\n \"Subsequent to exposing the biological sample, an appropriate reagent is applied in order to permit cell conditioning of the biological sample to be subsequently stained using histological or cytological techniques.\",\n \"The reagents used for cell conditioning can be the same as those for exposing the embedded biological sample.\",\n \"For example, for DNA targets, a cell conditioning solution may be a solution of SSC; a common temperature setting may be 95° C. for a duration ranging from 2-90 minutes.\",\n \"For protein targets, a cell conditioning solution may be a solution of phosphate buffer; a common temperature setting may be in excess of 100° C. for a duration ranging from 2-90 minutes.\",\n \"For RNA targets, a cell conditioning solution may be a solution of SSC; a common temperature setting may be 75° C. for a duration ranging from 2-90 minutes.\",\n \"For histochemical reactions, such as a Trichrome stain, a cell conditioning solution may be Bouins; a common temperature may range from 60-80° C. for a duration of 2-30 minutes.\",\n \"In yet another method of the present invention, cell conditioning is accomplished without the biological sample being exposed.\",\n \"In this method of the present invention, a biological sample is placed on a glass microscope slide and the microscope slide placed on a heating element within an automated staining instrument.\",\n \"A reagent is placed on the biological sample and the temperature of the heating element may or may not be increased.\",\n \"Cell conditioning of the biological sample may be performed prior to being stained using histological or cytological techniques.\",\n \"The reagents used for cell conditioning can be the same as those for exposing the embedded biological sample.\",\n \"For example, for DNA targets, a cell conditioning solution may be a solution of sodium citrate; a common temperature setting may be 90° C. for a duration ranging from 2-90 minutes.\",\n \"For protein targets, a cell conditioning solution may be a solution of urea; a common temperature setting may be in excess of 100° C. for a duration ranging from 2-90 minutes.\",\n \"For whole cells, a cell conditioning solution may be a solution of methanol; a common temperature setting may be ambient for a duration ranging from 4-10 minutes.\",\n \"For histochemical reactions, such as an Acid Fast Bacilli (AFB) stain, a cell conditioning solution may be peanut oil; a common temperature may range from 60-70° C. for a duration of 30-60 minutes.\",\n \"The present invention also comprises cell conditioning of cytological preps, such as fine needle aspirations (FNA) smears, touch preps, Ficoll, Cytospins®, Thins Preps®, cervical-vaginal pap smears, blood or body fluid films, etc., that are neither fixed with an aldehyde nor embedded in a matrix, such as paraffin.\",\n \"Many are fixed in an alcohol, such as methanol or ethanol, others will be sprayed with hair spray or other aerosol fixative and dried, and still others will be placed in cytological fixatives, which may include carbowax and Saccomanno's (organic or inorganic) reagent among others.\",\n \"The cells are either centrifuged or filtered to a slide or directly touched to a glass slide and smeared in some cases (PAP's) or applied directly against the slide (touch preps).\",\n \"The term “Biological sample” includes, but is not limited to, any collection of cells (either loose or in tissue) that can be mounted on a standard glass microscope slide including, without limitation, sections of organs, tumors sections, bodily fluids, smears, frozen sections, blood, cytology preps, microorganisms, tissue arrays and cell lines.\",\n \"The term “Stain” includes, but is not limited to, any biological or chemical entity which, when applied to targeted molecules in biological sample, renders the molecules detectable under microscopic examination.\",\n \"Stains include without limitation detectable nucleic acid probes, antibodies, and other reagents which in combination or by themselves result in a colored end product (by bright field or fluorescence).\",\n \"The terms “Reagent”, “Buffer”, “Additive”, “Component” and “Solution” as used herein for exposing or deparaffinizing (i.e., the process of deparaffinization) may comprise the following component or components, all of which are available from Sigma Chemical, unless otherwise noted: de-ionized water, de-ionized water with about 0.1% Triton X-100, 10 mM phosphate at around pH 6.1, 10 mM phosphate with about 0.1% Triton X-100 at around pH 6.1, 10 mM citrate at around pH 6, 10 mM citrate with about 0.1% Triton X-100, 2×SSC, 10 mM Tris[hydroxymethyl]aminomethane chloride (i.e., Tris-Cl) at around pH 8.2, 10 mM Tris-CJ with about 0.1% Triton X-100 at around pH 8.2.A person skilled in the art to which this invention pertains will recognize that the concentration or concentrations of the component or components listed above may be varied without altering the characteristics of the reagent, buffer, additive or solution for exposing or deparaffinizing.\",\n \"The terms “Reagent”, “Buffer”, “Additive”, “Component”, “Solution” and “Cell Conditioner” as used herein for cell conditioning may comprise the following component or components, all of which are available from Sigma Chemical, unless otherwise noted: 5 mM citrate at around pH 6, 5 mM citrate with about 0.5% sodium dodecyl sulfate (SDS) at around pH 6, 10 mM citrate at around pH 6, 10 mM citrate with about 0.5% SDS at around pH 6, 20 mM citrate at around pH 6, 20 mM citrate with about 0.5% SDS at around pH 6, 50 mM citrate at around pH 6, 50 mM citrate with about 0.5% SDS at around pH 6, 1 mM ethylene diamine tetraacetic acid (EDTA) at around pH 8, 1 mM EDTA with about 0.075% SDS at around pH 8, 10 mM EDTA at around pH 8, 10 mM EDTA with about 0.075% SDS at around pH 8, 20 mM EDTA at around pH 8, 20 mM EDTA with about 0.075% SDS at around pH 8, 50 mM EDTA at around pH 8, 50 mM EDTA with about 0.075% SDS at around pH 8, 10 mM citrate with about 0.5% SDS and about 1% ethylene glycol at around pH 6, 10 mM citrate with about 0.5% SDS and about 5% ethylene glycol at around pH 6, 10 mM citrate with about 0.5% SDS and about 10% ethylene glycol at around pH 6, 1 mM EDTA with about 0.075% SDS and about 1% ethylene glycol at around pH 8, 1 mM EDTA with about 0.075% SDS and about 5% ethylene glycol at around pH 8, 1 mM EDTA with about 0.075% SDS and about 10% ethylene glycol at around pH 8, phosphate/citrate/EDTA at about pH 9, 10 mM citrate with about 10 mM urea at around pH 6, 10 mM citrate with about 1 mM urea at around pH 6.2, 10 mM sodium citrate with about 1.4 mM MgCl2 and about 0.1% SDS at around pH 7, 10 mM sodium citrate with about 1.4 mM MgCl2 and about 0.1% SDS at around pH 7.99, 10 mM Tris-Cl at around pH 8, 10 mM Tris-Cl with about 20% formamide at around pH 8, 10 mM citrate with about 5% dimethyl sulfoxide (DMSO) at around pH 6, 10 mM citrate with about 0.1% Triton X-100 and about 20% formamide at around pH 6, 10 mM phosphate with 5×SSC and about 2.5% chrondroitin A at around pH 7, 10 mM Tris-Cl with about 10 mM EDTA and about 0.1% Triton X-100 and about 20% formamide at around pH 8.2, 10 mM citrate with about 20% glycerol at around pH 6, 10 mM citrate with about 0.1% Triton X-100 and about 10 mM glycine at around pH 6, 1 mM EDTA with about 1 mM citrate and about 0.25% SDS at around pH 7.8, Norpar/mineral oil (high temperature coverslip), PAG-100 oil, 10 mM citrate with about 2% SDS at a pH of around 6 to around 6.2, 10 mM citrate with about 1% SDS at a pH of around 6 to around 6.2, 10 mM citrate with about 0.5% SDS at a pH of around 6 to around 6.2, 10 mM citrate with about 0.25% SDS at a pH of around 6 to around 6.2, 1 mM EDTA with about 2% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 1% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.5% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.25% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.1% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.075% SDS at a pH of around 7.5 to around 8, 0.5 mM EDTA with about 0.25% SDS at around pH 8, 10 mM EDTA with about 0.5% SDS at around pH 9.6.A person skilled in the art to which this invention pertains will recognize that the concentration or concentrations of the component or components listed above may be varied without altering the characteristics of the reagent, buffer, additive or solution for cell conditioning.\",\n \"A preferred embodiment of the present invention is the basic formulation comprising 10 mM Tris base, 7.5 mM boric acid, 1 mM EDTA (disodium salt), 0.05% ProClin™ 300 (Supelco, Inc., Bellefonte, Pa.) at pH 8.5.Other embodiments contemplated include the basic formulation wherein the Tris base concentration ranges from about 5 mM to about 20 mM, wherein the boric acid concentration ranges from about 5 mM to about 40 mM, wherein the EDTA concentration ranges from about 0.5 mM to about 2 mM or wherein the pH ranges from around pH 7 to around pH 9.Temperature ranges contemplated are from around 95° C. to around 100° C. Without intending to be construed as a limitation, it should be noted that no significant difference is seen when the concentration of Tris base or EDTA varies over the range specified above, significantly better results were obtained when the concentration of boric acid was 10 mM as opposed to 20 mM or 40 mM, slightly better results were obtained when the concentration of boric acid was 10 mM as opposed to 5 mM, conditioning is optimal at a pH of around 8 to around 8.6.Comparing 10 mM Tris+20 mM boric acid+1 mM EDTA+0.5% Brij 35 with 10 mM Tris+20 mM boric acid+0.5% Brij 35, both reagents displayed strongest conditioning at 100° C. with significantly weaker conditioning at 95° C. Other embodiments contemplated include, but are not limited to, citrate buffer (a combination of sodium citrate trisodium salt dihydrate and citric acid monohydrate hydrate), 10 mM Tris+20 mM boric acid+1 mM EDTA, 10 mM Tris+20 mM boric acid, 10 mM Tris+1 mM EDTA, 10 mM Tris, 20 mM boric acid, 1 mM EDTA, 20 mM Tris+20 mM boric acid+1 mM EDTA, 5 mM Tris+20 mM boric acid+1 mM EDTA, 10 mM Tris+20 mM boric acid+2 mM EDTA, 10 mM Tris+20 mM boric acid+0.5 mM EDTA, 10 mM Tris+40 mM boric acid+1 mM EDTA, 10 mM Tris+10 mM boric acid+1 mM EDTA, 10 mM Tris+5 mM boric acid+1 mM EDTA, 10 mM Tris+7.5 mM boric acid+1 mM EDTA, 10 mM Tris+20 mM boric acid+1 mM EDTA+5% ethylene glycol, 10 mM Tris+20 mM boric acid+5% ethylene glycol, 10 mM Tris+20 mM boric acid+1 mM EDTA+0.1% SDS, 10 mM Tris+20 mM boric acid+0.1% SDS, 10 mM Tris+20 mM boric acid+1 mM EDTA+5% DMSO, 10 mM Tris+20 mM boric acid+5% DMSO, 10 mM Tris+20 mM boric acid+1 mM EDTA+10% DMSO, 10 mM Tris+20 mM boric acid+10% DMSO, 10 mM Tris+20 mM boric acid+1 mM EDTA+5% formamide, 10 mM Tris+20 mM boric acid+5% formamide, 10 mM Tris+20 mM boric acid+1 mM EDTA+10% formamide, 10 mM Tris+20 mM boric acid+10% formamide, 10 mM Tris+20 mM boric acid+1 mM EDTA+0.5% Brij 35, 10 mM Tris+20 mM boric acid+0.5% Brij 35, 10 mM Tris+20 mM boric acid+1 mM EDTA+0.1% Brij 35, 10 mM Tris+20 mM boric acid+0.1% Brij 35, 10 mM Tris+20 mM boric acid+1 mM EDTA+0.5% Triton X-100, 10 mM Tris+20 mM boric acid+0.5% Triton X-100.The term “10×SSC” refers to a 10 molar concentration of sodium chrloide/sodium citrate solution, comprising deionized water as needed to make a liter solution, 87.66 g NaCl, 44.12 g citric acid trisodium salt, dihydrate, adjusted to pH 7.0 with HCl or NaOH, as appropriate.\",\n \"0.5 ml ProClin 300 is added as preservative.\",\n \"For all phosphate buffers prepared at any concentration (X molar), prepare X molar solutions of (1) HPO4−2 using K2HPO4 or NaHPO4, and (2) H2PO4− using KH2PO4 or NaH2PO4.Another preferred embodiment of the cell conditioner (cell conditioning solution 2 or CC2) comprises about 10 mM citrate buffer at about pH 6, about 5% ethylene glycol, about 1 mM sodium metabisulfite (Morphosave™, Ventana Medical Systems, Inc., Tucson, Ariz.; U.S. Pat.\",\n \"No.\",\n \"5,432,056) and about 0.3% sodium dodecyl sulfate (SDS).\",\n \"The concentration of citrate may range from about 5 mM to about 50 mM.\",\n \"The pH of the buffer may range from about 4 to about S. The concentration of ethylene glycol may range from about 1% to about 10%.\",\n \"The concentration of sodium metabisulfite may range from about 0.1 mM to about 10 mM, preferably from about 0.5 mM to about 1.5 mM.\",\n \"The concentration of SDS may range from about 0.1% to about 1%, preferably from about 0.25% to about 0.5%.\",\n \"The following examples are presented for illustrative purposes only and are not intended, nor should they be construed, as limiting the invention in any way.\",\n \"Those skilled in the art will recognize that variations on the following can be made without exceeding the spirit or scope of the invention.\",\n \"All patents, patent applications, and other publications are hereby incorporated by reference in their entirety.\",\n \"EXAMPLES Example 1 Automated “Exposing” and “Cell Conditioning” with Biological Samples Stained with H&E Biological samples, including breast, stomach, brain, tonsil and kidney, that had been embedded in paraffin were exposed according to the following procedure: slides containing the above referenced biological sample were placed on an automated instrument (Ventana Medical Systems, Inc., Tucson, Ariz.) and subjected to the exposing protocol described below.\",\n \"Generally, the slides containing paraffin embedded biological samples were dry heated to 65° C. for six (6) minutes then rinsed with any of the following: 1) 1× citrate buffer, 2) de-ionized water, 3) 10 mM phosphate buffer (pH=6.3), or 4) 10 mM Tris-HCl buffer (pH=7.4) each containing 0.1% Triton X-100.Exposing Protocol 1 1.Incubate for 2 minutes 2.Rinse slide 3.Adjust slide volume and apply LIQUID COVERSLIPLIQUID COVERSLIP™ 4.Incubate for 6 minutes 5.Rinse slide 6.Adjust slide volume and apply LCS™ 7.Increase temperature to 65.0° C. 8.Rinse slide 9.Adjust slide volume and apply LCS™ 10.Incubate for 4 minutes 11.Adjust slide volume and apply LCS™ 12.Incubate for 4 minutes 13.Adjust slide volume and apply LCS™ 14.Incubate for 4 minutes 15.Rinse slide 16.Decrease temperature to 42.0° C. 17.Adjust slide volume and apply LCS™ 18.Incubate for 4 minutes 19.Rinse slide 20.Decrease temperature to 42.0° C. 21.Adjust slide volume and apply LCS™ 22.Incubate for 4 minutes 23.Rinse slide After automated exposing, the biological sample was stained with hematoxylin and eosin by the following method.\",\n \"Slides were placed in hematoxylin 1 (Richard Allen Scientific, Kalamazoo, Mich.) for 1.5 minutes and then rinsed with running de-ionized water for one minute.\",\n \"Slides were then placed in acid alcohol clarifier (Richard Allen Scientific) for one minute and then rinsed with running de-ionized water for one minute.\",\n \"Slides were then placed in diluting ammonia-bluing reagent for one minute (Richard Allen Scientific, Kalamazoo, Mich.) and then rinsed in running de-ionized water for one minute.\",\n \"Slides were then rinsed in 95% ethanol, and then placed in 2.5% eosin Y (Richard Allen Scientific, Kalamazoo, Mich.) for 2.5 minutes.\",\n \"The biological samples on the slides were dehydrated by exposing the biological sample to a 100% ethanol bath for one minute.\",\n \"This process was repeated three times followed by exposure of the biological sample to a xylene bath for three minutes, twice.\",\n \"After the dehydration step the biological sample was covered with a coverslip.\",\n \"Control biological samples were deparaffinized by a traditional solvent-based deparaffinization technique.\",\n \"Paraffin-embedded biological samples placed on microscope slides and preserved in paraffin were completely submersed in a xylene bath for five minutes.\",\n \"Slides containing biological samples were placed in a second xylene bath for five minutes.\",\n \"After removal from the second xylene bath, the slides were placed in a 100% ethanol bath for three minutes.\",\n \"Slides were then placed in a second 100% ethanol bath for three minutes and then placed in a 90% ethanol solution for two minutes.\",\n \"The slides were then placed in 80% ethanol for one minute followed by complete immersion in distilled water for one to three minutes.\",\n \"After deparaffinization, the biological samples were stained with hematoxylin and eosin as described above.\",\n \"The biological samples that were deparaffinized by the solvent technique and by the automated heating technique were compared after staining by hematoxylin and eosin.\",\n \"Morphology on all sets of slide was acceptable and essentially equivalent The tonsil and brain biological samples that were exposed by the automated heating method showed more intensified hematoxylin staining than the biological samples deparaffinized by standard solvent techniques.\",\n \"Example 2 Automated “Exposing” of Biological Samples with Simultaneous “Cell Conditioning” Biological samples of kidney and tonsil that had been formalin fixed and embedded in paraffin were exposed according to the protocol described in Example 1.After automated exposing, the biological sample was subjected to the automated DAB paraffin protocol used for immunohistochemical staining.\",\n \"The protocol for DAB staining is described below: DAB Protocol 1.Incubate for 2 minutes 2.Rinse slide 3.Adjust slide volume and apply LCS™ 4.Rinse slide 5.Adjust slide volume and apply LCS™ 6.Rinse slide 7.Adjust slide volume and apply LCS™ 8.Apply one drop of inhibitor 9.Incubate for 4 minutes 10.Adjust slide volume and apply LCS™ 11.Apply one drop of primary antibody 12.Incubate for 32 minutes 13.Adjust slide volume and apply LCS™ 14.Apply one drop of Biotinylated Ig 15.Incubate for 8 minutes 16.Rinse slide 17.Adjust slide volume and apply LCS™ 18.Apply one drop of Avidin-HRPO 19.Incubate for 8 minutes 20.Rinse slide 21.Adjust slide volume and apply LCS™ 22.Apply one drop of DAB and one drop DAB H2O2 23.Incubate for 8 minutes 24.Rinse slide 25.Adjust slide volume and apply LIQUID COVERSLIP™ 26.Apply one drop of Copper 27.Incubate for 4 minutes 28.Rinse slide The primary antibody used for the kidney biological sample was Anti-CD15 (Ventana Medical Systems, Inc. Tucson, Ariz., Catalogue no.\",\n \"250-2504).\",\n \"The primary antibody used for the tonsil biological sample was Anti-CD45RO (Ventana Medical Systems, Inc. Tucson, Ariz., Catalogue no.\",\n \"250-2563).\",\n \"The DAB staining kit used was obtained from Ventana Medical Systems, Inc. Tucson, Ariz., Catalogue no.\",\n \"250-001.Control biological samples were deparaffinized by a traditional solvent-based deparaffinization technique, as described in Example 1.After deparaffinization the biological samples were placed in a pressure cooker (Model #62104 Nordic Ware, Minneapolis, Minn.) containing 1.5 L 1× citrate buffer.\",\n \"The pressure cooker was then sealed and placed in a microwave oven (Model #MQSO836E, Matsushita, Franklin Park, Ill.).\",\n \"With the microwave oven set on “high,” the samples were subjected to microwave heating for approximately 30 minutes.\",\n \"After microwaving the samples were then “cured” for 30 minutes in the pressure cooker with the lid securely fastened.\",\n \"After curing the biological samples were placed in 1× citrate buffer for two minutes.\",\n \"The biological samples were then removed from the citrate buffer and the end of the slides blotted to removed excess citrate buffer.\",\n \"After blotting, the slides were placed on the automated instrument and immunohistochemically stained as described above.\",\n \"The biological samples deparaffinized by the solvent technique and by the automated exposing and simultaneous cell conditioning technique were compared after immunohistochemical staining.\",\n \"Morphology and staining on all sets of slides was acceptable and essentially equivalent.\",\n \"Example 3 Two Step Automated “Exposing” and “Cell Conditioning” Biological samples of tonsil and breast that had been preserved in paraffin and treated with formaldehyde were treated by the following protocol: Exposing and Cell Conditioning Protocol 1.Incubate for 2 minutes 2.Increase thermofoil temperature to 65.0° C. 3.Incubate for 6 minutes 4.Rinse slide and apply LCS™ 5.Incubate for 6 minutes 6.Rinse slide and apply LCS™ 7.Increase thermofoil temperature to 100.0° C. 8.Adjust slide volume and apply LCS™ 9.Rinse slide 10.Adjust slide volume and apply LCS™ 11.Incubate for 4 minutes 12.Adjust slide volume and apply LCS™ 13.Incubate for 4 minutes 14.Adjust slide volume and apply LCS™ 15.Incubate for 4 minutes 16.Adjust slide volume and apply LCS™ 17.Incubate for 4 minutes 18.Adjust slide volume and apply LCS™ 19.Incubate for 4 minutes 20.Adjust slide volume and apply LCS™ 21.Incubate for 4 minutes 22.Adjust slide volume and apply LCS™ 23.Incubate for 4 minutes 24.Adjust slide volume and apply LCS™ 25.Incubate for 4 minutes 26.Adjust slide volume and apply LCS™ 27.Incubate for 4 minutes 28.Rinse slide 29.Decrease temperature to 42.0° C. 30.Adjust slide volume and apply LCS™ 31.Incubate for 4 minutes 32.Rinse slide 33.Decrease temperature to 20.0° C. 34.Adjust slide volume and apply LCS™ 35.Incubate for 4 minutes 36.Rinse slide The buffer used in the protocol was SSC buffer with either 20% formamide or 0.1% Triton.\",\n \"After the biological sample was subjected to the above protocol, the DAB paraffin protocol used for immunohistochemical staining of Example 2 was applied.\",\n \"Tonsil biological sample was treated with anti-Ki67 as a primary antibody.\",\n \"Breast samples were treated with anti-estrogen receptor (6F11) or anti-progesterone receptor (1A6) as a primary antibody.\",\n \"All primary antibodies are available through Ventana.\",\n \"Control biological samples were deparaffinized by a traditional solvent-based de-paraffinization technique, as described in Example 1.After deparaffinization the biological samples were placed in a pressure cooker (Model #62104 Nordic Ware, Minneapolis, Minn.) containing 1.5 L 1× citrate buffer.\",\n \"The pressure cooker was then sealed and placed in a microwave oven (Model #MQSO836E, Matsushita, Franklin Park, Ill.).\",\n \"With the microwave oven set on “high”, the samples were subjected to microwave heating for approximately 30 minutes.\",\n \"After microwaving the samples were then “cured” for 30 minutes in the pressure cooker with the lid securely fastened.\",\n \"After curing the biological samples were placed in 1× citrate buffer for two minutes.\",\n \"The biological samples were then removed from the citrate buffer and the end of the slides were blotted to removed excess citrate buffer.\",\n \"After blotting the slide were placed on the automated instrument and immunohistochemically stained as described above.\",\n \"The biological samples deparaffinized by solvent technique and by the automated heating technique were compared after immunohistochemical staining.\",\n \"Morphology on all sets of slide was acceptable and essentially equivalent.\",\n \"Staining with Ki67 on all sets of slides was equivalent.\",\n \"ER (6F11) and PR (1A6) staining was slightly weaker with automated cell conditioning indicating the process does work but more development and optimization is required.\",\n \"Example 4 Automated Cell Conditioning of Non Paraffin Embedded Cell Lines for in situ hybridization Thin Preps™ Hela (ATCC lot # 980427H), Caski (ATCC lot # 980416C) and Siha (ATCC lot # 980416S) cell lines stored in Cytyk preparation solution (lot # 01139Q) were deposited on microscope slides using the Cytyk 2000 instrument.\",\n \"After deposition the slides were placed in alcohol to keep moist until use on the Discovery® In-Situ staining module (Ventana Medical Systems Inc., Tucson, Ariz.).\",\n \"Slides were loaded into the instrument and wetted with 2×SSC made from 20×SSC (Ventana P/N 650-012).\",\n \"Slides were run through a cell conditioning protocol currently referred to as Depar 30 where the slides are rinsed with 2×SSC and the temperature of the slides is increased to 95° C. for a period of approximately 30 minutes.\",\n \"The slides are then cooled to 37° C. and rinsed with APK Wash® prior to the in-situ staining run.\",\n \"Using the protocol Blue Swap ISH the cell lines were stained for HPV 16/18 (Enzo HPV 16/18 Bio Probe cat # 32874).\",\n \"Prior to probe application the cell lines are enzymatically digested with Protease 2 (Ventana P/N 250-2019).\",\n \"After the probe application the probe and biological sample are denatured simultaneously at 95° C. for 8 minutes.\",\n \"The non-specifically bound probe is washed off with stringency washes of 2×SSC at 55° C. The probe is then detected with Streptavidin Alk Phos and NBT/BCIP.\",\n \"The cell lines were dehydrated after staining with a one-minute exposure to 95% ethanol and a one-minute exposure to 100% ethanol repeated 2 times.\",\n \"Following the ethanol the slides were exposed to xylene for 3 minutes twice.\",\n \"After dehydration the slides were coverslipped.\",\n \"The stained cell lines after conditioning showed acceptable morphology, weak staining and there was high background on these slides indicating a need for the process to be developed more.\",\n \"Depar 30 Protocol Wet Load Slides 1.Skip Application & Incubate for 2 minutes 2.Rinse Slides (2×SSC Buffer) (Warm Slides to 65° C.) 3.Adjust Slide Volume, then apply LCS™ 4.Skip Application & Incubate 6 minutes 5.Rinse Slides (2×SSC Buffer) (Warm Slides to 95° C.) 6.Adjust Slide Volume, then Apply LCS™ 7.Rinse Slides 8.Adjust Slide Volume, then Apply LCS™ 9.Skip Application & Incubate for 4 minutes 10.Adjust Slide Volume, then Apply LCS™ 11.Skip Application & Incubate for 4 minutes 12.Adjust Slide Volume, then Apply LCS™ 13.Skip Application & Incubate for 4 minutes 14.Adjust Slide Volume, then Apply LCS™ 15.Skip Application & Incubate for 4 minutes 16.Adjust Slide Volume, then Apply LCS™ 17.Skip Application & Incubate for 4 minutes 18.Adjust Slide Volume, then Apply LCS™ 19.Skip Application & Incubate for 4 minutes 20.Adjust Slide Volume, then Apply LCS™ 21.Skip Application & Incubate for 4 minutes 22.Rinse Slides (2×SSC Buffer) (Warm Slides to 37° C.) 23.Adjust Slide Volume, then Apply LCS™ 24.Skip Application & Incubate for 4 minutes 25.Rinse Slides (APK Wash) 26.Adjust Slide Volume, then Apply LCS™ Example 5 Automated “Exposing” and “Cell Conditioning” for Single Copy DNA Detection Slides containing formalin fixed, paraffin embedded cell lines Caski (R96-1050A) and Siha (R96-96-C2), both generously provided by Dr. Raymond Tubbs, Cleveland Clinic Pathology Dept., Cleveland Ohio, were stained on Ventana target slides.\",\n \"Slides were dry loaded onto the instrument and the slide temperature was increased to 65° C. The Depar 30 protocol was run wherein the slides are rinsed with 2×SSC Buffer while at 65° C. then the heat is increased to 95° C. for about 40 minutes.\",\n \"The slides were then cooled to 37° C. and rinsed with APK wash. At this time the following in situ protocol was run: In situ Protocol: Tubbs 1 (Ventana APK Wash was used for all rinse steps) Protease Digestion: Protease 2, 4 minutes, 37° C. Inhibitor Step: Ventana Inhibitor from DAB kit 32 minutes 37° C. Probe: Enzo HPV Bio Probe 16/18 Control Probe: Enzo HPV Bio Probe 6/11 Denaturation: 95° C., 8 minutes Hybridization: 37° C., 64 minutes 2 Stringency Washes 2×SSC, 60° C., 8 minutes each 3rd Stringency Wash 2×SSC, 37° C., 4 minutes Probe Detection: Streptavidin HRPO (Dako GenPoint Cat.\",\n \"#K0620) Amplification: Biotinyl Tyramide (Dako GenPoint Cat.\",\n \"# K0620) Detection: Streptavidin HRPO (Dako GenPoint Cat.\",\n \"#K0620) or Streptavidin Alk Phos (Vector Cat.\",\n \"# SA5100) Chromogen DAB (Dako Gen Point Cat.\",\n \"# K0620) or Ventana NBT/BCIP (Kit P/N250-060) or Ventana Naphthol/Fast Red (Kit P/N250-030) Example 6 Automated “Cell Conditioning” for Non-paraffin Embedded Samples The protocol for DAB staining as described in Example 2 was used in this Example.\",\n \"The cell conditioning steps for these antibodies was done after using a Cytyk 2000® instrument to make ThinPreps® of cell lines.\",\n \"The ThinPreps® were stained using antibodies to ER, PgR, Ki67, P53 on Ventana ES instruments, NexES instruments and a manual procedure (Cytyk, Inc.).\",\n \"A duplicate group of slides have been stained on the NexES Insita module, allowing the cell conditioning steps to be performed by automation.\",\n \"Although the example stated above is specific to the Cytyk® instrument and staining of the ThinPreps®, the experience is not limited to that mode of making cytological preps.\",\n \"Example 7 “Cell conditioning” of Frozen Biological Sample Frozen tonsil blocks were prepared by cutting six sections from each block and placing the sample on microscope slides.\",\n \"Four slides from each block were placed on the Discovery™ in situ module and put through protocol Depar 10.Slides are dry heated to 65° for 6 minutes then rinsed with 0.1M EDTA buffer pH 8.After rinsing, the slide is incubated at 65° for 20 minutes.\",\n \"Slides were then cooled to 37° C. and rinsed with APK Wash. Two slides from each block were left untreated as controls.\",\n \"Following the Depar 10 treatment two treated slides from each block and one untreated slide were stained for H & E as described in Example 1.Two treated slides from each block and one untreated from each block are stained for LCA.\",\n \"Run outcomes: for both the H & E and antibody staining there was no staining difference between the treated and untreated slides.\",\n \"Example 8 Automated “Exposing” and “Cell Conditioning” for Immunohistochemistry Various antibodies were assayed in the NexES Plus® (Ventana Medical Systems, Inc., Tuscon, Ariz.) automated environment according to the following protocol and flow chart: Protocol: (1) ***** Select EZ Prep ***** (2) ***** Start Timed Steps ***** (3) If deparaffinization is selected (4) Warm slide to 75° C. and incubate for 4 minutes (5) Adjust volume (6) Apply LCS™ (7) Incubate for 8 minutes (8) Rinse slide (9) Adjust volume (10) Apply LCS™ (11) Warm slide to 42° C. and incubate for 2 minutes (12) If cell conditioning is selected (13) If conditioner #1 is selected (14) Rinse slide (15) Adjust slide volume (16) Apply LCS™ (17) Warm slide to 42° C. and incubate for 2 minutes (18) Apply cell conditioning coverslip (19) Warm slide to 100° C. and incubate for 2 minutes (20) Apply cell conditioner #1 (21) Apply LCS™ (22) If standard is selected (23) Incubate for 6 minutes (24) Apply cell conditioner #1 (25) Apply LCS™ (26) Repeat (21)-(23) 9 times (27) Warm slide to 42° C. and incubate for 2 minutes (28) If mild is selected (29) Incubate for 6 minutes (30) Apply cell conditioner #1 (31) Apply LCS™ (32) Repeat (27)-(29) 4 times (33) Warm slide to 42° C. and incubate for 2 minutes (34) If extended is selected (35) Incubate for 6 minutes (36) Apply cell conditioner #1 (37) Apply LCS™ (38) Repeat (33)-(35) 14 times (39) Warm slide to 42° C. and incubate for 2 minutes (40) If conditioner #1 is not selected (41) If conditioner #2 is selected (42) Rinse slide (43) Adjust slide volume (44) Apply LCS™ (45) Warm slide to 42° C. and incubate for 2 minutes (46) Apply cell conditioning coverslip (47) Warm slide to 100° C. and incubate for 2 minutes (48) Apply cell conditioner #2 (49) Apply LCS™ (50) If standard is selected (51) Incubate for 6 minutes (52) Apply cell conditioner #1 (53) Apply LCS™ (54) Repeat (49)-(51) 9 times (55) Warm slide to 42° C. and incubate for 2 minutes (56) If mild is selected (57) Incubate for 6 minutes (58) Apply cell conditioner #1 (59) Apply LCS™ (60) Repeat (55)-(57) 4 times (61) Warm slide to 42° C. and incubate for 2 minutes (62) If extended is selected (63) Incubate for 6 minutes (64) Apply cell conditioner #1 (65) Apply LCS™ (66) Repeat (61)-(63) 14 times (67) Warm slide to 42° C. and incubate for 2 minutes (68) Rinse slide (69) Adjust slide volume (70) Apply LCS™ (71) Disable slide heater (72) ***** Select Reaction Buffer ***** Table I summarizes the results from automated immunohistochemistry assays performed on the NexES Plus (also known as the BENCHMARK™ IHC) using the automated deparaffinization and cell conditioning protocols described herein.\",\n \"The slides from the automated immunohistochemistry assays were compared to slides prepared using manual deparaffinization/cell conditioning protocols on a NexES instrument.\",\n \"Specific antibody slides were prepared in triplicate.\",\n \"Negative control slides were prepared singly.\",\n \"Slides from both assays were analyzed by persons skilled in the art and assigned ratings for specific staining intensity and non-specific background staining intensity.\",\n \"The ratings for all of the slides from the NexES Plus fully automated assays exceeded the ratings for the manually deparaffinized and cell conditioned NexES slides in both specific staining intensity and non-specific background intensity.\",\n \"Slides were analyzed and rated by a minimum of two independent persons skilled in the art and average staining intensities were determined.\",\n \"The column marked “Qualified Tissue” in Table 1 represents the standard control tissue used to standardize the antibody used on it.\",\n \"The standardized antibody is given a rating of 4.0.As can be seen in the column marked “Average Staining Intensity,” most of the automated deparaffinized/cell conditioned slides met or exceeded those results.\",\n \"TABLE 1 Antibody and Cell Conditioning Results Incubation Average Qualified Time Negative Staining Negative Antibody Clone Tissue Pretreatment (minutes) Control Intensity Control anti-PR 1A6 Endo11299#2 60 min.\",\n \"cell conditioning 32 NC Ig 4 0 anti-ER 6F11 UT31698V 60 min.\",\n \"cell conditioning 32 NC Ig 4 0 anti-Pan Keratin AE1, AE3, PCK26 SK197A 4 min.\",\n \"Protease 1 16 NC Ig 4 0 anti-C-erbB-2 CB11 TMC197G 60 min.\",\n \"cell conditioning 32 NC Ig 4 0 anti-S100 polyclonal p19 None 16 Rbt NC 4 0 anti-CD20 L26 T1398H None 16 NC Ig 3.9 0 anti-Vimentin 3B4 245A 8 min.\",\n \"Protease 2 16 NC Ig 4 0 anti-LCA RP2/18 T31099B None 16 NC Ig 4 0 anti-Melanosome HMB45 97-715C None 16 NC Ig 4 0 anti-CEA TF-3H8-1 CCA98157 None 16 NC Ig 4 0 anti-CD45RO A6 T9810163 None 16 NC Ig 3.6 0 anti-Chromogratin LK2H10 pN None 16 NC Ig 4 0 anti-EMA Mc5 BRCA98139BC None 16 NC Ig 3.75 0 anti-Kappa Polyclonal T4282 4 min.\",\n \"Protease 1 16 Rbt NC 4 0 anti-Lambda Polyclonal 51099 4 min.\",\n \"Protease 1 16 Rbt NC 4 0 anti-CD15 MMA K121698 None 16 NC Ig 4 0 anti-Desmin DE-R-11 SI173BC 16 min Protease 2 16 NC Ig 4 0 anti-Muscle Actin HUC1-1 DIA8699A None 16 NC Ig 4 0 anti-PSA Polyclonal 271A None 16 Rbt NC 4 0 anti-Ki67 MM1 P92-3622 60 min.\",\n \"cell conditioning 16 NC Ig 3.9 0 anti-p53 bp-53-11 EV246B 60 min.\",\n \"cell conditioning 16 NC Ig 4 0 anti-Keratin 5D3 L103CB 8 min Protease 1 16 NC Ig 4 0 anti-NSE BBS/NC/VI-H14 #2 None 16 NC Ig 3.9 0 anti-bcl-2 bcl-2/100/D5 Block6 60 min.\",\n \"cell conditioning 32 NC Ig 4 0 anti-CD30 1G12 HodgkinsI912696C 60 min.\",\n \"cell conditioning 16 NC Ig 3.75 0 anti-CD43 L60 T11698H None 16 NC Ig 3.75 0 anti-GFAP Polyclonal CBR9872E None 16 Rbt NC 4 0 anti-Synaptophysin 27G12 406A ND 16 ND 4 0 NOTE: In Table 1, ND stands for No Data.\",\n \"The primary antibodies (available from Ventana Medical Systems, Inc., Tuscon, Ariz.) referred to in Table 1 (above) are described below, in alphabetical order: anti-bcl-2: Clone bcl-2/100/D5 is a monoclonal antibody used to detect bcl-2, which is an inhibitor of apoptosis.\",\n \"This antibody may be an aid to distinguish between reactive and neoplastic follicular proliferation.\",\n \"Control: Tonsil.\",\n \"(VMSI # 760-2693) anti-C-erbB-2 (HER2/neu): Clone CB11 is a monoclonal antibody to c-erb-B2, which is localized on the cell membrane, and occasionally, in the cytoplasm of some neoplastic cells.\",\n \"(VMSI # 760-2994) anti-CD15: Clone MMA is a monoclonal antibody used to aid in the identification of cells of the granulocytic lineage and/or Reed-Sternberg differentiation.\",\n \"Control: Hodgkin's Lymphoma.\",\n \"(VMSI # 760-2504) anti-CD20: Clone L26 is a monoclonal antibody used to aid in the identification of cells of the B lymphocytic lineage.\",\n \"Contol: Tonsil.\",\n \"(VMSI # 760-2531/760-2137) anti-CD43: Clone L60 is a monoclonal antibody that specifically binds to antigens located in the plasma membrane of normal granulocytes and T lymphocytes.\",\n \"Control: Tonsil.\",\n \"(VMSI # 760-2511) anti-CD45RA: Clone X148 is a monoclonal antibody that specifically binds to antigens located in the plasma membrane of normal B lymphocytes and a subset of T lymphocytes.\",\n \"Control: Tonsil.\",\n \"(VMSI # 76-2510) anti-CD45RO: Clone A6 is a monoclonal antibody that binds to the plasma membrane of cells of the T lineage and a subset of B-cells.\",\n \"Control: Tonsil.\",\n \"(VMSI # 760-2563) anti-CEA: Clone TF-3H8-1 specifically binds to antigens located in the plasma membrane and cytoplasmic regions of mucosal epithelial cells.\",\n \"Control: Colon Carcinoma.\",\n \"(VMSI # 760-2507/760-2141) anti-Chromogranin: Clone LK2H10 is a monoclonal antibody that binds the chromogranin protein located in the secretory granules of normal and neoplastic neuroendocrine cells.\",\n \"Control: Pancreas.\",\n \"(VMSI # 760-2519/760-2140) anti-Desmin: Clone DE-R-11 is a monoclonal antibody used to aid in the identification of cells of the myocytic lineage.\",\n \"Desmin is an intermediate filament found in mature smooth, striated and cardiac muscle.\",\n \"Control: Vas Deferens.\",\n \"(VMSI # 760-2513) anti-EGFR: Clone 31G7 is directed against a transmembrane glycoprotein present on a variety of cells.\",\n \"(VMSI # 760-2548) anti-EMA: Clone Mc5 is a monoclonal antibody used to aid in the identification of cells of epithelial lineage.\",\n \"EMA is of value in distinguishing both large-cell anaplastic carcinoma from diffuse histiocytic lymphoma, and small-cell anaplastic carcinoma from well and poorly differentiated lymphocytic lymphomas.\",\n \"Control: Carcinoma.\",\n \"(VMSI # 760-2508) anti-ER: Clone 6F11 is a monoclonal antibody used to detect the presence of estrogen receptor.\",\n \"(VMSI # 760-2596/760-2132) Clone CC4-5 is a monoclonal antibody also used to detect the presence of estrogen receptor.\",\n \"(VMSI #760-2546/760-2138) anti-GFAP: GFAP polyclonal antibody is directed against glial fibrillary acidic protein present in the cytoplasm of most human astrocytes and ependymal cells.\",\n \"This reagent may be used to aid in the identification of cells of the glial lineage.\",\n \"Control: Brain.\",\n \"(VMSI # 760-2516) anti-Kappa: Kappa light chains are expressed by cells of the B-cell lineage.\",\n \"Light chain production by lymphoid cells is genetically restricted such that the immunoglobulin molecules produced by an individual cell will only contain a single light chain class.\",\n \"This clonal restriction may be used to indicate the polyclonal or monoclonal nature of B-cell and plasma cell populations.\",\n \"Control: Plasmacytoma.\",\n \"(VMSI # 760-2514) anti-Keratin: Clone 5D3 is a monoclonal antibody raised against human epidermal keratins.\",\n \"This antibody may be used to aid in the identification of cells of the epithelial lineage.\",\n \"This antibody reacts with cytokeratins 8 and 18.Control: Carcinoma.\",\n \"(VMSI # 760-2501) Clone AE1 is a monoclonal antibody raised against human epidermal keratins.\",\n \"This antibody may be used to aid in the identification of cells of the ductal epithelial lineage.\",\n \"AE1 reacts with cytokeratins 10, 13, 14, 15 and 19.Control: Liver.\",\n \"(VMSI # 760-2521) anti-Pan Keratin: Pan Keratin is a cocktail of monoclonal antibody clones AE1/AE3/PCK26 used to aid in the identification of cells of the epithelial lineage.\",\n \"This antibody cocktail reacts with all cytokeratins except 9, 11 and 12.Control: Skin.\",\n \"(VMSI # 760-2595/760-2135) anti-Ki67: Clone MM1 is a monoclonal antibody that specifically binds to nuclear antigens associated with cell proliferation that are present throughout the active cell cycle (G1, S, G2 and M phases), but absent in resting (G0 phase) cells.\",\n \"This reagent may be used to aid in the identification of proliferating cells.\",\n \"Control: Tonsil.\",\n \"(VMSI # 760-2520) anti-Lambda: Lambda light chains are expressed by cells of the B-cell lineage.\",\n \"Light chain production by lymphoid cells is genetically restricted, such that the immunoglobulin molecules produced by an individual cell will only contain a single light chain class.\",\n \"This clonal restriction may be an aid to indicate the polyclonal or monoclonal nature of B-cell and plasma cell populations.\",\n \"Control: Plasmacytoma.\",\n \"(VMSI # 760-2515) anti-LCA: Clone RP2/18 is a is a monoclonal antibody used to aid in the identification of cells of lymphocytic descent.\",\n \"This antibody specifically binds to antigens located predominantly in the plasma membrane and cytoplasmic rim of lymphocytes with variable reactivity to monocytes/histocytes and polymorphs.\",\n \"Control: Lymphoma.\",\n \"(VMSI # 760-2505/760-2136) anti-Melanosome: Clone HMB45 monoclonal antibody is used to aid in the identification of cells of the melanocytic lineage.\",\n \"It reacts with an antigen expressed in abnormal melanocytes and melanoma cells.\",\n \"Control: Melanoma.\",\n \"(VMSI # 760-2518M60-2139) anti-Muscle Actin: Clone HUC1-1 is a monoclonal antibody used to aid in the identification of cells of myocytic descent.\",\n \"Muscle actin is expressed in cells of the striated, smooth and cardiac muscle lineage.\",\n \"Control: Ileum.\",\n \"(VMSI # 760-2502) anti-NSE: Clone BBS/NC/VI-H14 is a monoclonal antibody that reacts with an antigen that may be expressed in the cytoplasm of neurons, neuro-endocrine cells, endocrine tumors, carcinoids and Merkel cell tumors.\",\n \"Control: Pancreas.\",\n \"(VMSI # 76-2517) anti-p53: Clone Bp-53-11 is a monoclonal antibody directed against the p53 protein.\",\n \"This reagent may be used to aid in the identification of abnormally proliferating cells in neoplastic cell populations.\",\n \"Control: Carcinoma.\",\n \"(VMSI # 760-2540) anti-PCNA: Clone PC10 is a monoclonal antibody that may be used to aid in the identification of proliferating cells in various cell populations.\",\n \"The PCNA antigen is expressed in all proliferating cells in the G1, S, G2 and M phases.\",\n \"Control: Tonsil.\",\n \"(VMSI # 760-2503) anti-PSA: PSA polyclonal antibody reacts with the secretory protein expressed in the cytoplasm of prostate epithelial cells.\",\n \"Control: Prostate.\",\n \"(VMSI # 760-2506) anti-PSAP: Clone PASE/4LJ is a monoclonal antibody used to aid in the identification of cells of the prostate lineage.\",\n \"PSAP specifically binds to antigens located in the cytoplasmic regions of the normal prostate epithelial cells.\",\n \"Control: Prostate.\",\n \"(VMSI # 760-2509) anti-PR: Clone 1A6 is a monoclonal antibody used to aid in the identification of the progesterone receptor in human tissue.\",\n \"(VMSI # 760-2547/760-2133) anti-S100: S100 polyclonal antibody is used to aid in the identification of cells of normal and abnormal neuro-endocrine descent.\",\n \"Control: Skin.\",\n \"(VMSI # 760-2523/760-2133) anti-Vimentin: Clone 3B4 is used to aid in the identification of cells of mesenchymal origin.\",\n \"Control: Vas Deferens.\",\n \"(VMSI # 760-2512/760-2134) Negative Control: Polyclonal serum applied to negative tissue controls as part of quality control procedures for polyclonal antibodies.\",\n \"(VMSI # 760-1023) Negative Control Ig: Clone MOPC-21 is applied to negative tissue controls as part of quality control procedures for monoclonal antibodies.\",\n \"(VMSI # 760-2014) Example 9 Automated “Cell Conditioning” with or without “Exposing” for Immunohistochemistry (IHC), Immunocytochemistry (ICC) or In situ Hybribidization (ISH) The CC2 solution may be used on automated immunohistochemical, immunocytochemical or nz situ hybridization instruments (e.g., BenchMark™ ISH, Ventana Medical Systems, Inc., Tucson, Ariz.; BenchMark™ IHC, Ventana Medical Systems, Inc., Tucson, Ariz.; etc.)\",\n \"with reagents designed to detect the c-erb2/HER-2/neu gene or protein(s) expressed therefrom (e.g., INFORM® HER-2/neu DNA test, Ventana Medical Systems, Inc., Tucson, Ariz.; PATHWAY™ HER-2/neu (CB11) protein test, Ventana Medical Systems, Inc., Tucson, Ariz.; etc.).\",\n \"For an ISH assay, the CC2 solution may preferably be used in an automated pretreatment step following deparaffinization with the CC2 solution applied to the tissue at about 90° C. for about 10 minutes prior to application of the DNA probe.\",\n \"The heat treatment in conjunction with the CC2 solution allows the genomic DNA to become better suited for binding with the c-erb2/HER-2/neu DNA probe.\",\n \"For an IHC assay, the CC2 solution may preferably be used in an automated cell conditioning step following deparaffinization with the CC2 solution applied to the tissue at about 95° C. for about 30 minutes.\",\n \"The high temperature in conjunction with the CC2 solution allows the c-erb2/HER-2/neu protein (antigen or epitope) to become better suited for binding with the anti-c-erb2/HER-2/neu antibody.\",\n \"These are representative contexts in which the CC2 solution is preferably used.\",\n \"The CC2 solution is also useful in many other assays, techniques, protocols or procedures designed to detect the presence or absence of targeted.\",\n \"DNA or protein molecules under consideration.\",\n \"From the foregoing detailed description, it will be appreciated that numerous changes and modifications can be made to the aspects of the invention without departure from the true spirit and scope of the invention.\",\n \"This true spirit and scope of the invention is defined by the appended claims, to be interpreted in light of the foregoing specification.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471126"}}},{"rowIdx":577,"cells":{"text":{"kind":"list like","value":[["Tightening device for cables and similar","The aim of the invention is to produce a tightening device for cables or similar for controlling the transmission of force.","Said device comprises a clamping disk (6) and a deflector (10) engaging therebetween.","As a result, the cable (4) is guided between the deflector (10) and a guiding element (20) arranged on the periphery of the clamping disk (6)."],["1.A tightening apparatus for cables, comprising a clamping disk and a deflector engaging there between, characterized in that the cable is guided between the deflector and guiding element arranged on the circumference of the clamping disk, wherein the tightening apparatus is arranged at the base of a mast of a windsurfing sail, and wherein the guiding element is provided with a pipe-like or tubular configuration in which the loose part of the cable is guided.","2.A tightening apparatus as claimed in claim 1, characterized in that the guiding element is arranged in a fixed way, especially in the form of a spring steel tongue.","3.A tightening apparatus as claimed in claim 1, characterized in that the guiding element is arranged in a movable way.","4.A tightening apparatus as claimed in claim 3, characterized in that the guiding element is arranged in a swivelable way on a lever.","5.A tightening apparatus as claimed in claim 1, characterized in that the guiding element is arranged as a roller.","6.A tightening apparatus as claimed in claim 1, characterized in that the guiding element engages between wedge surfaces of the clamping disk and acts on the loose part of the cable.","7.","(canceled) 8.A tightening apparatus as claimed in claim 1, characterized in that a ratchet lever, a crank or an electric drive is provided for driving the clamping disk.","9.A tightening apparatus as claimed in claim 1, characterized in that the clamping disk is provided on its wedge surfaces with a profiling.","10.","(canceled)"],["A tightening device for cables and the like have always played an important role in easing the workload for humanity.","Cable winches and pulley blocks should be mentioned in particular.","Cable winches wind up a cable and are of large design and have a high weight depending on the cable length to be wound up.","Although pulley blocks do not wind up cables, they also have a high weight through several deflection rollers and are of large design.","In many areas where cables are currently used there is a desire for a tightening apparatus with minute dimensions and compact size (e.g.","in the construction industry, sports climbing, emergency services, shipping, windsurfing and sailing sports).","A tightening apparatus is known from DE 2500731 A1 for ropes, cables or wires, wherein a clamping disk with mutually opposite conical surfaces is described into which a deflector projects.","The clamping disk can be driven by means of a crank and can be provided with profilings for the purpose of increasing the frictional resistance.","The tightening apparatus is to be especially suitable as a winch for sailing yachts in order to allow a rapid tightening of a rope or cable.","A controlled loosening of the cable (e.g.","a rewind) is not possible in this case because the cable would detach from the clamping disk.","The invention is therefore based on the object of providing a tightening apparatus for cables and the like which allows a controlled transmission of force and a small, compact design.","This object is achieved by the features as mentioned in claim 1.Further advantageous embodiments of the invention are stated in the dependent claims.","The invention ensures that cables are tightened and relieved in a defined manner as in cable winches.","However, the cable is not wound up.","The cable is merely threaded into the clamping disk and tightens the cable via the clamping disk until the cable is tightened.","Once the tensile force becomes too high, a drive of the clamping disk is used, e.g.","a lever apparatus.","Depending on the lever length it is possible to transmit forces which far exceed the weight of the exerting person, For example, a climber hanging from the cable can lever himself upwardly all by himself.","As a result of the guiding element which is provided in accordance with the invention which is substantially opposite to the deflector on the circumference of the clamping disk, a secure wrap is ensured even during the reverse motion of the cable, so that the loose end of the cable or the like is guided into the clamping disk.","As a result, a tightening and relaxing is possible in both rotational directions of the tightening apparatus, e.g.","for the tightening of a windsurfing sail which when necessary can be slightly slackened for the purpose of adjustment to the respective wind conditions for example and can later be tightened again for adjustment to the wind conditions.","The cable, rope or rope end is held securely in the clamping disk by the guiding element.","The guiding element can be fixedly arranged on the circumference of the clamping disk, e.g.","in the form of a spring steel tongue aligned in the direction towards the center of the clamping disk.","The guiding element can also be provided with a movable arrangement, especially swivelable on a lever.","In order to adapt as precisely as possible to the course of the loose end of the cable the guiding element is arranged as a roller which can also contribute to the reduction of the wear and tear on the cable.","For this purpose the guiding element can also engage on the circumference of the clamping disk between the wedge surfaces and act upon the loose end of the cable.","The guiding element can also be provided with a tubular or loop-like shape.","In the possible application on the base of a mast of a windsurfing sail the guiding element can also be arranged within the base of the mast, thus leading to an especially compact design.","The scope of application can be extended by using different drives for the clamping disk.","Depending on the respective application, the drive of the clamping disk can be provided by a ratchet mechanism, a crank or other electric or mechanical drive.","A preferably provided ribbing or profiling of the clamping disk leads to the advantage that the cable is grasped tightly during the tightening and allows an easy rolling off of the cable end.","The security of the transmission of force is thus increased.","Moreover, the cable is treated with care and its life is increased.","An embodiment is now explained by reference to the enclosed drawings, wherein: FIG.","1 shows a tightening apparatus with load; FIG.","2 shows a partial side view in the plane of the clamping disk which illustrates the course of the cable, and FIG.","3 shows a partial side view in the plane of the ratchet drive which explains the lever mechanism.","FIG.","1 shows an example of a tightening apparatus.","The tightening apparatus is fastened to a holding device 2 with an eye 15, e.g.","to a climbing harness.","The eye 15 is preferably arranged in the same vertical plane as the course of the cable in order to avoid any moments of tilt.","For this purpose the holding device 2 is bent in the region of eye 15 relative to the clamping disk 6.The cable 4 is fastened to a load 1 and is guided on a clamping disk 6.In this way the load 1 is fixed to the holding device 2.The holding device 2 comprises a shaft 3.In addition to the clamping disk 6, a ratchet toothed disk 7 and a lever 5 are mounted on the shaft 3 (cf.","FIG.","3).","A ratchet element 8 is fixed on a lever 5 and a latching element 9 is held on the holding device 2.FIG.","2 clearly shows the course of the cable.","The load 1 is fastened to the cable 4.The cable 4 is guided on the clamping disk 6.The cable 4 is wedged by the tensile force at the loose end on the clamping disk 6.The cable 4 is wound up by rotation of the clamping disk 6.After approx.","300° the deflector 10 unwedges the cable 4.The cable 4 is thus rolled off and becomes loose.","The wedging surfaces on the clamping disk 6 are configured in such a way that the cable 4 will clamp securely depending on the coefficient of friction and allows unwinding without damaging the cable.","The relevant aspect is the arrangement of a guiding element 20 on the circumference of the clamping disk 6, so that the guiding element 20 is substantially opposite to the deflector 10.The guiding element 20 can be arranged fixedly, especially in the manner of a tongue as is indicated in the lower region of the holding device 2.The guiding element 20 can also be arranged movably on a lever 21.In this way the guiding element 20 can thus even engage between the wedge surfaces of the clamping disk 6 and conform especially precisely to the course of the cable.","Preferably, the lever 21 carries a roller 22 in the corner region, which roller rests on the loose end of the cable 4.In this way it is possible to achieve a precise pressing on and clinging to the course of the cable.","The guiding element 20 which is shown here in two alternatives (fixed and swivelably movable) can also be provided with a pipe-like or tubular arrangement.","In this case the loose end of the cable 4 would extend in said guiding element 20.The guiding element 20 lies substantially opposite of the deflector 10, so that the loose end of the cable 4 is guided precisely between said components 10 and 20.As indicated above, the term “cable” shall also relate to wires, ropes and the like, especially to rope ends used in sailing sports.","FIG.","3 shows a type of a drive for the clamping disk 6, which in this case is lever mechanism.","The said clamping disk 6 is fixedly connected with the ratchet toothed wheel 7.The clamping disk 6 is blocked in the left direction (counter-clockwise) by the ratchet element 9 with a spring 13 and can only be moved in the right direction (clockwise).","The transmission of force of the lever 5 onto the ratchet toothed wheel 7 occurs via the ratchet element 8 which is also pressed by a spring.","In the right direction the transmission of force occurs for tightening, whereas in the left direction the cable 4 can be loosened or relaxed in a defined manner in order to be brought optionally into the initial position again with the lever 5 for the purpose of renewed tightening.","The relevant aspect is that a defined tightening and relaxing of the cable 4 is possible in stages, as is desirable by the respective user.","As a result of the guiding element as proposed herein it is securely prevented that the cable 4 can detach in an uncontrolled way from the clamping disk 6.Rather, the wrap is definitely ensured because during the reverse motion of the cable the loose end is guided by the guiding element 20 into the clamping disk 6.As a result of the movable configuration of the guiding element 20, especially on a lever and/or in the form of a roller, a cling or pressing to the course of the cable is achieved, so that the wear and tear of the cable and the frictional losses during the drive are reduced."]],"string":"[\n [\n \"Tightening device for cables and similar\",\n \"The aim of the invention is to produce a tightening device for cables or similar for controlling the transmission of force.\",\n \"Said device comprises a clamping disk (6) and a deflector (10) engaging therebetween.\",\n \"As a result, the cable (4) is guided between the deflector (10) and a guiding element (20) arranged on the periphery of the clamping disk (6).\"\n ],\n [\n \"1.A tightening apparatus for cables, comprising a clamping disk and a deflector engaging there between, characterized in that the cable is guided between the deflector and guiding element arranged on the circumference of the clamping disk, wherein the tightening apparatus is arranged at the base of a mast of a windsurfing sail, and wherein the guiding element is provided with a pipe-like or tubular configuration in which the loose part of the cable is guided.\",\n \"2.A tightening apparatus as claimed in claim 1, characterized in that the guiding element is arranged in a fixed way, especially in the form of a spring steel tongue.\",\n \"3.A tightening apparatus as claimed in claim 1, characterized in that the guiding element is arranged in a movable way.\",\n \"4.A tightening apparatus as claimed in claim 3, characterized in that the guiding element is arranged in a swivelable way on a lever.\",\n \"5.A tightening apparatus as claimed in claim 1, characterized in that the guiding element is arranged as a roller.\",\n \"6.A tightening apparatus as claimed in claim 1, characterized in that the guiding element engages between wedge surfaces of the clamping disk and acts on the loose part of the cable.\",\n \"7.\",\n \"(canceled) 8.A tightening apparatus as claimed in claim 1, characterized in that a ratchet lever, a crank or an electric drive is provided for driving the clamping disk.\",\n \"9.A tightening apparatus as claimed in claim 1, characterized in that the clamping disk is provided on its wedge surfaces with a profiling.\",\n \"10.\",\n \"(canceled)\"\n ],\n [\n \"A tightening device for cables and the like have always played an important role in easing the workload for humanity.\",\n \"Cable winches and pulley blocks should be mentioned in particular.\",\n \"Cable winches wind up a cable and are of large design and have a high weight depending on the cable length to be wound up.\",\n \"Although pulley blocks do not wind up cables, they also have a high weight through several deflection rollers and are of large design.\",\n \"In many areas where cables are currently used there is a desire for a tightening apparatus with minute dimensions and compact size (e.g.\",\n \"in the construction industry, sports climbing, emergency services, shipping, windsurfing and sailing sports).\",\n \"A tightening apparatus is known from DE 2500731 A1 for ropes, cables or wires, wherein a clamping disk with mutually opposite conical surfaces is described into which a deflector projects.\",\n \"The clamping disk can be driven by means of a crank and can be provided with profilings for the purpose of increasing the frictional resistance.\",\n \"The tightening apparatus is to be especially suitable as a winch for sailing yachts in order to allow a rapid tightening of a rope or cable.\",\n \"A controlled loosening of the cable (e.g.\",\n \"a rewind) is not possible in this case because the cable would detach from the clamping disk.\",\n \"The invention is therefore based on the object of providing a tightening apparatus for cables and the like which allows a controlled transmission of force and a small, compact design.\",\n \"This object is achieved by the features as mentioned in claim 1.Further advantageous embodiments of the invention are stated in the dependent claims.\",\n \"The invention ensures that cables are tightened and relieved in a defined manner as in cable winches.\",\n \"However, the cable is not wound up.\",\n \"The cable is merely threaded into the clamping disk and tightens the cable via the clamping disk until the cable is tightened.\",\n \"Once the tensile force becomes too high, a drive of the clamping disk is used, e.g.\",\n \"a lever apparatus.\",\n \"Depending on the lever length it is possible to transmit forces which far exceed the weight of the exerting person, For example, a climber hanging from the cable can lever himself upwardly all by himself.\",\n \"As a result of the guiding element which is provided in accordance with the invention which is substantially opposite to the deflector on the circumference of the clamping disk, a secure wrap is ensured even during the reverse motion of the cable, so that the loose end of the cable or the like is guided into the clamping disk.\",\n \"As a result, a tightening and relaxing is possible in both rotational directions of the tightening apparatus, e.g.\",\n \"for the tightening of a windsurfing sail which when necessary can be slightly slackened for the purpose of adjustment to the respective wind conditions for example and can later be tightened again for adjustment to the wind conditions.\",\n \"The cable, rope or rope end is held securely in the clamping disk by the guiding element.\",\n \"The guiding element can be fixedly arranged on the circumference of the clamping disk, e.g.\",\n \"in the form of a spring steel tongue aligned in the direction towards the center of the clamping disk.\",\n \"The guiding element can also be provided with a movable arrangement, especially swivelable on a lever.\",\n \"In order to adapt as precisely as possible to the course of the loose end of the cable the guiding element is arranged as a roller which can also contribute to the reduction of the wear and tear on the cable.\",\n \"For this purpose the guiding element can also engage on the circumference of the clamping disk between the wedge surfaces and act upon the loose end of the cable.\",\n \"The guiding element can also be provided with a tubular or loop-like shape.\",\n \"In the possible application on the base of a mast of a windsurfing sail the guiding element can also be arranged within the base of the mast, thus leading to an especially compact design.\",\n \"The scope of application can be extended by using different drives for the clamping disk.\",\n \"Depending on the respective application, the drive of the clamping disk can be provided by a ratchet mechanism, a crank or other electric or mechanical drive.\",\n \"A preferably provided ribbing or profiling of the clamping disk leads to the advantage that the cable is grasped tightly during the tightening and allows an easy rolling off of the cable end.\",\n \"The security of the transmission of force is thus increased.\",\n \"Moreover, the cable is treated with care and its life is increased.\",\n \"An embodiment is now explained by reference to the enclosed drawings, wherein: FIG.\",\n \"1 shows a tightening apparatus with load; FIG.\",\n \"2 shows a partial side view in the plane of the clamping disk which illustrates the course of the cable, and FIG.\",\n \"3 shows a partial side view in the plane of the ratchet drive which explains the lever mechanism.\",\n \"FIG.\",\n \"1 shows an example of a tightening apparatus.\",\n \"The tightening apparatus is fastened to a holding device 2 with an eye 15, e.g.\",\n \"to a climbing harness.\",\n \"The eye 15 is preferably arranged in the same vertical plane as the course of the cable in order to avoid any moments of tilt.\",\n \"For this purpose the holding device 2 is bent in the region of eye 15 relative to the clamping disk 6.The cable 4 is fastened to a load 1 and is guided on a clamping disk 6.In this way the load 1 is fixed to the holding device 2.The holding device 2 comprises a shaft 3.In addition to the clamping disk 6, a ratchet toothed disk 7 and a lever 5 are mounted on the shaft 3 (cf.\",\n \"FIG.\",\n \"3).\",\n \"A ratchet element 8 is fixed on a lever 5 and a latching element 9 is held on the holding device 2.FIG.\",\n \"2 clearly shows the course of the cable.\",\n \"The load 1 is fastened to the cable 4.The cable 4 is guided on the clamping disk 6.The cable 4 is wedged by the tensile force at the loose end on the clamping disk 6.The cable 4 is wound up by rotation of the clamping disk 6.After approx.\",\n \"300° the deflector 10 unwedges the cable 4.The cable 4 is thus rolled off and becomes loose.\",\n \"The wedging surfaces on the clamping disk 6 are configured in such a way that the cable 4 will clamp securely depending on the coefficient of friction and allows unwinding without damaging the cable.\",\n \"The relevant aspect is the arrangement of a guiding element 20 on the circumference of the clamping disk 6, so that the guiding element 20 is substantially opposite to the deflector 10.The guiding element 20 can be arranged fixedly, especially in the manner of a tongue as is indicated in the lower region of the holding device 2.The guiding element 20 can also be arranged movably on a lever 21.In this way the guiding element 20 can thus even engage between the wedge surfaces of the clamping disk 6 and conform especially precisely to the course of the cable.\",\n \"Preferably, the lever 21 carries a roller 22 in the corner region, which roller rests on the loose end of the cable 4.In this way it is possible to achieve a precise pressing on and clinging to the course of the cable.\",\n \"The guiding element 20 which is shown here in two alternatives (fixed and swivelably movable) can also be provided with a pipe-like or tubular arrangement.\",\n \"In this case the loose end of the cable 4 would extend in said guiding element 20.The guiding element 20 lies substantially opposite of the deflector 10, so that the loose end of the cable 4 is guided precisely between said components 10 and 20.As indicated above, the term “cable” shall also relate to wires, ropes and the like, especially to rope ends used in sailing sports.\",\n \"FIG.\",\n \"3 shows a type of a drive for the clamping disk 6, which in this case is lever mechanism.\",\n \"The said clamping disk 6 is fixedly connected with the ratchet toothed wheel 7.The clamping disk 6 is blocked in the left direction (counter-clockwise) by the ratchet element 9 with a spring 13 and can only be moved in the right direction (clockwise).\",\n \"The transmission of force of the lever 5 onto the ratchet toothed wheel 7 occurs via the ratchet element 8 which is also pressed by a spring.\",\n \"In the right direction the transmission of force occurs for tightening, whereas in the left direction the cable 4 can be loosened or relaxed in a defined manner in order to be brought optionally into the initial position again with the lever 5 for the purpose of renewed tightening.\",\n \"The relevant aspect is that a defined tightening and relaxing of the cable 4 is possible in stages, as is desirable by the respective user.\",\n \"As a result of the guiding element as proposed herein it is securely prevented that the cable 4 can detach in an uncontrolled way from the clamping disk 6.Rather, the wrap is definitely ensured because during the reverse motion of the cable the loose end is guided by the guiding element 20 into the clamping disk 6.As a result of the movable configuration of the guiding element 20, especially on a lever and/or in the form of a roller, a cling or pressing to the course of the cable is achieved, so that the wear and tear of the cable and the frictional losses during the drive are reduced.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471143"}}},{"rowIdx":578,"cells":{"text":{"kind":"list like","value":[["Quantitative analysis, visualization and movement correction in dynamic processes","The invention relates to the quantitative analysis and/or visualization (virtual or real) of moving processes and to the detection, description, correction and the comparison of global movements inside the image space.","The invention particularly relates to a method and device for precisely and, while being limited to few parameters, quantitatively describing the global and local movement occurring in the image space and for the single representation of the movement of the quantitative parameters with regard to the image space.","A rough to fine recording and a detection of and compensation for global movements occurs whereby enabling a tracking and a corrected visualization.","The detection of the movement, which corresponds as precisely as possible to the real movement occurring in the image space is effected by a splines a plaques minces recording technique that enables the number of movement parameters to be kept low.","A fully automatic execution of all partial steps is made possible."],["1.A method for the quantitative and visual analysis of motion in a time sequence of images comprising the steps of: a) determination of the motion vectors for each point in the image space between images of directly or not directly subsequent time steps (i.e., the transformation of the corresponding image space) preferably by means of rigid, affine and/or “elastic” (including local deformations) overlapping/registration of images of an image sequence and calculation of the transformation parameter comprising the following further method steps: a1) for the non-rigid registration, local transformation types are given stepwise from coarse to fine, first global and then increasingly more local; a2) for more then two time steps, in order to obtain the transformation for not directly subsequent timesteps, the transformation is not only calculated in pairs for subsequent image pairs, but also a correction-registration step is further added thereto; a3) from the transformation result directly the local, defined for each point in the image/time space, quantities velocity, acceleration, warp, tension, deviation from the finest registration (i.e., distance between the point transformed by the selected transformation type and by the most local fineness level), etc.","as well as the global quantities warp energy for the entire motion as well as for each “principal warp” separately (Bookstein 1989), entropy, “directedness” of the motion and the transformation parameters for various fineness levels of the transformation; b) reconstruction of the space-time-structure within the image space and visualization thereof by means of b1) (optionally interactive) determination of a point (or a region; or according to a regular scheme selected point in the space or on the surface of a region) for a selectable moment, the automatic calculation of its (their) position for all other moments by means of the under a) calculated transformation (for not directly subsequent time steps) as well as the interpolated representation as 3D rendered (surface rendering) path (or “tube” for regions); and/or b2) a 3D rendered reference grid, deformed according to the transformation calculated in a), animated (interpolation of intermediate time steps) or not (animation also continuously over several time steps), the image space overlapped (inserted) or not; in an image record having several color channels, the reference grids corresponding to the various separately treated color channels can be represented in different color but simultaneously; and/or b3) color/pattern encoding (including gray value encoding) of quantities (quantities having vector values by means of absolute values), which are assigned to a point in the image/time space as described under a), particularly for (i) all points, lying within an interactively selectable level of the image space, (ii) interactively (or regularly) selected points or surface points of interactively selected regions/shapes/surfaces (iii) the points of the under b1) determined path (or tubes) of the time space; and/or b4) a motion corrected representation of the paths (or tubes), corrected by a rigid, affine or a selected level of the non-global transformation, or by means of a motion corrected playing of the original image sequence, particularly the color encoding can be back projected onto the original image or any other given moment.","2.The method according to claim 1, which extracts in a pre-processing step a0) critical (characteristic) points of the image space or surface points of regions in the image space and uses these in the steps b1), b3) and b4) instead of interactively selected points, whereby in step b2) the (critical/surface) points of the original space can be used instead of the reference grid.","3.The method according to claim 2, which uses for the preprocessing the confinement tree technique (CTT), whereby a) for the visualization of a region (a confiner), the user clicks either on knot in the tree or on a point in the image, upon which the surface/shape of the smallest confiners (in the filtered tree) containing said point will be shown; and/or b) the registration is performed on the basis of the extracted points (confiner-surface points or critical points, e.g., confiner center points) by means of a point registration algorithm, whereby confiners corresponding to each other are pre-determined in the original and target image or an algorithm based on the minimization of an “iconic” similarity measure follow: and/or c) transformed confiner of the original image overlapped with non-transformed confiners of the target image can be represented whereby the confiner surfaces replace the parts of the reference grid in 1b2), and to the quantities from 1a3) the distance from one point to the next adjacent point extracted in the other image is added.","4.A method for registration by means of CTT, which either a) uses confiner center points (CS) or confiner density maxima (CD) as critical points for registration and by using the method of structural outlier determination (by means of relPos, see Mattes and Demongeot 2001) starting from an initial overlapping (optionally improved by a direct use of a point registration method, see page 6) ) first determines corresponding confiner pairs and thereby only maintains pairs with best relPos (optionally according to 6(i)), on the basis of this (CD, CS or confiner shapes of which) determines the best fitting (in the sense of the error functional, see 6) as well as Fieres et al.","2001) transformation, exerts on the data confiner and repeats this process iteratively, whereby the such determined landmarks can be used for the establishment of a “point distribution model”; or b) corresponding confiners are determined as follows: for the knots, which occur after a bifurcation in the CT of one of the images, the knots/confiner having the lowest relPos value in the CT of the other image are searched, the point outliers are thereby suppressed as in claim 6, then the relPos distribution for all confiner pairs are examined, and the confiner outlier are eliminated temporarily as described in 6)(i) for point outliers, finally a point registration method under consideration of corresponding confiners are used, by an optional usage of first highly cut-down CTs and then increasingly less highly cut-down CTs a coarse to fine effect can be achieved in a) and b), which can also be combined with/replaced by the following method(s) and the images are first highly (i.e., on great scales), (e.g., Gauss-) smoothed (or presented in a high pixel resolution), and in the proceeding of the method increasingly less highly smoothed (or represented in increasingly finer resolution).","5.The method according to claim 3 using the CTT as in claim 4.6.The method according to claim 3, which instead of the pre-processing by means of CTT identifies objects in the image space in a segmentation step and calculates the surface and/or center point of which, the objects replace the confiners, in case only (non-coherent) points are identified on the object surface (e.g., by means of a Canny edge-detector), corresponding confiner/objects cannot be pre-determined according to 3b) and particularly for the segmented objects can also individual translations and rotation or deformations be determined and the object paths can individually corrected by selected transformation types.","7.A method for the registration of a data point (DP) set on a reference point (RP) set, which determines iteratively for a series of given transformation types, which allow an increasing number of local deformations, its free parameters in such a manner, that an error functional is minimized, which compares the positions of the transformed data points with those of the reference points, thereby selecting as transformation types first a rigid (rotation and translation), then an affine transformation and then transformation types, defined by a number of checkpoints, which can freely be displaced (the free parameter of the transformation) and between the displacement vectors of which with thin-plate splines is interpolated (Bookstein 1989) and, the number of checkpoints is iteratively increased (in order to allow an increasing number of local deformation), and the initial position of the checkpoints is adapted to the form of the data points and/or the reference points or the relative position thereof, particularly for the determination of the initial position of the data point set—whereby each point can be weight depending on its distance to the next reference point—a point-lustering-algorithm can be exerted und the cluster center points (or the point with the highest density within each cluster, etc.)","can be selected as the initial position, particularly also the sum of the quadratic distances of each DP to the next RP (to this term the distances of each reference point to the next data point can be added and/or a regularization term which smoothes too high warp) can be selected as error functional, outliers can be suppressed in several ways: (i) starting from the distribution of the distances between the data points and the next reference point, all data points/reference points having higher distances as the median or average+standard deviation of the distribution are eliminated or (ii) instead of the quadratic distances another function of the distance is used (e.g., amount function or see Miesmator in Press et al.","1992).","8.The method according to claim 3, 4 and/or 6, using the method of claim 7 for point registration.","9.The method according to claim 1 to 3, 4, 5, 6 and/or 8, that automatically (also partially) compares the transformation determined in ia) (or corresponding) with idealized dynamic reference types (in time and space) as, for example, spatial linear dynamic, diffusion dynamic, Hamilton dynamic, elastic/harmonic vibration etc., whereby this is performed by the steps: 0 search for the best fitting reference dynamic und determination of its parameters and of the pointwise and absolute error with reference to the transformation; 1 optionally alternatively to 1b): color/pattern encoded representation of the (pointwise) error at the surface/in the image volume.","10.A method for the partition of the space into regions of different, internally homogeneous motion, whereby the dynamic parameters are separately determined for the various regions and for this purpose first for the point set of a series of time steps a “point distribution model” is established, for which the dominating main components of motion are selected, and then each point of the space is coordinated to the component which contributes the dominating part to its displacement during the time step under examination, the various region are represented by different colors.","11.A method for the continuous numerical description of the motion by means of statistic techniques, like clustering and histogram analysis, which simplifies the comparison of motions, application of these techniques on the pointwise determined local quantities (e.g.","on the in 1b3) (i) selected level), in order to mark regions, in which a certain quantity occurs especially intensive or in which particularly those values occur, which are assumed especially often (clustering on the basis of the histogram).","12.Computer program comprising a program code means for performing a method according to any one of the preceding claims, if the computer program is executed by a computer.","13.A computer program product comprising a program code means which is stored on a computer readable medium, in order to perform a method according to any one of claims 1 to 11, if the program product is executed by a computer.","14.A data processing system particularly for performing the method according to any one of claims 1 to 11."],["The invention relates to the quantitative analysis and/or visualization (virtual and real) of moved processes, as well as the registration, description, correction and the comparison of global motion mechanisms within the image space (time space).","In particular, it relates to a method and an apparatus for a precise and limited to a few parameters (concise) quantitative description of the global and local motions taking place in the image space (not only of objects but also within and between the objects) and for a catchy representation of the motions and the quantitative parameters in relation to the image space.","Here, a virtual moved process is understood as the sequencing of images (or point sets) of comparable objects (e.g., same organs of different individuals).","The quantitative analysis, visualization and motion correction in dynamic processes facilitates to develop an improved understanding of the reasons of the motions, to predict and to find the interrelation between motion parameters and conditions.","An example is the investigation of elasticity properties of cell structures.","Due to the availability of imaging apparatuses, new possibilities have developed to compare object (more general: materials/substances) or to analyze different conditions or motions of an object and to visually present them in a catchy way (easily to understand).","This can be performed by a computer and be automated.","Great advances have been achieved in the extraction and presentation of surfaces and also in the calculation of velocity fields.","For images showing multiple objects lying side by side, new methods were suggested, to track the individual objects and to visualize the path of said objects by 3D rendered (surface rendering) lines or tubes.","Global motion mechanisms overruling the motion of the individual objects, will result in a wrong assignment of the object in the different images and thus in wrong paths.","Difficulties will also be created by great local displacements from one image to the next, which make the determination of the motions based only on gray values (e.g., optical flow methods) very difficult.","Both cases occur, e.g., if, in favor of the space resolution, the time resolution was chosen not particularly fine.","The precise and concise quantitative description of the motion is a further difficulty which goes far beyond the calculation of velocity fields.","Here it is to detect the global motion type and to describe it quantitatively with a few parameters as well as to detect (especially if no overruling global dynamic prevails) only a few locally different motion types and to separate these spatially; and finally to evaluate local phenomena.","The concise description simplifies the comparison of different motions in different image series.","It is a special problem in images having only a reduced structure, i.e., having only at a few parts of the surface of only a few objects a non-uniform gray value structure, to reconstruct the motion which corresponds to the real motion taking place in the image space.","If (non-rigid but) relatively global deformations occur, it is a problem to define a motion model which does not destroy local structures of the image/the surface shape and being tolerant against noise or points, which do not belong to the investigated object.","In the prior art, it is suggested to use methods for the determination of the optical flow, particle tracking as well as surface tracking and registration for analyzing and visualizing moved processes.","Optical flow methods (for an overview see Barron J. L. et al., Performance of optical flow techniques, Int.","J. Comp.","Vision, 12: 43-77, 1994) are based on the assumption that the optical densities (intensities) remain unchanged over the time.","They calculate for each image point a motion vector and thus allow to quantify the velocities for each point in space and time and to visualize it in a velocity vector field.","But they do not allow a continuous visualization (over more than 2 images) and they require that the difference in time is small in comparison to the local changes of the structures in the image as well as no changes in the illumination or other disturbances occur over the time and they are locally formulated (using a regularization term).","An exception is described in F. Germain et al.","(Characterization of Cell Deformation and Migration using a parametric estimation of image motion, IEEE Trans.","Biomed.","Eng., 46, 584-600, 1999) wherein the parameters of an affine motion model is calculated and analyzed.","This calculation and analysis is effected only locally over the time, i.e., it is separately performed for each two time steps.","Thus, it is, e.g., not possible to quantify the difference if objects are deformed continuously in the same direction or each in different directions.","But this is essential for the resulting deformation of the object.","The present invention allows a continuous quantitative analysis and furthermore provides the opportunity to visualize the deviation with respect to the reference dynamic for each point—also for global deformations being described with more degrees of freedom than for a an affine image, and for local motions.","In particle tracking (Eils, Tvarusko und Bentele, Zeitaufgelöste Analyse und/oder Visualisierung dynamischer Prozesse in dynamischen biologischen Systemen, publication GPTO 199 30 598.6, 1998/9) beforehand extracted object (or object centers) are tracked within series of images, the dynamic of said objects is quantified and their paths are continuously shown.","For this purpose, it is essential to detect the individual objects and to identify (to relocate) them in subsequent images.","This is facilitated by a combination of the critera of object proximity and object similarity (similar area, average gray value etc.).","These methods (especially the method according to Eils et al.)","fail, if all objects are similar, if for a time step an object has not been segmented (detected) and if the dynamic of the individual particle is overruled by a global dynamic.","This situation is shown in FIG.","1.The method of the present invention which also comprises a registration step allows a correct detection of the motion also in these cases and presents in the part of visualization and quantification new and improved possibilities especially for complicated image structures (animated, 3D rendered reference grid, use of characteristic image/surface points the 3D rendered paths of which are shown, the path through an interactively choosable point which can be located at any arbitrary point in the time-space, the determination of homogenous or locally especially strong motions etc.).","In the following the words “matching” (and “to match”) are used in the sense of registration, i.e., the determination of parameters of a motion model, by which an image (or an object) is transformed in a manner that it transforms as best as possible into another, and not as in Eils et al.","in the sense of the identification (relating) of points from two sets of points, e.g., surface points of two surfaces.","In J. M. Odobez and P. Bbuthemy, Detection of multiple moving objects using multiscale MRF with camera motion compensation, ICIP'94, a method for the analysis (object detection) of moving processes is suggested, which includes the compensation of the global camera motion.","Here the global motion is compensated only implicitly in order to calculate the optical flux field without presenting the opportunities of a motion compensated visualisation or to visualize the object paths (neither with correction of the global motion nor object paths at all) and without comprising a two-step-strategy by which the compensation of the global motion only facilitates the local identification of objects in a second step.","The correction of motions can also be necessary if areas with changed gray values (e.g., in angiograms) in images of moving objects (e.g., patients) should be detected.","I.","A. Boesnach et al.","(Compensation of motion artifacts in MR mammography by elastic deformation, SPIE Medical Image 2001) suggest an approach of local registration for compensating the patient's motions, which allows to distinguish areas of increased concentration of radiopaque material from areas of changed gray values caused by motions.","This approach also comprises no continuous visualization and furthermore only a local registration and thus has no means to quantify, visualize and compensate global (reference) dynamics.","It is also not suitable and not designed for the tracking of image series which also contain global dynamics.","For the visualization of dynamic parameters during the motion of surfaces, the surfaces are encolored in such a manner that the length of the motion vectors are shown in a color-encoded manner (Ferrant et al., Real-time simulation and visualization of volumetric brain deformation for image guided neurosurgery, SPIE Medical Imaging 2001).","During registration, only the surface but not the entire space has been transformed.","Here, we do not only suggest a more complete visualization concept, which includes the entire image space, but also visualizes parameters which can only be obtained as a result of the entire method.","In order to overlap data sets of different steps in time, often point data are extracted from image data (in case point data are not already available, as with contact scanners), on the basis of which the overlapping is determined.","Mattes and Demongeot (Structural outliner detection for automatic landmark extraction, SPIE Medical Imaging 2001, Vol.","4322 I: 602610) extract the shapes from confiners.","These are defined for a given gray value density function as the maximum cohering partial sets of the level-sets.","They define (by amount inclusion) a tree-structure (confinement-tree) if they are extracted for various gray value levels including the zero-level.","Irrelevant confiners can then be deleted by screening the tree.","For two gray value images first pairwise corresponding confiners are searched, the correspondence of the confiners of a pair is evaluated and then the pairs having a too low correspondence are deleted.","On the basis of the remaining pairs (e.g., of their centers or shapes) the images are overlapped.","Up to now, there is no evaluation for an iterative use of this method, which could also investigate the importance of the parameters (e.g., the number of deleted confiner pairs or the number of cut-down confiners).","In order not to rigidly overlap the points extracted in that manner, a motion model has to be given.","Szeliski and Lavallèe (IJCV 1996) use trilinear B-splines the control points of which are arranged around the extracted point set on an octree grid, having a higher resolution near to the surface.","By means of the various levels in the octree first a few and then increasingly more check points can be used, thus leading to an registration from “coarse to fine”.","In order to precisely overlap the point sets, however, (due to the insufficient smoothness of the trilinear B-splines) a lot of check points are necessary (i.e., a lot of degrees of freedom of the transformation are necessary), which additionally have to be arranged in accordance with the regular octree scheme and cannot be arbitrarily distributed in the space.","The registration with a too high number of degrees of freedom incorporates the risk to destroy local structures and to obtain very sensitive regularization parameters.","Chui and Rangarajan (CVPR 2000) use thin-plate spines and each extracted point as check point.","But this is, due to the calculation time needed, only sensible for small point sets.","We will suggest a method for setting check points which allows to precisely find the desired motion with particularly few degrees of freedom.","It has the advantage, besides the reduced calculation time, that, on the one hand, the destroying of local form characteristics and the fitting of noise can be avoided in a better way and, on the other hand, the regularization parameters have less importance (smaller sensitivity), as the transformation with less parameters is smoother, anyway.","Additionally, the user receives a more concise description of the motion, which can also be advantageous for subsequent steps, e.g., establishing an active shape model (see below) on the basis of the initial and final positions of the check points.","In summary, the present invention has the advantage besides the (afore-described) better and more concise quantitative detection of the motion, especially if extended local deformations occur, to facilitate a continuous time-space-visualization even within complex objects (and at their surfaces) and to avoid thereby the error source of object detection.","The method described herein also facilitates the detection of regions of homogeneous motion.","The “active shape model” (ASM) or point distribution model (PDM) was introduced by T. F. Cootes, C. J. Taylor, D. H. Cooper and J. Graham, Active shape models—their training and their application, Computer Vision and Image Understanding 61, 38-59, 1995, in order to be able to introduce previous statistical knowledge into the object segmentation.","Here for a number of pre-determined surfaces, which present varations of a shape, a surface model is issued by means of n landmarks, which are determined for each surface in such a manner that each landmark is present on each surface.","For an object, a landmark vector is then determined, which contains all space coordinates for all n landmarks and is thus of the dimension 3 n. The set of all landmark vectors forms a point distribution in R3n.","This point distribution is submitted to a main component analysis (in especially inhomogeneous distributions also a multimodal mix model a or even a kernel analysis) and the point distribution is characterized by means of a few proper values and proper spaces, which only represent a partial space of the original space.","For the use of the model based segmentation the optimization problem is then solved which finds the object surface in the such determined partial space which adapts best possible to the image data.","A further use is the extrapolution of registered data by means of the model, in order to rebuild best possible the surface of an object from few data (M. Fleute, S. Lavallée, Building a Complete Surface Model from sparse data using statistical shape models: application to computer assisted knee surgery, MICCAI'98, 880-887, LNCS Springer-Verlag, 1998).","The “Active Shape Model” also allows to clearly visualize the differences of different objects of the same kind.","But it has not been used for the quantitative analysis in (virtual or real) moving processes, neither for the automatic segmentation of the surface (or the space) in regions of homogeneous (spaciously linear) dynamic.","The present invention avoids the afore-mentioned disadvantages each alone or more preferred all together.","The present invention relates to a method and an apparatus for the precise and concise quantitative description of the global and local motion taking place in the image space and for the plausible presentation of the motion and the quantitative parameters with regard to the image space, the registration, the object/point tracking, the visualization and the determination of quantitative parameters of motion models and motion characteristics.","In particular, the present invention relates to a method and an apparatus which allows to determine quantitatively the motion taken place, to quantify (in space and in time) and to visualize the conformity of object motion and reference dynamics as well as to detect and to spatially separate a few different motion types and to evaluate local motion phenomena.","Furthermore, it allows due to a coarse-to-fine registration the detection and compensation of global motion mechanisms, which only allows the tracking in the embodiment shown (FIG.","1) and also permits a corrected visualization.","The registration of the motion, which corresponds as best as possible to the real motion in the image space (in the entire space volume) is realized by a “splines à plaques minces” registration, which additionally allows to keep the number of motion parameters low.","An entirely automatic cycle of all partial steps is possible.","The method according to the present invention comprises three parts or modules: module 1 “image pre-processing/point extraction”, module 2 “registration” and module 3 “visualization/quantitative analysis”, whereby module 2 and 3 may use different point sets extracted in module 1 and optionally module 2 may not use points (module 1).","Module 3 uses the quantitative data determined by module 2.Image Pre-Processing; Point Extraction During the image preprocessing, structures of the image space are extracted as image points.","In highly noisy images, the noise is first eliminated without destroying essential structures of the image.","Depending on the structure of the image objects, a reaction-diffusion-operator (G. H. Cottet and L. Germain, Image processing through reaction combined with non-linear diffusion, Math.","Comp., 61 (1993), pp.","659-673 or as an discrete equivalent; J. Mattes, D. Tysram and J. Demongeot, Parallel image processing using neural networks; applications in contrast enhancement of medical images, Parallel Processing Letters, 8: 63-76, 1998) or an operator of the anisotropic diffusion with an edge stop function on the basis of the tuckney standard (M. J.","Black N S D. Heeger, IEEE Trans.","on Image Processing 7, 421 (1998)) is used, by which the image is segmented into homogeneous regions.","Other smoothing methods are the Gaussian smoothing or methods based on wavelets.","The preferred technique for the extraction of structures is the confinement-tree-technique (Mattes and Demongeot, Tree representation and implicit tree matching for a coarse to fine image matching algorithm MICCAI, 1999).","Here, a gray value image is represented as tree structure.","Here, each knot of the tree corresponds to a region (called confiner) of the image, which is defined by means of a given gray value level (as one of the coherence components of the set of all image points having an elevated gray value).","The connection between the knots is determined by the subset relation between the regions, whereby only directly consecutive gray value levels are considered.","According to criteria like too small area, gray value mass, etc.","knots are deleted (filtration of the tree), among other, as they may be noise artifacts but also in order to reduce the number of points.","As points either all gravity centers and/or all shape points of all confiners are extracted.","In order to reduce the number of points, only those gravity centers may further be used, which correspond to knots, which follow directly on a bifurcation of the tree.","Alternatives for the point extraction are (i) using a Canny-Deriche edge-detector (R. Deriche, Using Canny's criteria to derive a recursively implemented optimal edge detector; Int.","J. Comput.","Vision, 1987, 167-187).","(ii) extracting crestridge-lines (O. Monga, S. Benayoun and O. Faugeras, From partial derivatives of 3D density images to ridge lines, IEEE CVPR'92, 354-359, Champaign, Ill., 1992) or (iii) extremity points (J.","-P. Thirion, New feature points based on geometric invariants for 3D image registration, Int.","J. Comp, Vision, 18:121-137, 1996, K. Rohr, On 3D differential operators of detecting point landmarks, Image and Vision Computing, 15,219-233, 1997).","It is also possible, if this is considered to be helpful for the desired quantification, visualization or tracking by the user, to conduct an entire segmentation of the objects, by which objects are identified and shape points are assigned to the object.","Therefore, as described in the following paragraph, a start form, e.g., a globe, is registered with the beforehand extracted points (in this case points which have been found by the Canny-Deriche edge-detector are to be preferred).","For the segmentation of objects which have a clear contrast to their surrounding, it is sufficient to select a confiner, which fulfills one additional criteria, as, e.g., a maximum value (average gray value)/(number of the shape(boundary) pixels) or (area)/(number of shape pixels)2.For the segmentation of small objects in highly noisy images, we proceed as Eils et al.","in that a step of edge completion is succeeding a step of edge extraction (see above).","Finally, a point extraction is not necessary if the recording device already delivers points.","This is the case for, e.g., laser scanners.","Following the point extraction, the user has the opportunity to delete points or to select regions interactively.","Registration The space in which images or point sets, or—more general—spatial data sets are present is called image space.","For the registration of two images (/spatial data sets) A and B, a parametrized transformation is exerted on one of the images (namely A in our terminology).","The parameters are then determined in such a manner that the transformed image resembles as exactly as possible the other image and additionally a realistic deformation is described, which describes as well as possible the real deformation of the objects of the image space.","Here, the similarity of one image to the other one is described by a functional (error functional).","The functional is a function which assigns an error value to a given transformation, the error value decreases the better the images resemble each other.","By an optimization algorithm the transformation parameter is determined such that the error functional becomes a local minimum.","Confiner assignment, evaluation and selection.","The functional is preferably based on the points extracted during the image pre-processing.","When the confiner shapes and confiner gravity points are calculated in both images, according to J. Mattes and J. Demongeot (SPIE Medical Image 2001) the corresponding confiners in the other image are searched for the confiners in the first image, whereby either the confiner with the least structural error (see also Mattes and Demongeot 2001, where the value of the structural error is referred to as relPos) or the one with the nearest gravity point is selected.","Then pairs with too little similarities are deleted as being outliers (in order to determine this similarity relPos can be chosen again, but here it is possible to choose in relPos 1 as normalization factor; prior to the use of relPos, the confiners can be made locally affine).","On the basis of the remaining pairs (e.g., their gravity points or shapes) the images are then overlapped.","This process can be repeated iteratively, as due to the overlapping for a given confiner a better selection of the confiner corresponding to said given confiner has become possible.","In a first evaluation of the iterative use of the method, the better results were achieved the less confiners were deleted during cutting down (which then lead to an increased calculation time) and for only 15% to 20% of the remaining confiner pairs (see FIG.","2) for the registration on the basis of the confiner gravity points.","If the confiner shapes are used for registration, the individual identification of the confiner shapes has the advantage, that in the following steps only the next adjacent neighbor within the corresponding confiner shape has to be searched for a shape point.","This identification, however, has to be repeated (step by step) for a part of the confiners, in case the following calculated transformation allows further assignments or a correction of the existing assignments becomes necessary.","According to the evalution of the confiner pairs by means of relPos (whereby the normalization factor in relPos can here be selected to be 1, too) the various confiner pairs in the error functional can be weighted differently (see also below).","Error functional.","For the definition of the error functional (or error measure) the sets of extracted points of A and B (these sets are also indicated with A and B) are differentiated: The points of the set B are referred to as model points and the others as data point ai (1≦i≦N).","The transformation prototype T is defined as T: R3×Π→R3, which depends on a parameter vector P ε Π (Π is the parameter space), and for each P a transformation T(P) of the space is obtained.","The euclidic distance of a point x to the next model point is referred to as d(x, B).","The summed-up quadratic distances represent the provisory error functional Cprov: Cprov(P)=(1/σ)Σi=1 .",".",".","Nσi(d(T(P, ai),B))2.Here σ=Σi=1 .",".",".","N σi.","The weight factors σi facilitate the handling of outliers.","The value of σi depends on the distribution of all distances dj of the transformed data points aj to B and becomes 1 if di is smaller than the average distance <dj>, for higher values, this value decreases like a Gaussian aroung <dj>.","The standard deviation of this Gaussian is chosen as <dj>2/σd whereby σd is the standard deviation of the distances dj.","In order to avoid a repeated checking of all model points, a distance map can be established beforehand (Lavallée and Szeliski, IEEE Trans.","PAMI, 1995) and be stored in an octree (as well as corresponding gradients of the such defined distance function, see below).","Other alternatives are based on k-d-trees or the Voronoi diagram.","A symmetric error measure is also possible, in which not only the distances of each data point to the model set is taken into account but also the distances of each model point to the data set, The use of a symmetric error measure leads in many cases to better results, but cannot be preferred in all cases due to its calculation complexity.","Alternatively, so-called iconic or gray value based error measures may be used (see, e.g., Roche, Malandaine, Ayache, Prima, MICCAI'99, LNCS Springer Verlag, 1999) which work without image processing and which determine the quality of a registration on the basis of a comparison of the overlapped gray values for a given transformation.","This makes sense and is advantageous if the change of position between the various images is relatively small and the subsequent visualization and quantitative analysis are not based on extracted or segmented data.","Motion model and optimization (Fieres et al A point set registration algorithm using a motion model based on thin-plate splines and point clustering, in Pattern recognition, DAGM 2001, vol.","2191 of LNCS, 76-83, Springer-Verlag, 2001).","The minimization of the above-described error functional alone is, however, not a criteria for a good overlapping.","For example, the transformation, which transforms all data points into a certain model point, minimizes the (asymmetric) functional, but is not the desired solution.","Other examples can easily be imagined, in which the pure minimization even of the symmetric error measure leads to undesired solutions.","It is, however, the aim to calculate a deformation which comes as close as possible to the natural deformation/motion of the object/the substance/material.","Furthermore, after the registration, physical/anatomic corresponding points of the data and model set should overlap.","According to the present invention, there are several methods for limiting the solution space to realistic solutions.","One possibility is to select an as realistic as possible (spatial) motion model, that is to find a suitable transformation prototype.","For global transformations, preferred motion models are the rigid and affine transformation.","As described in the following, local deformations are described by the displacement of check points of volume splines.","In order to receive the transformation defined for the entire space, the check point displacement vectors are interpolated by the splines.","Thin-plate splines are preferred (“splines à plaques minces”, Duchon 1976, thin-plate splines, Bookstein 1989, see FIG.","3).","In non-affine transformations, transformation prototypes are preferred by which suitable start positions of the check points can be determined (in the original image) and by which realistic (volume) transformations can be produced (see FIG.","3).","These start positions are called A landmarks pk (1≦k≦n, whereby n the number of A landmarks is) and the transformation prototype defined by them as Tpk.","The final position qk of the check points (in the target image) are called B landmarks.","They are the free parameter of the transformation: P=(q1, .",".",".",", qn).","Thus, we have Tpk((q1, .",".",".",", qn), pi)=qi and Tpk(qi, .",".",".",", qn): R3→R3, x→Tpk((q1, .",".",".",", qn), x).","Tpk((q1, .",".",".",", qn), x) can be represented as a closed term, analytically derivable according to P=(q1, .",".",".",", qn), as stated in Bookstein (1989).","This facilitates to derive Cprov as well as C (see following paragraph) according to P, which is of high importance for the optimization.","The motion model is additionally determined by the regularization technique, which introduces an additional term into the error functional which forces the minimization of the warp.","The here used term ∪bend=∪bend(P) is the integral of the sum of all quadratic second derivations (with respect to the spatial coordinates x) of the transformation TpK(P,x), integrated over the space R3 (see Bookstein 1989).","Thus, the error functional is C=Cprov+α∪band.","As a second possibility the coarse-to-fine strategy is used whereby the number of free parameters of the transformation and thus also the accuracy of the overlapping is increased stepwise (see below).","This strategy avoids undesired minima, as the optimizing algorithm used searches for solutions near the initializing values.","In the optimization according to the present invention the preferred (quadratic) error functional of the parameter vector P* has to be determined, which fulfills C(P*)=minPεΠC(P).","Here, the parameters for the transformation are preferably determined by the Levenberg-Marquardt algorithm.","The herein needed gradient of the error functional according to P=(q1, .",".",".",", qn) can easily be calculated due to the simple algebraic representation of Tpk((q1, .",".",".",", qn), x) (see above).","This is true for the global as well as for the local registration.","For other functionals other optimizing strategies may be more sensible.","In this context the following are to be mentioned: the down-hill Simplex algorithm, Simulated Annealing and others (Press et al.","Numerical Recipes in C, 1992).","For rigid and affine registrations, the optimization can also be made by the ICP algorithm (BesI and McKay, IEEE Trans.","PAMI, 1992).","The registration procedure embraces three steps (coarse to fine), whereby the third step is further refined stepwise.","From step to step, the number of free parameters of the transformation to be determined and, thus, the accuracy of the overlapping increases.","First, a rigid registration is made by which the parameters for rotation and translation are determined.","In the second step, it is registered affinelinearly, in the third step also local deformations are allowed.","According to the present invention, the third transformation is limited to the above-described prototype which is determined by the A landmarks.","Principally, the A landmarks can be positioned everywhere.","According to the present invention, they are set automatically and their number is increased stepwise; first, the best overlapping for a relatively small number of check points is determined (8 in the 3D examples described below, 4 in the 2D examples), resulting in the still relatively global transformation T(1)=Tpk(1)(P1) (according to the definition, T(0) is the identity).","After the introduction of further check points (whereby the displacement vectors (i.e., B landmarks) are initialized according to T(1)) the next more local transformation T(2) is achieved, etc., until the desired degree of locality is achieved.","The automatic positioning of the A-landmarks can, e.g., be made by an octree representation of the data points, whereby each cube of the octree contains a check point in its center.","By varying the depth of the octree, the transformation can easily be refined stepwise.","The (preferred) automatic determination of the A-landmarks according to the present invention, however, consists in that each point aj of the data point set with its distance to the next point of the model set (or with the cube of this distance or any other monotone function of the distance) is weighted and that the k-means cluster-method (formular below, Fieres et al.","2001) is exerted to the such weight point set.","The number of clusters can be chosen arbitrarily.","It is increased stepwise in accordance with the coarse-to-fine principle.","For a given degree of fineness v of the transformation, the cluster gravity centers CSi are thus defined in each step of the k-means method: CSi=(Σjdid(T(v)(aj),B)3)/(Σjdid(T(v)(aj),B)3, whereby Ii the set of indices of the data points is which is the very next to the ith cluster center.","The cluster gravity centers CSi, which are received after the convergence of the method are used as check points.","They are in regions with still high distances between data and model points.","For starting the method, the 8 corner points of the bounding box around the data point set are used.","During our evaluations, the number of checkpoints has been increased stepwise by 1+3 v. FIGS.","4-7 show comparisons of both methods for setting the check points.","For the representation of the non-rigid transformation, also other methods can be considered, e.g, the octree spline by Szeliski and Lavallee (1994).","According to the present invention, in the third registration step—as described above—a weak regularization is made, i.e., a term is added to the error functional which punishes warp.","It has been shown that the suppressing of excessive warp as well as the coarse-to-fine method improve the quality of the overlapping considerably in most cases (e.g., the correct assignment of logically corresponding points).","Without these method steps, local shape characteristics are destroyed.","Reference dynamic.","Reference dynamics can be selected which either can be fitted to the motion (transformation) found or can directly be fitted to the data if the reference dynamic can be represented as a parametrized transformation.","Possible reference dynamics are, e.g., linear dynamic (affine transformation, with arbitrary time dependence), diffusion dynamic, Hamilton dynamic (e.g., hydrodynamic), elastic (harmonic) vibration.","These dynamics can also be combined, simultaneously or sequentially Transformation over several time steps.","The transformation (as well as the reference dynamic) is determined according to the present invention also between several time steps tn−i to tn to tn+k.","In order to avoid a summing up of errors, which would occur by a mere linking of the transformations between each two time steps, the following procedure is chosen: If the transformation to tn+k should be calculated starting from tn, the same is made as described for tn, tn+1, tn+2, tn+3.By registration, the transformations Tn1, Tn2*, Tn3* are calculated which register the images tn with image tn+1, tn+1 with tn+2 and tn+2 with tn+3.We transform the image tn (or the corresponding extracted point set) by means of Tn2*otn1 (Tn2* linked with Tn1), and register for correction purposes the resulting image again with the image from tn+2.This delivers the transformation Tn2 between the images tn and tn+2.Then we transform the image tn by means of Tn3*otn2 and register for correction purposes the resulting image again with the image from tn+3.This delivers Tn3.Similar steps are taken for the transformation from tn to tn−1, by using the method described for tn.","tn+1, tn+2, tn+3 for tn, tn−1, tn−2, tn−3.If new structures (optionally given as further points) appear in an image, e.g., tn+2, these, transformed by Tn3*, are added in a further preceding correction step to the image transformed by Tn3*otn2 before the correction registration with the image tn+3.Alternatively, e.g., in order to maintain Tn2, also image tn can be transformed by means of Tn1 and the thus achieved image (after the correction with new structures) can be registered with tn+2.And so on.","For a (optionally only global) motion corrected reproduction of objects/images from the time steps tn to tn+k an absolute reference point in time is necessary.","If tn is selected as such a time step, it is, in order to transform all images to tn, an opportunity to calculate each inverse (first for the transformation between tn and tn+1) In order to avoid the calculation of the inverses (at least in the non-rigid and non-affine case), it is in general the easiest way to register the points of tn+1 on such of tn.","Combined with the method described above, this can be made between two arbitrary time steps.","In order to effect this in real time, i.e., simultaneously with the recording of the images, registration should be effected only twice for each new image.","Therefore, the correction steps are performed as above, whereby first the transformation tn+1 and tn is calculated and then between tn+2 and tn (by using the first transformation), etc.","If this procedure is used in the case of a direct registration of the gray value images without a preceding point extraction, the gray values are transformed whereby the interpolation for the resampling for the reproduction on a regular discret grid (discretization) is used.","Regions of homogeneous motion.","For the above-described registered point sets of the time steps tn to tn+k, now the point distribution model already described in the part “description” is established Therefore, the main components are selected, which represent more than a % of the shape variations.","Typical values are a=95% or a=90%.","A given point on the surface/in the space is then coordinated to the main component which leads to the highest distance d between the point displaced by the positive standard deviation (from the average model) in the direction of the main component and the point displaced by the negative standard deviation.","Or alternatively leads to the highest value d/b whereby b is the eigen-value of the corresponding main component.","The points of each region of this segmentation can then be affin registered separately with the above-described deviation, in order to assign motion parameters to each sub region Object determination.","If the points extracted during the image processing represent different objects, which are identified as such, these can be individually tracked or even registered after the registration of the image space.","Therefore it is necessary to first identify each of the equal (or corresponding) objects in the different images.","Differently to Eils et al (1989/9), it is only necessary to use a structural similarity measure (Mattes and Demongeot, SPIE Medical Imaging 2001) as the registration already moved the individual objects towards each other.","The following individual registration is interesting, e.g., for images of cells in which the rotation or extension of the individual cells are of interest.","Visualization and Quantification The calculation of the gray value image series which represents all gray value images of the series corrected by the motion of the reference dynamic (or also corrected by the entire registered motion) is a first visualization embodiment.","The explicit calculation of the inverse is herein avoided stepwise as described above (see part “registration”).","The image series recalculated in such a manner can then be animated and represented as video.","But also all other methods of the analysis of image series and visualization can also be exerted thereon.","In particular, these corrected image series are used, in case the motion corrected paths of individual objects should be visualized, which were tracked as described in the part “registration”.","The present invention comprises several quantifying and visualization modules which can be used as listed below on its own or in combination.","According to the present invention, the motion of the object/image points should be visualized as well as the resulting quantitative values and these especially with regard to the image space.","This is achieved by encoding the points of the image space by color and/or by different patterns, which represent different values of quantifiable parameters (a continuous parameter is represented by a pattern becoming denser and denser).","Quantification and visualisation modules: 1) Segmented objects or selected confiners in the original image space are visualized as closed but transparent, 3D rendered (surface rendering) surfaces, in the target image space as triangulation grid (which has been transformed by the calculated transformation).","Both are before as well as after the registration overlapped and (in different colors) represented (see FIG.","8).","The only extracted points can also be represented in such a manner.","According to the transformation found, intermediate steps are interpolated by means of a segmentation of the parameter interval (of the transformation parameter), the interpolated transformations are exerted on the original object the motion of which can then be represented animatedly.","2) The interactive confiner selection (with subsequent visualization) is effected either by clicking on a knot of a confinement-tree or by entering a gray value level and clicking on a spot of an image by which the confiner, containing the spot, is specified on the given level (or optionally no confiner).","Without entering the gray value level the smallest confiner containing the spot in the filtered tree will be determined.","The visualized surfaces can then also be represented overlapped with the target gray value image.","In order not to cover the target gray value image, the parts of the surfaces above the examined level of the target gray value image can be cut off (see also 4c)).","The overlapped representation also facilitates a visual control of the quality of the registration 3) The deformation is represented by a 3D rendered reference grid (surface rendering).","This grid can be represented animatedly and can be overlapped with the surfaces or one of the gray value images (FIG.","8).","For different color channels, the reference grids (and optionally the extracted points, too) are represented overlapped in different colors.","4) The color/pattern encoded (including gray value encoded) representation of quantified values, which have been calculated for each point in the image space, is effected in the following manner: a) Objects are segmented or confiners are selected, then the 3D rendered surface points are color/pattern encoded.","b) Either characteristic points are determined by means of the original point set (see part Image pre-processing/point extraction and Mattes and Demongeot, SPIE Medical Imaging 2001 for the entire image space and Brett and Taylor, Image and Vision Computing 18:739-748, 2000 for points on the surface) or regular points are selected or the user clicks on at least one point in the space or on the segmented/confiner surface.","For this/these the path with (error) color(/pattern) encoding is then shown (see FIG.","10).","c) All points on a level through the space volume interactively determined by the user are represented color/pattern encoded.","In the case of 2D images, this is the entire image space.","FIG.","9 shows an example for the gray value encoded quantity velocity.","d) In a) to c) the following quantities are shown in this manner.","i) Error with regard to the reference dynamic (for extracted points) as an Euclidean distance between the point transformed with the reference dynamic and the next point in the target space (or from the originally same point transformed by means of the registration of the motion calculated; ii) standardized local warp (Bookstein, Principal Warps: Thin-plate splines and the decomposition of deformations, IEEE Trans.","PAMI 11: 567-585, 1995, see also Duchon, Interpolation des functions de deux variables suivant le principe de la flexion des plaques minces, RAIRO Analyse Numérique, 10: 5-12, 1976) integrated over a globe of a given radius around a given point in the image space; iii) ii) for each principal warp (Bookstein, IEEE Trans.","PAMI, 1995) and/or for different depths of the check point octree; iv) velocity, acceleration.","5) Visualization of the paths of tracked objects/confiners (color encoded).","6) Visualization of the paths (determined by registration) of surface points, which are defined (in the original image) either interactively or regularly distributed or as characteristic points (automatically calculated as in point 4b)).","7) Interactive deleting of points (e.g., in a bounding box) or surfaces or visualized paths.","8) Color encoding of the partitioning which according to the part registration assigns to each point in the space a motion main component (whereby regions with uniform motion type are achieved) and to each motion main component a color.","9) Visualization of the motion according to 1) for each main component, for each principal warp and/or for different depths of the check point octree.","10) Interpolation for animations and paths as described in 1).","11) Quantifying of global parameters, as global warp (particularly for each principal warp, but also for different depths of the check point octree), global errors with regard to the reference dynamic (particularly for each main component), parameter of the reference dynamic, for subsequent steps as well as over several time steps, as discussed in registration and bringing both values into a relation.","12) Local parameters, as velocity or acceleration, can be further analyzed with classic statistic methods, as e.g., histogram analysis (number of pixels for each value of the local parameter) or (density based) cluster analysis.","Here the confinement tree analysis can be exerted on, e.g., the gray value encoded level determined in 4 c).","This is shown in FIG.","9 and allows the abstracted description of the motion, which is suitable for a comparison of motions.","The homogeneity of the motion direction is characterized by the size of the cluster.","By means of a histogram, value ranges of local parameters can be identified (e.g., as 1D confiner), the values of which are particularly often in the image space.","The corresponding pixels can be marked in the image/on the surface (e.g., as colored regions).","EXAMPLES Examples for the use of visualization and of the methods for the quantitative analysis are: compensation of global microscope or cell nucleus motions (see FIG.","1), exemplarily visualization and quantitative analysis of the femur, see Fieres et al.","(DAGM 2001) and FIGS.","8-10; Usage for analyzing the cell membrane as well as the chromatin dynamic (FIG.","10), visualisation of paths of points in individual cells.","Dynamic of grained structures in metal during the heating process; analysis and visualization of the heart's, lung's motion; the growth of tumors but also in tissue microtomy; dynamic of the cell nucleus envelope (membrane and lamina) during mitosis (particularly during the start stadium of its break down (FIG.","9, Beaudouin et al.","Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina.","Cell Vol.","108, 83-96, 2002, Mattes et al.","New tools for visualization and quantification in dynamic processes: Application to the nuclear envelope dynamics during mitosis.","MICCAI 1323-1325, 2001)."]],"string":"[\n [\n \"Quantitative analysis, visualization and movement correction in dynamic processes\",\n \"The invention relates to the quantitative analysis and/or visualization (virtual or real) of moving processes and to the detection, description, correction and the comparison of global movements inside the image space.\",\n \"The invention particularly relates to a method and device for precisely and, while being limited to few parameters, quantitatively describing the global and local movement occurring in the image space and for the single representation of the movement of the quantitative parameters with regard to the image space.\",\n \"A rough to fine recording and a detection of and compensation for global movements occurs whereby enabling a tracking and a corrected visualization.\",\n \"The detection of the movement, which corresponds as precisely as possible to the real movement occurring in the image space is effected by a splines a plaques minces recording technique that enables the number of movement parameters to be kept low.\",\n \"A fully automatic execution of all partial steps is made possible.\"\n ],\n [\n \"1.A method for the quantitative and visual analysis of motion in a time sequence of images comprising the steps of: a) determination of the motion vectors for each point in the image space between images of directly or not directly subsequent time steps (i.e., the transformation of the corresponding image space) preferably by means of rigid, affine and/or “elastic” (including local deformations) overlapping/registration of images of an image sequence and calculation of the transformation parameter comprising the following further method steps: a1) for the non-rigid registration, local transformation types are given stepwise from coarse to fine, first global and then increasingly more local; a2) for more then two time steps, in order to obtain the transformation for not directly subsequent timesteps, the transformation is not only calculated in pairs for subsequent image pairs, but also a correction-registration step is further added thereto; a3) from the transformation result directly the local, defined for each point in the image/time space, quantities velocity, acceleration, warp, tension, deviation from the finest registration (i.e., distance between the point transformed by the selected transformation type and by the most local fineness level), etc.\",\n \"as well as the global quantities warp energy for the entire motion as well as for each “principal warp” separately (Bookstein 1989), entropy, “directedness” of the motion and the transformation parameters for various fineness levels of the transformation; b) reconstruction of the space-time-structure within the image space and visualization thereof by means of b1) (optionally interactive) determination of a point (or a region; or according to a regular scheme selected point in the space or on the surface of a region) for a selectable moment, the automatic calculation of its (their) position for all other moments by means of the under a) calculated transformation (for not directly subsequent time steps) as well as the interpolated representation as 3D rendered (surface rendering) path (or “tube” for regions); and/or b2) a 3D rendered reference grid, deformed according to the transformation calculated in a), animated (interpolation of intermediate time steps) or not (animation also continuously over several time steps), the image space overlapped (inserted) or not; in an image record having several color channels, the reference grids corresponding to the various separately treated color channels can be represented in different color but simultaneously; and/or b3) color/pattern encoding (including gray value encoding) of quantities (quantities having vector values by means of absolute values), which are assigned to a point in the image/time space as described under a), particularly for (i) all points, lying within an interactively selectable level of the image space, (ii) interactively (or regularly) selected points or surface points of interactively selected regions/shapes/surfaces (iii) the points of the under b1) determined path (or tubes) of the time space; and/or b4) a motion corrected representation of the paths (or tubes), corrected by a rigid, affine or a selected level of the non-global transformation, or by means of a motion corrected playing of the original image sequence, particularly the color encoding can be back projected onto the original image or any other given moment.\",\n \"2.The method according to claim 1, which extracts in a pre-processing step a0) critical (characteristic) points of the image space or surface points of regions in the image space and uses these in the steps b1), b3) and b4) instead of interactively selected points, whereby in step b2) the (critical/surface) points of the original space can be used instead of the reference grid.\",\n \"3.The method according to claim 2, which uses for the preprocessing the confinement tree technique (CTT), whereby a) for the visualization of a region (a confiner), the user clicks either on knot in the tree or on a point in the image, upon which the surface/shape of the smallest confiners (in the filtered tree) containing said point will be shown; and/or b) the registration is performed on the basis of the extracted points (confiner-surface points or critical points, e.g., confiner center points) by means of a point registration algorithm, whereby confiners corresponding to each other are pre-determined in the original and target image or an algorithm based on the minimization of an “iconic” similarity measure follow: and/or c) transformed confiner of the original image overlapped with non-transformed confiners of the target image can be represented whereby the confiner surfaces replace the parts of the reference grid in 1b2), and to the quantities from 1a3) the distance from one point to the next adjacent point extracted in the other image is added.\",\n \"4.A method for registration by means of CTT, which either a) uses confiner center points (CS) or confiner density maxima (CD) as critical points for registration and by using the method of structural outlier determination (by means of relPos, see Mattes and Demongeot 2001) starting from an initial overlapping (optionally improved by a direct use of a point registration method, see page 6) ) first determines corresponding confiner pairs and thereby only maintains pairs with best relPos (optionally according to 6(i)), on the basis of this (CD, CS or confiner shapes of which) determines the best fitting (in the sense of the error functional, see 6) as well as Fieres et al.\",\n \"2001) transformation, exerts on the data confiner and repeats this process iteratively, whereby the such determined landmarks can be used for the establishment of a “point distribution model”; or b) corresponding confiners are determined as follows: for the knots, which occur after a bifurcation in the CT of one of the images, the knots/confiner having the lowest relPos value in the CT of the other image are searched, the point outliers are thereby suppressed as in claim 6, then the relPos distribution for all confiner pairs are examined, and the confiner outlier are eliminated temporarily as described in 6)(i) for point outliers, finally a point registration method under consideration of corresponding confiners are used, by an optional usage of first highly cut-down CTs and then increasingly less highly cut-down CTs a coarse to fine effect can be achieved in a) and b), which can also be combined with/replaced by the following method(s) and the images are first highly (i.e., on great scales), (e.g., Gauss-) smoothed (or presented in a high pixel resolution), and in the proceeding of the method increasingly less highly smoothed (or represented in increasingly finer resolution).\",\n \"5.The method according to claim 3 using the CTT as in claim 4.6.The method according to claim 3, which instead of the pre-processing by means of CTT identifies objects in the image space in a segmentation step and calculates the surface and/or center point of which, the objects replace the confiners, in case only (non-coherent) points are identified on the object surface (e.g., by means of a Canny edge-detector), corresponding confiner/objects cannot be pre-determined according to 3b) and particularly for the segmented objects can also individual translations and rotation or deformations be determined and the object paths can individually corrected by selected transformation types.\",\n \"7.A method for the registration of a data point (DP) set on a reference point (RP) set, which determines iteratively for a series of given transformation types, which allow an increasing number of local deformations, its free parameters in such a manner, that an error functional is minimized, which compares the positions of the transformed data points with those of the reference points, thereby selecting as transformation types first a rigid (rotation and translation), then an affine transformation and then transformation types, defined by a number of checkpoints, which can freely be displaced (the free parameter of the transformation) and between the displacement vectors of which with thin-plate splines is interpolated (Bookstein 1989) and, the number of checkpoints is iteratively increased (in order to allow an increasing number of local deformation), and the initial position of the checkpoints is adapted to the form of the data points and/or the reference points or the relative position thereof, particularly for the determination of the initial position of the data point set—whereby each point can be weight depending on its distance to the next reference point—a point-lustering-algorithm can be exerted und the cluster center points (or the point with the highest density within each cluster, etc.)\",\n \"can be selected as the initial position, particularly also the sum of the quadratic distances of each DP to the next RP (to this term the distances of each reference point to the next data point can be added and/or a regularization term which smoothes too high warp) can be selected as error functional, outliers can be suppressed in several ways: (i) starting from the distribution of the distances between the data points and the next reference point, all data points/reference points having higher distances as the median or average+standard deviation of the distribution are eliminated or (ii) instead of the quadratic distances another function of the distance is used (e.g., amount function or see Miesmator in Press et al.\",\n \"1992).\",\n \"8.The method according to claim 3, 4 and/or 6, using the method of claim 7 for point registration.\",\n \"9.The method according to claim 1 to 3, 4, 5, 6 and/or 8, that automatically (also partially) compares the transformation determined in ia) (or corresponding) with idealized dynamic reference types (in time and space) as, for example, spatial linear dynamic, diffusion dynamic, Hamilton dynamic, elastic/harmonic vibration etc., whereby this is performed by the steps: 0 search for the best fitting reference dynamic und determination of its parameters and of the pointwise and absolute error with reference to the transformation; 1 optionally alternatively to 1b): color/pattern encoded representation of the (pointwise) error at the surface/in the image volume.\",\n \"10.A method for the partition of the space into regions of different, internally homogeneous motion, whereby the dynamic parameters are separately determined for the various regions and for this purpose first for the point set of a series of time steps a “point distribution model” is established, for which the dominating main components of motion are selected, and then each point of the space is coordinated to the component which contributes the dominating part to its displacement during the time step under examination, the various region are represented by different colors.\",\n \"11.A method for the continuous numerical description of the motion by means of statistic techniques, like clustering and histogram analysis, which simplifies the comparison of motions, application of these techniques on the pointwise determined local quantities (e.g.\",\n \"on the in 1b3) (i) selected level), in order to mark regions, in which a certain quantity occurs especially intensive or in which particularly those values occur, which are assumed especially often (clustering on the basis of the histogram).\",\n \"12.Computer program comprising a program code means for performing a method according to any one of the preceding claims, if the computer program is executed by a computer.\",\n \"13.A computer program product comprising a program code means which is stored on a computer readable medium, in order to perform a method according to any one of claims 1 to 11, if the program product is executed by a computer.\",\n \"14.A data processing system particularly for performing the method according to any one of claims 1 to 11.\"\n ],\n [\n \"The invention relates to the quantitative analysis and/or visualization (virtual and real) of moved processes, as well as the registration, description, correction and the comparison of global motion mechanisms within the image space (time space).\",\n \"In particular, it relates to a method and an apparatus for a precise and limited to a few parameters (concise) quantitative description of the global and local motions taking place in the image space (not only of objects but also within and between the objects) and for a catchy representation of the motions and the quantitative parameters in relation to the image space.\",\n \"Here, a virtual moved process is understood as the sequencing of images (or point sets) of comparable objects (e.g., same organs of different individuals).\",\n \"The quantitative analysis, visualization and motion correction in dynamic processes facilitates to develop an improved understanding of the reasons of the motions, to predict and to find the interrelation between motion parameters and conditions.\",\n \"An example is the investigation of elasticity properties of cell structures.\",\n \"Due to the availability of imaging apparatuses, new possibilities have developed to compare object (more general: materials/substances) or to analyze different conditions or motions of an object and to visually present them in a catchy way (easily to understand).\",\n \"This can be performed by a computer and be automated.\",\n \"Great advances have been achieved in the extraction and presentation of surfaces and also in the calculation of velocity fields.\",\n \"For images showing multiple objects lying side by side, new methods were suggested, to track the individual objects and to visualize the path of said objects by 3D rendered (surface rendering) lines or tubes.\",\n \"Global motion mechanisms overruling the motion of the individual objects, will result in a wrong assignment of the object in the different images and thus in wrong paths.\",\n \"Difficulties will also be created by great local displacements from one image to the next, which make the determination of the motions based only on gray values (e.g., optical flow methods) very difficult.\",\n \"Both cases occur, e.g., if, in favor of the space resolution, the time resolution was chosen not particularly fine.\",\n \"The precise and concise quantitative description of the motion is a further difficulty which goes far beyond the calculation of velocity fields.\",\n \"Here it is to detect the global motion type and to describe it quantitatively with a few parameters as well as to detect (especially if no overruling global dynamic prevails) only a few locally different motion types and to separate these spatially; and finally to evaluate local phenomena.\",\n \"The concise description simplifies the comparison of different motions in different image series.\",\n \"It is a special problem in images having only a reduced structure, i.e., having only at a few parts of the surface of only a few objects a non-uniform gray value structure, to reconstruct the motion which corresponds to the real motion taking place in the image space.\",\n \"If (non-rigid but) relatively global deformations occur, it is a problem to define a motion model which does not destroy local structures of the image/the surface shape and being tolerant against noise or points, which do not belong to the investigated object.\",\n \"In the prior art, it is suggested to use methods for the determination of the optical flow, particle tracking as well as surface tracking and registration for analyzing and visualizing moved processes.\",\n \"Optical flow methods (for an overview see Barron J. L. et al., Performance of optical flow techniques, Int.\",\n \"J. Comp.\",\n \"Vision, 12: 43-77, 1994) are based on the assumption that the optical densities (intensities) remain unchanged over the time.\",\n \"They calculate for each image point a motion vector and thus allow to quantify the velocities for each point in space and time and to visualize it in a velocity vector field.\",\n \"But they do not allow a continuous visualization (over more than 2 images) and they require that the difference in time is small in comparison to the local changes of the structures in the image as well as no changes in the illumination or other disturbances occur over the time and they are locally formulated (using a regularization term).\",\n \"An exception is described in F. Germain et al.\",\n \"(Characterization of Cell Deformation and Migration using a parametric estimation of image motion, IEEE Trans.\",\n \"Biomed.\",\n \"Eng., 46, 584-600, 1999) wherein the parameters of an affine motion model is calculated and analyzed.\",\n \"This calculation and analysis is effected only locally over the time, i.e., it is separately performed for each two time steps.\",\n \"Thus, it is, e.g., not possible to quantify the difference if objects are deformed continuously in the same direction or each in different directions.\",\n \"But this is essential for the resulting deformation of the object.\",\n \"The present invention allows a continuous quantitative analysis and furthermore provides the opportunity to visualize the deviation with respect to the reference dynamic for each point—also for global deformations being described with more degrees of freedom than for a an affine image, and for local motions.\",\n \"In particle tracking (Eils, Tvarusko und Bentele, Zeitaufgelöste Analyse und/oder Visualisierung dynamischer Prozesse in dynamischen biologischen Systemen, publication GPTO 199 30 598.6, 1998/9) beforehand extracted object (or object centers) are tracked within series of images, the dynamic of said objects is quantified and their paths are continuously shown.\",\n \"For this purpose, it is essential to detect the individual objects and to identify (to relocate) them in subsequent images.\",\n \"This is facilitated by a combination of the critera of object proximity and object similarity (similar area, average gray value etc.).\",\n \"These methods (especially the method according to Eils et al.)\",\n \"fail, if all objects are similar, if for a time step an object has not been segmented (detected) and if the dynamic of the individual particle is overruled by a global dynamic.\",\n \"This situation is shown in FIG.\",\n \"1.The method of the present invention which also comprises a registration step allows a correct detection of the motion also in these cases and presents in the part of visualization and quantification new and improved possibilities especially for complicated image structures (animated, 3D rendered reference grid, use of characteristic image/surface points the 3D rendered paths of which are shown, the path through an interactively choosable point which can be located at any arbitrary point in the time-space, the determination of homogenous or locally especially strong motions etc.).\",\n \"In the following the words “matching” (and “to match”) are used in the sense of registration, i.e., the determination of parameters of a motion model, by which an image (or an object) is transformed in a manner that it transforms as best as possible into another, and not as in Eils et al.\",\n \"in the sense of the identification (relating) of points from two sets of points, e.g., surface points of two surfaces.\",\n \"In J. M. Odobez and P. Bbuthemy, Detection of multiple moving objects using multiscale MRF with camera motion compensation, ICIP'94, a method for the analysis (object detection) of moving processes is suggested, which includes the compensation of the global camera motion.\",\n \"Here the global motion is compensated only implicitly in order to calculate the optical flux field without presenting the opportunities of a motion compensated visualisation or to visualize the object paths (neither with correction of the global motion nor object paths at all) and without comprising a two-step-strategy by which the compensation of the global motion only facilitates the local identification of objects in a second step.\",\n \"The correction of motions can also be necessary if areas with changed gray values (e.g., in angiograms) in images of moving objects (e.g., patients) should be detected.\",\n \"I.\",\n \"A. Boesnach et al.\",\n \"(Compensation of motion artifacts in MR mammography by elastic deformation, SPIE Medical Image 2001) suggest an approach of local registration for compensating the patient's motions, which allows to distinguish areas of increased concentration of radiopaque material from areas of changed gray values caused by motions.\",\n \"This approach also comprises no continuous visualization and furthermore only a local registration and thus has no means to quantify, visualize and compensate global (reference) dynamics.\",\n \"It is also not suitable and not designed for the tracking of image series which also contain global dynamics.\",\n \"For the visualization of dynamic parameters during the motion of surfaces, the surfaces are encolored in such a manner that the length of the motion vectors are shown in a color-encoded manner (Ferrant et al., Real-time simulation and visualization of volumetric brain deformation for image guided neurosurgery, SPIE Medical Imaging 2001).\",\n \"During registration, only the surface but not the entire space has been transformed.\",\n \"Here, we do not only suggest a more complete visualization concept, which includes the entire image space, but also visualizes parameters which can only be obtained as a result of the entire method.\",\n \"In order to overlap data sets of different steps in time, often point data are extracted from image data (in case point data are not already available, as with contact scanners), on the basis of which the overlapping is determined.\",\n \"Mattes and Demongeot (Structural outliner detection for automatic landmark extraction, SPIE Medical Imaging 2001, Vol.\",\n \"4322 I: 602610) extract the shapes from confiners.\",\n \"These are defined for a given gray value density function as the maximum cohering partial sets of the level-sets.\",\n \"They define (by amount inclusion) a tree-structure (confinement-tree) if they are extracted for various gray value levels including the zero-level.\",\n \"Irrelevant confiners can then be deleted by screening the tree.\",\n \"For two gray value images first pairwise corresponding confiners are searched, the correspondence of the confiners of a pair is evaluated and then the pairs having a too low correspondence are deleted.\",\n \"On the basis of the remaining pairs (e.g., of their centers or shapes) the images are overlapped.\",\n \"Up to now, there is no evaluation for an iterative use of this method, which could also investigate the importance of the parameters (e.g., the number of deleted confiner pairs or the number of cut-down confiners).\",\n \"In order not to rigidly overlap the points extracted in that manner, a motion model has to be given.\",\n \"Szeliski and Lavallèe (IJCV 1996) use trilinear B-splines the control points of which are arranged around the extracted point set on an octree grid, having a higher resolution near to the surface.\",\n \"By means of the various levels in the octree first a few and then increasingly more check points can be used, thus leading to an registration from “coarse to fine”.\",\n \"In order to precisely overlap the point sets, however, (due to the insufficient smoothness of the trilinear B-splines) a lot of check points are necessary (i.e., a lot of degrees of freedom of the transformation are necessary), which additionally have to be arranged in accordance with the regular octree scheme and cannot be arbitrarily distributed in the space.\",\n \"The registration with a too high number of degrees of freedom incorporates the risk to destroy local structures and to obtain very sensitive regularization parameters.\",\n \"Chui and Rangarajan (CVPR 2000) use thin-plate spines and each extracted point as check point.\",\n \"But this is, due to the calculation time needed, only sensible for small point sets.\",\n \"We will suggest a method for setting check points which allows to precisely find the desired motion with particularly few degrees of freedom.\",\n \"It has the advantage, besides the reduced calculation time, that, on the one hand, the destroying of local form characteristics and the fitting of noise can be avoided in a better way and, on the other hand, the regularization parameters have less importance (smaller sensitivity), as the transformation with less parameters is smoother, anyway.\",\n \"Additionally, the user receives a more concise description of the motion, which can also be advantageous for subsequent steps, e.g., establishing an active shape model (see below) on the basis of the initial and final positions of the check points.\",\n \"In summary, the present invention has the advantage besides the (afore-described) better and more concise quantitative detection of the motion, especially if extended local deformations occur, to facilitate a continuous time-space-visualization even within complex objects (and at their surfaces) and to avoid thereby the error source of object detection.\",\n \"The method described herein also facilitates the detection of regions of homogeneous motion.\",\n \"The “active shape model” (ASM) or point distribution model (PDM) was introduced by T. F. Cootes, C. J. Taylor, D. H. Cooper and J. Graham, Active shape models—their training and their application, Computer Vision and Image Understanding 61, 38-59, 1995, in order to be able to introduce previous statistical knowledge into the object segmentation.\",\n \"Here for a number of pre-determined surfaces, which present varations of a shape, a surface model is issued by means of n landmarks, which are determined for each surface in such a manner that each landmark is present on each surface.\",\n \"For an object, a landmark vector is then determined, which contains all space coordinates for all n landmarks and is thus of the dimension 3 n. The set of all landmark vectors forms a point distribution in R3n.\",\n \"This point distribution is submitted to a main component analysis (in especially inhomogeneous distributions also a multimodal mix model a or even a kernel analysis) and the point distribution is characterized by means of a few proper values and proper spaces, which only represent a partial space of the original space.\",\n \"For the use of the model based segmentation the optimization problem is then solved which finds the object surface in the such determined partial space which adapts best possible to the image data.\",\n \"A further use is the extrapolution of registered data by means of the model, in order to rebuild best possible the surface of an object from few data (M. Fleute, S. Lavallée, Building a Complete Surface Model from sparse data using statistical shape models: application to computer assisted knee surgery, MICCAI'98, 880-887, LNCS Springer-Verlag, 1998).\",\n \"The “Active Shape Model” also allows to clearly visualize the differences of different objects of the same kind.\",\n \"But it has not been used for the quantitative analysis in (virtual or real) moving processes, neither for the automatic segmentation of the surface (or the space) in regions of homogeneous (spaciously linear) dynamic.\",\n \"The present invention avoids the afore-mentioned disadvantages each alone or more preferred all together.\",\n \"The present invention relates to a method and an apparatus for the precise and concise quantitative description of the global and local motion taking place in the image space and for the plausible presentation of the motion and the quantitative parameters with regard to the image space, the registration, the object/point tracking, the visualization and the determination of quantitative parameters of motion models and motion characteristics.\",\n \"In particular, the present invention relates to a method and an apparatus which allows to determine quantitatively the motion taken place, to quantify (in space and in time) and to visualize the conformity of object motion and reference dynamics as well as to detect and to spatially separate a few different motion types and to evaluate local motion phenomena.\",\n \"Furthermore, it allows due to a coarse-to-fine registration the detection and compensation of global motion mechanisms, which only allows the tracking in the embodiment shown (FIG.\",\n \"1) and also permits a corrected visualization.\",\n \"The registration of the motion, which corresponds as best as possible to the real motion in the image space (in the entire space volume) is realized by a “splines à plaques minces” registration, which additionally allows to keep the number of motion parameters low.\",\n \"An entirely automatic cycle of all partial steps is possible.\",\n \"The method according to the present invention comprises three parts or modules: module 1 “image pre-processing/point extraction”, module 2 “registration” and module 3 “visualization/quantitative analysis”, whereby module 2 and 3 may use different point sets extracted in module 1 and optionally module 2 may not use points (module 1).\",\n \"Module 3 uses the quantitative data determined by module 2.Image Pre-Processing; Point Extraction During the image preprocessing, structures of the image space are extracted as image points.\",\n \"In highly noisy images, the noise is first eliminated without destroying essential structures of the image.\",\n \"Depending on the structure of the image objects, a reaction-diffusion-operator (G. H. Cottet and L. Germain, Image processing through reaction combined with non-linear diffusion, Math.\",\n \"Comp., 61 (1993), pp.\",\n \"659-673 or as an discrete equivalent; J. Mattes, D. Tysram and J. Demongeot, Parallel image processing using neural networks; applications in contrast enhancement of medical images, Parallel Processing Letters, 8: 63-76, 1998) or an operator of the anisotropic diffusion with an edge stop function on the basis of the tuckney standard (M. J.\",\n \"Black N S D. Heeger, IEEE Trans.\",\n \"on Image Processing 7, 421 (1998)) is used, by which the image is segmented into homogeneous regions.\",\n \"Other smoothing methods are the Gaussian smoothing or methods based on wavelets.\",\n \"The preferred technique for the extraction of structures is the confinement-tree-technique (Mattes and Demongeot, Tree representation and implicit tree matching for a coarse to fine image matching algorithm MICCAI, 1999).\",\n \"Here, a gray value image is represented as tree structure.\",\n \"Here, each knot of the tree corresponds to a region (called confiner) of the image, which is defined by means of a given gray value level (as one of the coherence components of the set of all image points having an elevated gray value).\",\n \"The connection between the knots is determined by the subset relation between the regions, whereby only directly consecutive gray value levels are considered.\",\n \"According to criteria like too small area, gray value mass, etc.\",\n \"knots are deleted (filtration of the tree), among other, as they may be noise artifacts but also in order to reduce the number of points.\",\n \"As points either all gravity centers and/or all shape points of all confiners are extracted.\",\n \"In order to reduce the number of points, only those gravity centers may further be used, which correspond to knots, which follow directly on a bifurcation of the tree.\",\n \"Alternatives for the point extraction are (i) using a Canny-Deriche edge-detector (R. Deriche, Using Canny's criteria to derive a recursively implemented optimal edge detector; Int.\",\n \"J. Comput.\",\n \"Vision, 1987, 167-187).\",\n \"(ii) extracting crestridge-lines (O. Monga, S. Benayoun and O. Faugeras, From partial derivatives of 3D density images to ridge lines, IEEE CVPR'92, 354-359, Champaign, Ill., 1992) or (iii) extremity points (J.\",\n \"-P. Thirion, New feature points based on geometric invariants for 3D image registration, Int.\",\n \"J. Comp, Vision, 18:121-137, 1996, K. Rohr, On 3D differential operators of detecting point landmarks, Image and Vision Computing, 15,219-233, 1997).\",\n \"It is also possible, if this is considered to be helpful for the desired quantification, visualization or tracking by the user, to conduct an entire segmentation of the objects, by which objects are identified and shape points are assigned to the object.\",\n \"Therefore, as described in the following paragraph, a start form, e.g., a globe, is registered with the beforehand extracted points (in this case points which have been found by the Canny-Deriche edge-detector are to be preferred).\",\n \"For the segmentation of objects which have a clear contrast to their surrounding, it is sufficient to select a confiner, which fulfills one additional criteria, as, e.g., a maximum value (average gray value)/(number of the shape(boundary) pixels) or (area)/(number of shape pixels)2.For the segmentation of small objects in highly noisy images, we proceed as Eils et al.\",\n \"in that a step of edge completion is succeeding a step of edge extraction (see above).\",\n \"Finally, a point extraction is not necessary if the recording device already delivers points.\",\n \"This is the case for, e.g., laser scanners.\",\n \"Following the point extraction, the user has the opportunity to delete points or to select regions interactively.\",\n \"Registration The space in which images or point sets, or—more general—spatial data sets are present is called image space.\",\n \"For the registration of two images (/spatial data sets) A and B, a parametrized transformation is exerted on one of the images (namely A in our terminology).\",\n \"The parameters are then determined in such a manner that the transformed image resembles as exactly as possible the other image and additionally a realistic deformation is described, which describes as well as possible the real deformation of the objects of the image space.\",\n \"Here, the similarity of one image to the other one is described by a functional (error functional).\",\n \"The functional is a function which assigns an error value to a given transformation, the error value decreases the better the images resemble each other.\",\n \"By an optimization algorithm the transformation parameter is determined such that the error functional becomes a local minimum.\",\n \"Confiner assignment, evaluation and selection.\",\n \"The functional is preferably based on the points extracted during the image pre-processing.\",\n \"When the confiner shapes and confiner gravity points are calculated in both images, according to J. Mattes and J. Demongeot (SPIE Medical Image 2001) the corresponding confiners in the other image are searched for the confiners in the first image, whereby either the confiner with the least structural error (see also Mattes and Demongeot 2001, where the value of the structural error is referred to as relPos) or the one with the nearest gravity point is selected.\",\n \"Then pairs with too little similarities are deleted as being outliers (in order to determine this similarity relPos can be chosen again, but here it is possible to choose in relPos 1 as normalization factor; prior to the use of relPos, the confiners can be made locally affine).\",\n \"On the basis of the remaining pairs (e.g., their gravity points or shapes) the images are then overlapped.\",\n \"This process can be repeated iteratively, as due to the overlapping for a given confiner a better selection of the confiner corresponding to said given confiner has become possible.\",\n \"In a first evaluation of the iterative use of the method, the better results were achieved the less confiners were deleted during cutting down (which then lead to an increased calculation time) and for only 15% to 20% of the remaining confiner pairs (see FIG.\",\n \"2) for the registration on the basis of the confiner gravity points.\",\n \"If the confiner shapes are used for registration, the individual identification of the confiner shapes has the advantage, that in the following steps only the next adjacent neighbor within the corresponding confiner shape has to be searched for a shape point.\",\n \"This identification, however, has to be repeated (step by step) for a part of the confiners, in case the following calculated transformation allows further assignments or a correction of the existing assignments becomes necessary.\",\n \"According to the evalution of the confiner pairs by means of relPos (whereby the normalization factor in relPos can here be selected to be 1, too) the various confiner pairs in the error functional can be weighted differently (see also below).\",\n \"Error functional.\",\n \"For the definition of the error functional (or error measure) the sets of extracted points of A and B (these sets are also indicated with A and B) are differentiated: The points of the set B are referred to as model points and the others as data point ai (1≦i≦N).\",\n \"The transformation prototype T is defined as T: R3×Π→R3, which depends on a parameter vector P ε Π (Π is the parameter space), and for each P a transformation T(P) of the space is obtained.\",\n \"The euclidic distance of a point x to the next model point is referred to as d(x, B).\",\n \"The summed-up quadratic distances represent the provisory error functional Cprov: Cprov(P)=(1/σ)Σi=1 .\",\n \".\",\n \".\",\n \"Nσi(d(T(P, ai),B))2.Here σ=Σi=1 .\",\n \".\",\n \".\",\n \"N σi.\",\n \"The weight factors σi facilitate the handling of outliers.\",\n \"The value of σi depends on the distribution of all distances dj of the transformed data points aj to B and becomes 1 if di is smaller than the average distance <dj>, for higher values, this value decreases like a Gaussian aroung <dj>.\",\n \"The standard deviation of this Gaussian is chosen as <dj>2/σd whereby σd is the standard deviation of the distances dj.\",\n \"In order to avoid a repeated checking of all model points, a distance map can be established beforehand (Lavallée and Szeliski, IEEE Trans.\",\n \"PAMI, 1995) and be stored in an octree (as well as corresponding gradients of the such defined distance function, see below).\",\n \"Other alternatives are based on k-d-trees or the Voronoi diagram.\",\n \"A symmetric error measure is also possible, in which not only the distances of each data point to the model set is taken into account but also the distances of each model point to the data set, The use of a symmetric error measure leads in many cases to better results, but cannot be preferred in all cases due to its calculation complexity.\",\n \"Alternatively, so-called iconic or gray value based error measures may be used (see, e.g., Roche, Malandaine, Ayache, Prima, MICCAI'99, LNCS Springer Verlag, 1999) which work without image processing and which determine the quality of a registration on the basis of a comparison of the overlapped gray values for a given transformation.\",\n \"This makes sense and is advantageous if the change of position between the various images is relatively small and the subsequent visualization and quantitative analysis are not based on extracted or segmented data.\",\n \"Motion model and optimization (Fieres et al A point set registration algorithm using a motion model based on thin-plate splines and point clustering, in Pattern recognition, DAGM 2001, vol.\",\n \"2191 of LNCS, 76-83, Springer-Verlag, 2001).\",\n \"The minimization of the above-described error functional alone is, however, not a criteria for a good overlapping.\",\n \"For example, the transformation, which transforms all data points into a certain model point, minimizes the (asymmetric) functional, but is not the desired solution.\",\n \"Other examples can easily be imagined, in which the pure minimization even of the symmetric error measure leads to undesired solutions.\",\n \"It is, however, the aim to calculate a deformation which comes as close as possible to the natural deformation/motion of the object/the substance/material.\",\n \"Furthermore, after the registration, physical/anatomic corresponding points of the data and model set should overlap.\",\n \"According to the present invention, there are several methods for limiting the solution space to realistic solutions.\",\n \"One possibility is to select an as realistic as possible (spatial) motion model, that is to find a suitable transformation prototype.\",\n \"For global transformations, preferred motion models are the rigid and affine transformation.\",\n \"As described in the following, local deformations are described by the displacement of check points of volume splines.\",\n \"In order to receive the transformation defined for the entire space, the check point displacement vectors are interpolated by the splines.\",\n \"Thin-plate splines are preferred (“splines à plaques minces”, Duchon 1976, thin-plate splines, Bookstein 1989, see FIG.\",\n \"3).\",\n \"In non-affine transformations, transformation prototypes are preferred by which suitable start positions of the check points can be determined (in the original image) and by which realistic (volume) transformations can be produced (see FIG.\",\n \"3).\",\n \"These start positions are called A landmarks pk (1≦k≦n, whereby n the number of A landmarks is) and the transformation prototype defined by them as Tpk.\",\n \"The final position qk of the check points (in the target image) are called B landmarks.\",\n \"They are the free parameter of the transformation: P=(q1, .\",\n \".\",\n \".\",\n \", qn).\",\n \"Thus, we have Tpk((q1, .\",\n \".\",\n \".\",\n \", qn), pi)=qi and Tpk(qi, .\",\n \".\",\n \".\",\n \", qn): R3→R3, x→Tpk((q1, .\",\n \".\",\n \".\",\n \", qn), x).\",\n \"Tpk((q1, .\",\n \".\",\n \".\",\n \", qn), x) can be represented as a closed term, analytically derivable according to P=(q1, .\",\n \".\",\n \".\",\n \", qn), as stated in Bookstein (1989).\",\n \"This facilitates to derive Cprov as well as C (see following paragraph) according to P, which is of high importance for the optimization.\",\n \"The motion model is additionally determined by the regularization technique, which introduces an additional term into the error functional which forces the minimization of the warp.\",\n \"The here used term ∪bend=∪bend(P) is the integral of the sum of all quadratic second derivations (with respect to the spatial coordinates x) of the transformation TpK(P,x), integrated over the space R3 (see Bookstein 1989).\",\n \"Thus, the error functional is C=Cprov+α∪band.\",\n \"As a second possibility the coarse-to-fine strategy is used whereby the number of free parameters of the transformation and thus also the accuracy of the overlapping is increased stepwise (see below).\",\n \"This strategy avoids undesired minima, as the optimizing algorithm used searches for solutions near the initializing values.\",\n \"In the optimization according to the present invention the preferred (quadratic) error functional of the parameter vector P* has to be determined, which fulfills C(P*)=minPεΠC(P).\",\n \"Here, the parameters for the transformation are preferably determined by the Levenberg-Marquardt algorithm.\",\n \"The herein needed gradient of the error functional according to P=(q1, .\",\n \".\",\n \".\",\n \", qn) can easily be calculated due to the simple algebraic representation of Tpk((q1, .\",\n \".\",\n \".\",\n \", qn), x) (see above).\",\n \"This is true for the global as well as for the local registration.\",\n \"For other functionals other optimizing strategies may be more sensible.\",\n \"In this context the following are to be mentioned: the down-hill Simplex algorithm, Simulated Annealing and others (Press et al.\",\n \"Numerical Recipes in C, 1992).\",\n \"For rigid and affine registrations, the optimization can also be made by the ICP algorithm (BesI and McKay, IEEE Trans.\",\n \"PAMI, 1992).\",\n \"The registration procedure embraces three steps (coarse to fine), whereby the third step is further refined stepwise.\",\n \"From step to step, the number of free parameters of the transformation to be determined and, thus, the accuracy of the overlapping increases.\",\n \"First, a rigid registration is made by which the parameters for rotation and translation are determined.\",\n \"In the second step, it is registered affinelinearly, in the third step also local deformations are allowed.\",\n \"According to the present invention, the third transformation is limited to the above-described prototype which is determined by the A landmarks.\",\n \"Principally, the A landmarks can be positioned everywhere.\",\n \"According to the present invention, they are set automatically and their number is increased stepwise; first, the best overlapping for a relatively small number of check points is determined (8 in the 3D examples described below, 4 in the 2D examples), resulting in the still relatively global transformation T(1)=Tpk(1)(P1) (according to the definition, T(0) is the identity).\",\n \"After the introduction of further check points (whereby the displacement vectors (i.e., B landmarks) are initialized according to T(1)) the next more local transformation T(2) is achieved, etc., until the desired degree of locality is achieved.\",\n \"The automatic positioning of the A-landmarks can, e.g., be made by an octree representation of the data points, whereby each cube of the octree contains a check point in its center.\",\n \"By varying the depth of the octree, the transformation can easily be refined stepwise.\",\n \"The (preferred) automatic determination of the A-landmarks according to the present invention, however, consists in that each point aj of the data point set with its distance to the next point of the model set (or with the cube of this distance or any other monotone function of the distance) is weighted and that the k-means cluster-method (formular below, Fieres et al.\",\n \"2001) is exerted to the such weight point set.\",\n \"The number of clusters can be chosen arbitrarily.\",\n \"It is increased stepwise in accordance with the coarse-to-fine principle.\",\n \"For a given degree of fineness v of the transformation, the cluster gravity centers CSi are thus defined in each step of the k-means method: CSi=(Σjdid(T(v)(aj),B)3)/(Σjdid(T(v)(aj),B)3, whereby Ii the set of indices of the data points is which is the very next to the ith cluster center.\",\n \"The cluster gravity centers CSi, which are received after the convergence of the method are used as check points.\",\n \"They are in regions with still high distances between data and model points.\",\n \"For starting the method, the 8 corner points of the bounding box around the data point set are used.\",\n \"During our evaluations, the number of checkpoints has been increased stepwise by 1+3 v. FIGS.\",\n \"4-7 show comparisons of both methods for setting the check points.\",\n \"For the representation of the non-rigid transformation, also other methods can be considered, e.g, the octree spline by Szeliski and Lavallee (1994).\",\n \"According to the present invention, in the third registration step—as described above—a weak regularization is made, i.e., a term is added to the error functional which punishes warp.\",\n \"It has been shown that the suppressing of excessive warp as well as the coarse-to-fine method improve the quality of the overlapping considerably in most cases (e.g., the correct assignment of logically corresponding points).\",\n \"Without these method steps, local shape characteristics are destroyed.\",\n \"Reference dynamic.\",\n \"Reference dynamics can be selected which either can be fitted to the motion (transformation) found or can directly be fitted to the data if the reference dynamic can be represented as a parametrized transformation.\",\n \"Possible reference dynamics are, e.g., linear dynamic (affine transformation, with arbitrary time dependence), diffusion dynamic, Hamilton dynamic (e.g., hydrodynamic), elastic (harmonic) vibration.\",\n \"These dynamics can also be combined, simultaneously or sequentially Transformation over several time steps.\",\n \"The transformation (as well as the reference dynamic) is determined according to the present invention also between several time steps tn−i to tn to tn+k.\",\n \"In order to avoid a summing up of errors, which would occur by a mere linking of the transformations between each two time steps, the following procedure is chosen: If the transformation to tn+k should be calculated starting from tn, the same is made as described for tn, tn+1, tn+2, tn+3.By registration, the transformations Tn1, Tn2*, Tn3* are calculated which register the images tn with image tn+1, tn+1 with tn+2 and tn+2 with tn+3.We transform the image tn (or the corresponding extracted point set) by means of Tn2*otn1 (Tn2* linked with Tn1), and register for correction purposes the resulting image again with the image from tn+2.This delivers the transformation Tn2 between the images tn and tn+2.Then we transform the image tn by means of Tn3*otn2 and register for correction purposes the resulting image again with the image from tn+3.This delivers Tn3.Similar steps are taken for the transformation from tn to tn−1, by using the method described for tn.\",\n \"tn+1, tn+2, tn+3 for tn, tn−1, tn−2, tn−3.If new structures (optionally given as further points) appear in an image, e.g., tn+2, these, transformed by Tn3*, are added in a further preceding correction step to the image transformed by Tn3*otn2 before the correction registration with the image tn+3.Alternatively, e.g., in order to maintain Tn2, also image tn can be transformed by means of Tn1 and the thus achieved image (after the correction with new structures) can be registered with tn+2.And so on.\",\n \"For a (optionally only global) motion corrected reproduction of objects/images from the time steps tn to tn+k an absolute reference point in time is necessary.\",\n \"If tn is selected as such a time step, it is, in order to transform all images to tn, an opportunity to calculate each inverse (first for the transformation between tn and tn+1) In order to avoid the calculation of the inverses (at least in the non-rigid and non-affine case), it is in general the easiest way to register the points of tn+1 on such of tn.\",\n \"Combined with the method described above, this can be made between two arbitrary time steps.\",\n \"In order to effect this in real time, i.e., simultaneously with the recording of the images, registration should be effected only twice for each new image.\",\n \"Therefore, the correction steps are performed as above, whereby first the transformation tn+1 and tn is calculated and then between tn+2 and tn (by using the first transformation), etc.\",\n \"If this procedure is used in the case of a direct registration of the gray value images without a preceding point extraction, the gray values are transformed whereby the interpolation for the resampling for the reproduction on a regular discret grid (discretization) is used.\",\n \"Regions of homogeneous motion.\",\n \"For the above-described registered point sets of the time steps tn to tn+k, now the point distribution model already described in the part “description” is established Therefore, the main components are selected, which represent more than a % of the shape variations.\",\n \"Typical values are a=95% or a=90%.\",\n \"A given point on the surface/in the space is then coordinated to the main component which leads to the highest distance d between the point displaced by the positive standard deviation (from the average model) in the direction of the main component and the point displaced by the negative standard deviation.\",\n \"Or alternatively leads to the highest value d/b whereby b is the eigen-value of the corresponding main component.\",\n \"The points of each region of this segmentation can then be affin registered separately with the above-described deviation, in order to assign motion parameters to each sub region Object determination.\",\n \"If the points extracted during the image processing represent different objects, which are identified as such, these can be individually tracked or even registered after the registration of the image space.\",\n \"Therefore it is necessary to first identify each of the equal (or corresponding) objects in the different images.\",\n \"Differently to Eils et al (1989/9), it is only necessary to use a structural similarity measure (Mattes and Demongeot, SPIE Medical Imaging 2001) as the registration already moved the individual objects towards each other.\",\n \"The following individual registration is interesting, e.g., for images of cells in which the rotation or extension of the individual cells are of interest.\",\n \"Visualization and Quantification The calculation of the gray value image series which represents all gray value images of the series corrected by the motion of the reference dynamic (or also corrected by the entire registered motion) is a first visualization embodiment.\",\n \"The explicit calculation of the inverse is herein avoided stepwise as described above (see part “registration”).\",\n \"The image series recalculated in such a manner can then be animated and represented as video.\",\n \"But also all other methods of the analysis of image series and visualization can also be exerted thereon.\",\n \"In particular, these corrected image series are used, in case the motion corrected paths of individual objects should be visualized, which were tracked as described in the part “registration”.\",\n \"The present invention comprises several quantifying and visualization modules which can be used as listed below on its own or in combination.\",\n \"According to the present invention, the motion of the object/image points should be visualized as well as the resulting quantitative values and these especially with regard to the image space.\",\n \"This is achieved by encoding the points of the image space by color and/or by different patterns, which represent different values of quantifiable parameters (a continuous parameter is represented by a pattern becoming denser and denser).\",\n \"Quantification and visualisation modules: 1) Segmented objects or selected confiners in the original image space are visualized as closed but transparent, 3D rendered (surface rendering) surfaces, in the target image space as triangulation grid (which has been transformed by the calculated transformation).\",\n \"Both are before as well as after the registration overlapped and (in different colors) represented (see FIG.\",\n \"8).\",\n \"The only extracted points can also be represented in such a manner.\",\n \"According to the transformation found, intermediate steps are interpolated by means of a segmentation of the parameter interval (of the transformation parameter), the interpolated transformations are exerted on the original object the motion of which can then be represented animatedly.\",\n \"2) The interactive confiner selection (with subsequent visualization) is effected either by clicking on a knot of a confinement-tree or by entering a gray value level and clicking on a spot of an image by which the confiner, containing the spot, is specified on the given level (or optionally no confiner).\",\n \"Without entering the gray value level the smallest confiner containing the spot in the filtered tree will be determined.\",\n \"The visualized surfaces can then also be represented overlapped with the target gray value image.\",\n \"In order not to cover the target gray value image, the parts of the surfaces above the examined level of the target gray value image can be cut off (see also 4c)).\",\n \"The overlapped representation also facilitates a visual control of the quality of the registration 3) The deformation is represented by a 3D rendered reference grid (surface rendering).\",\n \"This grid can be represented animatedly and can be overlapped with the surfaces or one of the gray value images (FIG.\",\n \"8).\",\n \"For different color channels, the reference grids (and optionally the extracted points, too) are represented overlapped in different colors.\",\n \"4) The color/pattern encoded (including gray value encoded) representation of quantified values, which have been calculated for each point in the image space, is effected in the following manner: a) Objects are segmented or confiners are selected, then the 3D rendered surface points are color/pattern encoded.\",\n \"b) Either characteristic points are determined by means of the original point set (see part Image pre-processing/point extraction and Mattes and Demongeot, SPIE Medical Imaging 2001 for the entire image space and Brett and Taylor, Image and Vision Computing 18:739-748, 2000 for points on the surface) or regular points are selected or the user clicks on at least one point in the space or on the segmented/confiner surface.\",\n \"For this/these the path with (error) color(/pattern) encoding is then shown (see FIG.\",\n \"10).\",\n \"c) All points on a level through the space volume interactively determined by the user are represented color/pattern encoded.\",\n \"In the case of 2D images, this is the entire image space.\",\n \"FIG.\",\n \"9 shows an example for the gray value encoded quantity velocity.\",\n \"d) In a) to c) the following quantities are shown in this manner.\",\n \"i) Error with regard to the reference dynamic (for extracted points) as an Euclidean distance between the point transformed with the reference dynamic and the next point in the target space (or from the originally same point transformed by means of the registration of the motion calculated; ii) standardized local warp (Bookstein, Principal Warps: Thin-plate splines and the decomposition of deformations, IEEE Trans.\",\n \"PAMI 11: 567-585, 1995, see also Duchon, Interpolation des functions de deux variables suivant le principe de la flexion des plaques minces, RAIRO Analyse Numérique, 10: 5-12, 1976) integrated over a globe of a given radius around a given point in the image space; iii) ii) for each principal warp (Bookstein, IEEE Trans.\",\n \"PAMI, 1995) and/or for different depths of the check point octree; iv) velocity, acceleration.\",\n \"5) Visualization of the paths of tracked objects/confiners (color encoded).\",\n \"6) Visualization of the paths (determined by registration) of surface points, which are defined (in the original image) either interactively or regularly distributed or as characteristic points (automatically calculated as in point 4b)).\",\n \"7) Interactive deleting of points (e.g., in a bounding box) or surfaces or visualized paths.\",\n \"8) Color encoding of the partitioning which according to the part registration assigns to each point in the space a motion main component (whereby regions with uniform motion type are achieved) and to each motion main component a color.\",\n \"9) Visualization of the motion according to 1) for each main component, for each principal warp and/or for different depths of the check point octree.\",\n \"10) Interpolation for animations and paths as described in 1).\",\n \"11) Quantifying of global parameters, as global warp (particularly for each principal warp, but also for different depths of the check point octree), global errors with regard to the reference dynamic (particularly for each main component), parameter of the reference dynamic, for subsequent steps as well as over several time steps, as discussed in registration and bringing both values into a relation.\",\n \"12) Local parameters, as velocity or acceleration, can be further analyzed with classic statistic methods, as e.g., histogram analysis (number of pixels for each value of the local parameter) or (density based) cluster analysis.\",\n \"Here the confinement tree analysis can be exerted on, e.g., the gray value encoded level determined in 4 c).\",\n \"This is shown in FIG.\",\n \"9 and allows the abstracted description of the motion, which is suitable for a comparison of motions.\",\n \"The homogeneity of the motion direction is characterized by the size of the cluster.\",\n \"By means of a histogram, value ranges of local parameters can be identified (e.g., as 1D confiner), the values of which are particularly often in the image space.\",\n \"The corresponding pixels can be marked in the image/on the surface (e.g., as colored regions).\",\n \"EXAMPLES Examples for the use of visualization and of the methods for the quantitative analysis are: compensation of global microscope or cell nucleus motions (see FIG.\",\n \"1), exemplarily visualization and quantitative analysis of the femur, see Fieres et al.\",\n \"(DAGM 2001) and FIGS.\",\n \"8-10; Usage for analyzing the cell membrane as well as the chromatin dynamic (FIG.\",\n \"10), visualisation of paths of points in individual cells.\",\n \"Dynamic of grained structures in metal during the heating process; analysis and visualization of the heart's, lung's motion; the growth of tumors but also in tissue microtomy; dynamic of the cell nucleus envelope (membrane and lamina) during mitosis (particularly during the start stadium of its break down (FIG.\",\n \"9, Beaudouin et al.\",\n \"Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina.\",\n \"Cell Vol.\",\n \"108, 83-96, 2002, Mattes et al.\",\n \"New tools for visualization and quantification in dynamic processes: Application to the nuclear envelope dynamics during mitosis.\",\n \"MICCAI 1323-1325, 2001).\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471153"}}},{"rowIdx":579,"cells":{"text":{"kind":"list like","value":[["Mechanical seal without elastomers","A seal that provides sealing between a rotatable shaft and a housing has a stationary part for connection to the housing and a rotary part for rotation with the shaft.","The rotary part includes a sleeve for mounting on the shaft.","One end of the sleeve is provided with a non-elastometric sealing arrangement.","The sealing arrangement has sealing surfaces for contacting outer circumferential surfaces of the sleeve and the shaft as well as means for effecting sealing engagement between each of said sleeve and said shaft and a respective sealing surface."],["1.A seal that provides sealing between a rotatable shaft and a housing, comprising: a stationary part connected to the housing; and a rotary part for rotation with the shaft, said rotary part comprising a sleeve mounted on the shaft, one end of the sleeve comprising a non-elastomeric sealing arrangement, said sealing arrangement comprising sealing surfaces that contact the outer circumferential surfaces of the sleeve and the shaft and means for effecting sealing engagement between each of said sleeve and said shaft and a respective sealing surface.","2.A seal according to claim 1, wherein the seal is a mechanical seal.","3.","(canceled) 4.A seal according to claim 1, wherein said sealing arrangement is a single sealing member.","5.","(canceled) 6.","(canceled) 7.","(canceled) 8.","(canceled) 9.","(canceled) 10.A seal according to claim 1, wherein the radial space cross section between the rotatable shaft and housing is at least 0.312″ (7.9 mm) or larger.","11.A seal according to claim 1, wherein the sleeve is radially supported at one or more points along its axial length.","12.A seal according to claim 1, wherein the sleeve is positively driven at one or more points along its axial length.","13.A seal according to claim 1, wherein a component of the seal has a radially disposed relief directly adjacent to a packing member.","14.A seal according to claim 1, wherein a component of the seal is axially secured to another component of the seal, a first one of said components having an axially extending protrusion which radially locates in an axially extending recess, said first component supporting the other component in the vicinity of a packing member.","15.A seal according to claim 14, wherein said components comprise different materials.","16.A seal according to claim 1, wherein one component of the seal, located in contact with process media, has a radially disposed groove to accommodate a sacrificial component.","17.","(canceled)"],["This invention relates to mechanical seals and especially cartridge mechanical seals which use compressed packing materials and not elastomers to create secondary sealing points.","A Mechanical seal comprises a “floating” component which is mounted axially movably around the rotary shaft of, for example, a pump and a “static” component which is axially fixed, typically being secured to a housing.","The floating component has a flat annular end face, i.e.","its seal face, directed towards a complementary seal face of the static component.","The floating component is urged towards the static component to close the seal faces together to form a sliding face seal, usually by means of one or more spring members.","In use, one of the floating and static components rotates; this component is therefore referred to as the rotary component.","The other of the floating and static components does not rotate and is referred to as the stationary component.","Those seals whose floating component is rotary are described as rotary seals.","If the floating component is stationary, the seal is referred to as a stationary seal.","If the sliding seal between the Rotary and Stationary components are assembled and pre-set prior to despatch from the Mechanical seal manufacturing premises, the industry terminology for this is “cartridge seal”.","If the Rotary and Stationary components are despatched individually (unassembled) form the Mechanical Seal manufacturing premises, the industry terminology for this is “component seal”.","Mechanical seals often include elastomeric members to seal static and semi-dynamic surfaces.","In certain seal designs such elastomeric members may be replaced with packing materials, which form a compression seal between two or more surfaces.","For the clarity of this invention the term elastomer may be thought of as on o-ring, lip seal, wiper seal or a similar component which has an elastomeric membrane or structure.","Likewise, a packing material may be thought of as graphite, PTFE or a material, which may be compressed, often to change its density, to create a seal between two or more surfaces.","Such materials are considered to have no, or very little elastomeric membrane.","Mechanical seals are used in all types of industries to seal a variety of different process media and operating conditions.","Such operating conditions range for high temperatures, pressures or particularly aggressive chemicals.","In such cases it is imperative that the correct selection of materials, used in the mechanical seal construction, is made.","Elastomers are often the weakest link in the mechanical seal construction.","This is due to the fact that they need to be chemically compatible with the sealed media and withstand various operating conditions.","Furthermore, elastomers often degrade when subjected to high/low temperatures or pressures.","This often limits the mechanical seal life.","It is considered to be particularly advantageous to create a mechanical seal design with no elastomeric sealing members.","This removes the weakest link thereby helping to extend seal live.","Is is also considered extremely advantageous to create a mechanical seal design with no elastomeric sealing members that will physically fit inside a small radial cross sectional space in a cartridge seal format.","A prior art single cartridge mechanical seal design, which removes the elastomeric sealing member, is shown in FIG.","1.The rotary and axially floating seal face (1) is spring biased towards a static stationary seal face (2).","The rotary face (1) is allowed to slide on the static seal face (2).","The interface between the rotary seal face (1) and stationary seal face (2) forms sealing area (3).","This sealing area (3) is the primary seal that prevents the process media (4) from escaping from the process chamber (5).","At the process side of the deal, denoted as inboard, the detachable removable rotary seal assembly (6) is secured to a sleeve (7) with screws (8).","A compression packing seal (9) is formed between the clamp ring (10) and rotary seal face drive end (11) using screws (12).","The rotary seal assembly (6), screws (8 and 12), packing (9) and clamp ring (10) from the detachable assembly (13).","From FIG.","1 this detachable assembly (13) is fitted to the mechanical seal from the left-hand side due to the radially outwardly extending portion of sleeve (7) on the outboard side.","The rotary compression packing (9) creates the first of three secondary sealing areas.","The second secondary sealing area (14) is formed between stationary seal face (2) and stationary gland (15) using a gland compression packing (16).","The gland packing (16) is compressed in place by the spring bias of the rotary seal assembly (6) and by the hydraulic force established from the process media (4) pressure.","At atmospheric side of the seal, denoted as outboard, the sleeve (7) increases radially to accommodate a sleeve compression packing (17).","The packing (17) is compressed to the shaft (18) by the clamp ring (19) and screws (20) to from the third secondary sealing area.","Form FIG.","1 it is evident that when radial screws (8) are secured to sleeve (7), the sleeve (7) will radially deform.","This creates assembly difficulties.","The deformation can be limited it the radial cross section of the sleeve (7) is increased as shown in FIG.","1.A large proportion of standard process equipment is designed with either a 0.3125″ (8 mm), 0.375″ (10 mm) or 0.500″ (12 mm) radial cross section between the shaft (18) and the process chamber (5), depending on the shaft diameter.","Radially increasing the cartridge sleeve (7) as shown in FIG.","1 limits where the design can be physically applied.","The materials used in the design shown in FIG.","1 also suffer from chemical attack problems since the screws (8) and (12) are sited in the process media.","This could limit seal life and make the design commercially impractical as exotic alloy screw materials are not commonly available.","It is therefore considered that a seal design which removes the screws (8) and (12) out of the process media, removes all elastomeric sealing members and is offered to fit inside a radilly small cross sectional inboard opening is deemed considerably advantageous.","From FIG.","1, the screws (8) and (12) could be eliminated by a leak tight, permanent joint fixing the clamp ring (10) to the sleeve (7) and rotary seal face drive end (11).","However as the sleeve (7) increases radially on the outboard side to cater for the shaft packing (17) this would mean that the design could not be assembled or disassembled for seal repair.","The seal features described above are therefore not possible to accommodate with the existing prior art technology.","The present invention is described by way of example only with reference to the accompanying drawings, in which: FIG.","2.is a longitudinal cross section through a mechanical seal of the invention.","FIG.","3 corresponds to FIG.","2 and is an enlarged partial longitudinal cross section through a mechanical seal of the invention.","FIG.","4 corresponds to FIG.","3 and is an enlarged partial longitudinal cross section through the outboard side of a mechanical seal of the invention.","FIG.","5 corresponds to FIG.","3 and is an enlarged partial longitudinal cross section through the inboard side of a mechanical seal of the invention.","FIG.","6 shows an enlarged partial longitudinal cross section of an alternate inboard arrangement of the invention with a non-detachable inboard seal face assembly.","FIG.","7 shows a partial cross section of an alternate arrangement of the invention with a different outboard seal to shaft sealing arrangement without the sleeve supporting member.","FIG.","8 shows a partial cross section of an alternate arrangement of the invention with an optional inboard sleeve supporting member.","FIG.","9 shows a partial cross section of an alternate arrangement of the invention with a different outboard seal to shaft sealing arrangement.","FIG.","10 shows a partial cross section of an alternate arrangement of the invention with a different outboard seal to shaft sealing arrangement.","FIG.","11 shows a partial cross section through a double cartridge mechanical seal of the invention.","FIG.","12 shows a longitudinal cross section through a non-mechanical seal design.","FIG.","13 shows a partial cross section of an alternate arrangement of the invention with a different outboard seal to shaft sealing arrangement.","From FIG.","2, of the invention the rotary and axially floating seal face (1) is spring biased towards a static stationary seal face (2).","The rotary face (1) is allowed to slide an the static seal face (2).","The interface between the rotary seal face (1) and stationary seal face (2) forms sealing area (3).","This sealing area (3) is the primary seal that prevents the process media (4) from escaping from the process chamber (5).","At the process media (4) side of the seal, denoted as inboard, the detachable removable rotary seal assembly (6) is secured to a sleeve (7) with screws (12).","A compression packing seal (9) is formed between the sleeve (7) and rotary seal face drive end (11) using screws (12).","The rotary compression packing (9) creates the first of four secondary sealing areas.","The second secondary sealing area (14) is formed between stationary seal face (2) and stationary gland (15) using a gland compression packing (16).","The gland packing (16) is compressed in place by the spring bias of the rotary seal assembly (6) and by the hydraulic force established form the process media (4) pressure.","At atmospheric side of the seal, denoted as outboard, the sleeve (7) is terminated adjacent to the drive ring (21).","A draw ring (22) located radially outwardly of sleeve (7) is axially restrained by a circlip (23).","The compression packing (24) is located radially outwardly of sleeve (7) and is compressed between the draw ring (22), spacer ring (26) and drive ring (21) using screws (25).","The separate spacer ring (26) is used to facilitate component manufacture.","It is considered self evident that this could be omitted if the profile of the spacer ring (26) is machined into the drive ring (21) to accommodate the angle on the packing member (24).","The clamp ring (27) compresses the shaft packing (17) when it is secured to the drive ring (21) using screws (28).","The invention illustrated in FIG.","2 therefore offers a cartridge mechanical seal with no elastomeric members which will fit into small radial cross sections.","This is seen as a major advantage.","From FIG.","3 it should be noted that the drive ring (21) has a radial support lip (29) that locates into the recess (30) in sleeve (7).","As the packing (24) is compressed it exerts a radial force on the end of the sleeve (7).","The support lip (29) in drive ring (21) supports the end of the sleeve (7) helping to prevent sleeve (7) distortion.","The process media (4) is allowed enter the radial gap (31) between the sleeve (7) and shaft (18).","The process media (4) is sealed by packing members (24 and 17).","There are no other un-sealed joints, between the respective outboard parts, where the process media (4) contacts.","FIG.","4 illustrates the drive lug (32) in the drive (21) which locates into corresponding slots (33) in sleeve (7).","Rotational drive from the shaft (18) is transmitted via the redial screws (34) located in the clamp ring (19) through the screws (28) into the drive ring (21) through the drive lugs (32) into the sleeve (7), and then into the rotary seal assembly (6) via the screws (12).","This positive drive mechanism is thought to be particularly advantageous in certain applications.","Furthermore, the arrangement of the drive acting in the end of the sleeve (7) ensures that the thin cartridge sleeve (7) is not weakened by the introduction of radial slots away from the packing ling area (24).","Referring to FIG.","5, it should be noted that sleeve (7) has an elastomer groove (35) positioned at the inboard side in contact with the process media (4).","This allows a sacrificial component to be installed into groove (35), if the application dictates.","This helps to eliminate the possibility of process media (4) contamination in radial gap (31), therefore assisting the removal of the cartridge mechanical seal from the shaft (18).","FIG.","5 also illustrates a relief (36) extending radially outwardly in the bore of the sleeve (7).","Said relief is positioned in the vicinity of the packing member (9).","Any radial sleeve (7) deformation occurred when compressing packing member (9) using screws (12) is accommodated by the relief (36).","This is of particular importance when using packing (9) acting on a thin cross section cartridge sleeve (7).","From FIG.","6 an alternate arrangement of the invention may be viewed.","The inboard screws (12), packing (9) and rotary drive end (11) have been removed.","FIG.","6 illustrates, by way of example only, the rotary seal face assembly (6), in particular the metal bellows spring member (37) has been permanently fixed to the radially outwardly protruding section of the sleeve (7).","This design is seen to be particularly advantageous when chemical attack is of prime consideration as there are no screws (12) in the process media.","Therefore all materials in contact with the process media can be selected in one or more exotic alloy or non-metallic combinations.","A person skilled in the art of mechanical seal design will appreciate that the invention allows many different inboard seal configurations while allowing a mechanical cartridge seal, with no elastomers, to be fitted into a thin cross sectional radial space between the shaft (18) and process chamber (5).","It is considered self evident from FIG.","3 that the invention can be offered without the radial lip (29) on drive ring (21) and corresponding recess (30) in sleeve (7).","The invention without this supporting feature is shown in FIG.","7.If it is considered appropriate, the sleeve (7) deformation, as the result of radial force exerted by the packing (24) can be minimised by one or a combination of several factors.","Such factors may include changing the materials of construction, reducing the squeeze hence force applied on the packing (24) or designing sufficient radial relief in the inner diameter of the sleeve (7) to accommodate the deformation.","This principle has been illustrated in FIG.","5 for the inboard side of the sleeve (7) but could also be applied to the outboard side of the sleeve (7) if required.","FIG.","7 also illustrates a design without a positive drive mechanism.","The seal drive is transmitted through the metal to metal friction drive (54) by the screws (25) applying a compression load on sleeve (7) and drive ring (21).","Similarly, FIG.","8 illustrates the inboard end of sleeve (7) with a supporting end cap (38) clamping the sleeve (7) with packing (9) and the rotary seal face drive end (11) using screws (12).","Clamp ring (38) has an axially extending lip (39) which radially locates in recess (40) of sleeve (7).","This lip (39) supports the end of the sleeve (7) from radially deforming under the force exerted by the packing (9) when compressed.","This is particularly important when the cartridge mechanical seal is required to fit into a thin radial cross sectional space.","It has been surprising found that said end cap (38) could be offered in an exotic alloy material for increased chemical resistance.","If required a non-elastomeric gasket could be added in between the end cap (38) and drive end (11) in area (41) to protect the sleeve (7) further.","This offers a commercial advantage.","By way of example only, FIG.","9 shows a partial cross section of an alternate arrangement of the invention with a different outboard seal to shaft sealing arrangement.","From FIG.","9 the sleeve (7) terminates in the vicinity of the drive ring (42).","The dive ring (42) is secured to the sleeve (7) using radial screws (43).","If required an appropriate relief (63) could be included in the sleeve (7).","The packing (44) is compressed by the clamp ring (27) using screws (28).","When the clamp ring (27) reaches its installed axial position it is secured to the shaft (18) using radial screws (45).","The compressed packing (44) forms a leak tight seal between the shaft (18), the sleeve (7), drive ring (42) and clamp ring (27).","By way of example only, FIG.","10 shows a partial cross section of an alternate arrangement of the invention with a different outboard seal to shaft sealing arrangement.","From FIG.","10 the sleeve (7) terminates in the vicinity of the split drive ring (46).","The split drive ring (46) is radially supported by locking collar (47) which extends radially outwardly to the split drive ring (46).","This arrangement ensures that the radial inwardly protruding lug (48) in the split drive ring (46) engages in the radial grove (49) in the sleeve (7) and thereby provides axial positioning.","Spacer ring (50) radially engages into the drive ring (46) and acts to compress packing (51) when screws (52) are secured.","Screws (52) located in clamp ring (53).","By way of example only, FIG.","11 illustrates the invention applied to a double cartridge mechanical seal.","From FIG.","11 the stationary and axially floating seal face (2) is spring biased towards an axially static rotary seal face (1).","The rotary face (1) is allowed to slide on the stationary seal face (2).","The interface between the rotary face (1) and stationary seal face (2) forms sealing area (3).","This sealing area (3) is the primary seal that prevents the process media (4) from escaping from the process chamber (5).","The second primary seal is made in the barrier media chamber (55) on the outboard side of the mechanical seal.","The stationary and axially floating seal face (56) is spring biased towards an axially static rotary seal face (57).","The rotary face (57) is allowed to slide on the stationary seal face (56).","The interface between the rotary seal face (57) and stationary seal face (56) forms sealing area (58).","This sealing area (58) is the primary seal that prevents the barrier media from escaping from the barrier media chamber (55).","Secondary sealing members in contact with the process media include packing members (59, 60 and 61).","As described previously, the process media is allowed to travel in the radial gap (62) between the shaft (18) and the sleeve (7).","It then is sealed by two further secondary packing members (17 and 24) as described in detail previously.","FIG.","11 clearly illustrates the invention applies to yet another inboard seal configuration.","It is therefore clear that the invention can be applied to any arrangement with a very thin radial cross sectional sleeve.","For reference the invention could be applied to a sleeve with a radial cross section as small as 0.020″ (0.5 mm).","By way of example only, FIG.","12 illustrates the invention applied to the atmospheric side of a “non-mechanical” design.","One or more packing member's (63) are radially outwardly positioned on sleeve (7).","The packing member (63) forms a sliding contracting seal between the press chamber (5) and outer radial portion of the sleeve (7).","Said packing mnember's (63) are compressed in position by the gland (15) and bolts (64).","This arrangement prevents the process media (4) form escaping from the process chamber (5).","The sleeve (7) to shaft (18) area is sealed by the invention assembly (65) as described previously.","FIG.","13 illustrates an alternative arrangement of the invention, The split drive ring (46) has a radially inwardly protrusion (67) that locates into a radial recess (67) of sleeve (7).","The split drive ring (46) is radially restrained by support ring (47).","This arrangement ensures that the protrusion (67) can not disengage from recess (68).","The spilt drive ring (46) has an appropriate positive drive mechanism to transmit the rotational drive into the sleeve (7).","Radial screws (66) axially connect the split drive ring (46) to clamp ring (27).","The process media (4) is sealed when the packing member (44) is compressed by push ring (69) and axial screws (28).","It is considered self evident to the experienced reader that the invention may be employed for both Rotary seals and Stationary seals, single, double or triple mechanical seals, whether designed in a cartridge or component seal format.","It is also considered self evident that the invention may be used with metallic components as well as non-metallic components.","Some types of equipment rotate the housing and have a stationary shaft.","It is considered that the invention can be similarly applied to such designs.","The invention may also be applied to a non-mechanical seal design, an example of which is illustrated in FIG.","12."]],"string":"[\n [\n \"Mechanical seal without elastomers\",\n \"A seal that provides sealing between a rotatable shaft and a housing has a stationary part for connection to the housing and a rotary part for rotation with the shaft.\",\n \"The rotary part includes a sleeve for mounting on the shaft.\",\n \"One end of the sleeve is provided with a non-elastometric sealing arrangement.\",\n \"The sealing arrangement has sealing surfaces for contacting outer circumferential surfaces of the sleeve and the shaft as well as means for effecting sealing engagement between each of said sleeve and said shaft and a respective sealing surface.\"\n ],\n [\n \"1.A seal that provides sealing between a rotatable shaft and a housing, comprising: a stationary part connected to the housing; and a rotary part for rotation with the shaft, said rotary part comprising a sleeve mounted on the shaft, one end of the sleeve comprising a non-elastomeric sealing arrangement, said sealing arrangement comprising sealing surfaces that contact the outer circumferential surfaces of the sleeve and the shaft and means for effecting sealing engagement between each of said sleeve and said shaft and a respective sealing surface.\",\n \"2.A seal according to claim 1, wherein the seal is a mechanical seal.\",\n \"3.\",\n \"(canceled) 4.A seal according to claim 1, wherein said sealing arrangement is a single sealing member.\",\n \"5.\",\n \"(canceled) 6.\",\n \"(canceled) 7.\",\n \"(canceled) 8.\",\n \"(canceled) 9.\",\n \"(canceled) 10.A seal according to claim 1, wherein the radial space cross section between the rotatable shaft and housing is at least 0.312″ (7.9 mm) or larger.\",\n \"11.A seal according to claim 1, wherein the sleeve is radially supported at one or more points along its axial length.\",\n \"12.A seal according to claim 1, wherein the sleeve is positively driven at one or more points along its axial length.\",\n \"13.A seal according to claim 1, wherein a component of the seal has a radially disposed relief directly adjacent to a packing member.\",\n \"14.A seal according to claim 1, wherein a component of the seal is axially secured to another component of the seal, a first one of said components having an axially extending protrusion which radially locates in an axially extending recess, said first component supporting the other component in the vicinity of a packing member.\",\n \"15.A seal according to claim 14, wherein said components comprise different materials.\",\n \"16.A seal according to claim 1, wherein one component of the seal, located in contact with process media, has a radially disposed groove to accommodate a sacrificial component.\",\n \"17.\",\n \"(canceled)\"\n ],\n [\n \"This invention relates to mechanical seals and especially cartridge mechanical seals which use compressed packing materials and not elastomers to create secondary sealing points.\",\n \"A Mechanical seal comprises a “floating” component which is mounted axially movably around the rotary shaft of, for example, a pump and a “static” component which is axially fixed, typically being secured to a housing.\",\n \"The floating component has a flat annular end face, i.e.\",\n \"its seal face, directed towards a complementary seal face of the static component.\",\n \"The floating component is urged towards the static component to close the seal faces together to form a sliding face seal, usually by means of one or more spring members.\",\n \"In use, one of the floating and static components rotates; this component is therefore referred to as the rotary component.\",\n \"The other of the floating and static components does not rotate and is referred to as the stationary component.\",\n \"Those seals whose floating component is rotary are described as rotary seals.\",\n \"If the floating component is stationary, the seal is referred to as a stationary seal.\",\n \"If the sliding seal between the Rotary and Stationary components are assembled and pre-set prior to despatch from the Mechanical seal manufacturing premises, the industry terminology for this is “cartridge seal”.\",\n \"If the Rotary and Stationary components are despatched individually (unassembled) form the Mechanical Seal manufacturing premises, the industry terminology for this is “component seal”.\",\n \"Mechanical seals often include elastomeric members to seal static and semi-dynamic surfaces.\",\n \"In certain seal designs such elastomeric members may be replaced with packing materials, which form a compression seal between two or more surfaces.\",\n \"For the clarity of this invention the term elastomer may be thought of as on o-ring, lip seal, wiper seal or a similar component which has an elastomeric membrane or structure.\",\n \"Likewise, a packing material may be thought of as graphite, PTFE or a material, which may be compressed, often to change its density, to create a seal between two or more surfaces.\",\n \"Such materials are considered to have no, or very little elastomeric membrane.\",\n \"Mechanical seals are used in all types of industries to seal a variety of different process media and operating conditions.\",\n \"Such operating conditions range for high temperatures, pressures or particularly aggressive chemicals.\",\n \"In such cases it is imperative that the correct selection of materials, used in the mechanical seal construction, is made.\",\n \"Elastomers are often the weakest link in the mechanical seal construction.\",\n \"This is due to the fact that they need to be chemically compatible with the sealed media and withstand various operating conditions.\",\n \"Furthermore, elastomers often degrade when subjected to high/low temperatures or pressures.\",\n \"This often limits the mechanical seal life.\",\n \"It is considered to be particularly advantageous to create a mechanical seal design with no elastomeric sealing members.\",\n \"This removes the weakest link thereby helping to extend seal live.\",\n \"Is is also considered extremely advantageous to create a mechanical seal design with no elastomeric sealing members that will physically fit inside a small radial cross sectional space in a cartridge seal format.\",\n \"A prior art single cartridge mechanical seal design, which removes the elastomeric sealing member, is shown in FIG.\",\n \"1.The rotary and axially floating seal face (1) is spring biased towards a static stationary seal face (2).\",\n \"The rotary face (1) is allowed to slide on the static seal face (2).\",\n \"The interface between the rotary seal face (1) and stationary seal face (2) forms sealing area (3).\",\n \"This sealing area (3) is the primary seal that prevents the process media (4) from escaping from the process chamber (5).\",\n \"At the process side of the deal, denoted as inboard, the detachable removable rotary seal assembly (6) is secured to a sleeve (7) with screws (8).\",\n \"A compression packing seal (9) is formed between the clamp ring (10) and rotary seal face drive end (11) using screws (12).\",\n \"The rotary seal assembly (6), screws (8 and 12), packing (9) and clamp ring (10) from the detachable assembly (13).\",\n \"From FIG.\",\n \"1 this detachable assembly (13) is fitted to the mechanical seal from the left-hand side due to the radially outwardly extending portion of sleeve (7) on the outboard side.\",\n \"The rotary compression packing (9) creates the first of three secondary sealing areas.\",\n \"The second secondary sealing area (14) is formed between stationary seal face (2) and stationary gland (15) using a gland compression packing (16).\",\n \"The gland packing (16) is compressed in place by the spring bias of the rotary seal assembly (6) and by the hydraulic force established from the process media (4) pressure.\",\n \"At atmospheric side of the seal, denoted as outboard, the sleeve (7) increases radially to accommodate a sleeve compression packing (17).\",\n \"The packing (17) is compressed to the shaft (18) by the clamp ring (19) and screws (20) to from the third secondary sealing area.\",\n \"Form FIG.\",\n \"1 it is evident that when radial screws (8) are secured to sleeve (7), the sleeve (7) will radially deform.\",\n \"This creates assembly difficulties.\",\n \"The deformation can be limited it the radial cross section of the sleeve (7) is increased as shown in FIG.\",\n \"1.A large proportion of standard process equipment is designed with either a 0.3125″ (8 mm), 0.375″ (10 mm) or 0.500″ (12 mm) radial cross section between the shaft (18) and the process chamber (5), depending on the shaft diameter.\",\n \"Radially increasing the cartridge sleeve (7) as shown in FIG.\",\n \"1 limits where the design can be physically applied.\",\n \"The materials used in the design shown in FIG.\",\n \"1 also suffer from chemical attack problems since the screws (8) and (12) are sited in the process media.\",\n \"This could limit seal life and make the design commercially impractical as exotic alloy screw materials are not commonly available.\",\n \"It is therefore considered that a seal design which removes the screws (8) and (12) out of the process media, removes all elastomeric sealing members and is offered to fit inside a radilly small cross sectional inboard opening is deemed considerably advantageous.\",\n \"From FIG.\",\n \"1, the screws (8) and (12) could be eliminated by a leak tight, permanent joint fixing the clamp ring (10) to the sleeve (7) and rotary seal face drive end (11).\",\n \"However as the sleeve (7) increases radially on the outboard side to cater for the shaft packing (17) this would mean that the design could not be assembled or disassembled for seal repair.\",\n \"The seal features described above are therefore not possible to accommodate with the existing prior art technology.\",\n \"The present invention is described by way of example only with reference to the accompanying drawings, in which: FIG.\",\n \"2.is a longitudinal cross section through a mechanical seal of the invention.\",\n \"FIG.\",\n \"3 corresponds to FIG.\",\n \"2 and is an enlarged partial longitudinal cross section through a mechanical seal of the invention.\",\n \"FIG.\",\n \"4 corresponds to FIG.\",\n \"3 and is an enlarged partial longitudinal cross section through the outboard side of a mechanical seal of the invention.\",\n \"FIG.\",\n \"5 corresponds to FIG.\",\n \"3 and is an enlarged partial longitudinal cross section through the inboard side of a mechanical seal of the invention.\",\n \"FIG.\",\n \"6 shows an enlarged partial longitudinal cross section of an alternate inboard arrangement of the invention with a non-detachable inboard seal face assembly.\",\n \"FIG.\",\n \"7 shows a partial cross section of an alternate arrangement of the invention with a different outboard seal to shaft sealing arrangement without the sleeve supporting member.\",\n \"FIG.\",\n \"8 shows a partial cross section of an alternate arrangement of the invention with an optional inboard sleeve supporting member.\",\n \"FIG.\",\n \"9 shows a partial cross section of an alternate arrangement of the invention with a different outboard seal to shaft sealing arrangement.\",\n \"FIG.\",\n \"10 shows a partial cross section of an alternate arrangement of the invention with a different outboard seal to shaft sealing arrangement.\",\n \"FIG.\",\n \"11 shows a partial cross section through a double cartridge mechanical seal of the invention.\",\n \"FIG.\",\n \"12 shows a longitudinal cross section through a non-mechanical seal design.\",\n \"FIG.\",\n \"13 shows a partial cross section of an alternate arrangement of the invention with a different outboard seal to shaft sealing arrangement.\",\n \"From FIG.\",\n \"2, of the invention the rotary and axially floating seal face (1) is spring biased towards a static stationary seal face (2).\",\n \"The rotary face (1) is allowed to slide an the static seal face (2).\",\n \"The interface between the rotary seal face (1) and stationary seal face (2) forms sealing area (3).\",\n \"This sealing area (3) is the primary seal that prevents the process media (4) from escaping from the process chamber (5).\",\n \"At the process media (4) side of the seal, denoted as inboard, the detachable removable rotary seal assembly (6) is secured to a sleeve (7) with screws (12).\",\n \"A compression packing seal (9) is formed between the sleeve (7) and rotary seal face drive end (11) using screws (12).\",\n \"The rotary compression packing (9) creates the first of four secondary sealing areas.\",\n \"The second secondary sealing area (14) is formed between stationary seal face (2) and stationary gland (15) using a gland compression packing (16).\",\n \"The gland packing (16) is compressed in place by the spring bias of the rotary seal assembly (6) and by the hydraulic force established form the process media (4) pressure.\",\n \"At atmospheric side of the seal, denoted as outboard, the sleeve (7) is terminated adjacent to the drive ring (21).\",\n \"A draw ring (22) located radially outwardly of sleeve (7) is axially restrained by a circlip (23).\",\n \"The compression packing (24) is located radially outwardly of sleeve (7) and is compressed between the draw ring (22), spacer ring (26) and drive ring (21) using screws (25).\",\n \"The separate spacer ring (26) is used to facilitate component manufacture.\",\n \"It is considered self evident that this could be omitted if the profile of the spacer ring (26) is machined into the drive ring (21) to accommodate the angle on the packing member (24).\",\n \"The clamp ring (27) compresses the shaft packing (17) when it is secured to the drive ring (21) using screws (28).\",\n \"The invention illustrated in FIG.\",\n \"2 therefore offers a cartridge mechanical seal with no elastomeric members which will fit into small radial cross sections.\",\n \"This is seen as a major advantage.\",\n \"From FIG.\",\n \"3 it should be noted that the drive ring (21) has a radial support lip (29) that locates into the recess (30) in sleeve (7).\",\n \"As the packing (24) is compressed it exerts a radial force on the end of the sleeve (7).\",\n \"The support lip (29) in drive ring (21) supports the end of the sleeve (7) helping to prevent sleeve (7) distortion.\",\n \"The process media (4) is allowed enter the radial gap (31) between the sleeve (7) and shaft (18).\",\n \"The process media (4) is sealed by packing members (24 and 17).\",\n \"There are no other un-sealed joints, between the respective outboard parts, where the process media (4) contacts.\",\n \"FIG.\",\n \"4 illustrates the drive lug (32) in the drive (21) which locates into corresponding slots (33) in sleeve (7).\",\n \"Rotational drive from the shaft (18) is transmitted via the redial screws (34) located in the clamp ring (19) through the screws (28) into the drive ring (21) through the drive lugs (32) into the sleeve (7), and then into the rotary seal assembly (6) via the screws (12).\",\n \"This positive drive mechanism is thought to be particularly advantageous in certain applications.\",\n \"Furthermore, the arrangement of the drive acting in the end of the sleeve (7) ensures that the thin cartridge sleeve (7) is not weakened by the introduction of radial slots away from the packing ling area (24).\",\n \"Referring to FIG.\",\n \"5, it should be noted that sleeve (7) has an elastomer groove (35) positioned at the inboard side in contact with the process media (4).\",\n \"This allows a sacrificial component to be installed into groove (35), if the application dictates.\",\n \"This helps to eliminate the possibility of process media (4) contamination in radial gap (31), therefore assisting the removal of the cartridge mechanical seal from the shaft (18).\",\n \"FIG.\",\n \"5 also illustrates a relief (36) extending radially outwardly in the bore of the sleeve (7).\",\n \"Said relief is positioned in the vicinity of the packing member (9).\",\n \"Any radial sleeve (7) deformation occurred when compressing packing member (9) using screws (12) is accommodated by the relief (36).\",\n \"This is of particular importance when using packing (9) acting on a thin cross section cartridge sleeve (7).\",\n \"From FIG.\",\n \"6 an alternate arrangement of the invention may be viewed.\",\n \"The inboard screws (12), packing (9) and rotary drive end (11) have been removed.\",\n \"FIG.\",\n \"6 illustrates, by way of example only, the rotary seal face assembly (6), in particular the metal bellows spring member (37) has been permanently fixed to the radially outwardly protruding section of the sleeve (7).\",\n \"This design is seen to be particularly advantageous when chemical attack is of prime consideration as there are no screws (12) in the process media.\",\n \"Therefore all materials in contact with the process media can be selected in one or more exotic alloy or non-metallic combinations.\",\n \"A person skilled in the art of mechanical seal design will appreciate that the invention allows many different inboard seal configurations while allowing a mechanical cartridge seal, with no elastomers, to be fitted into a thin cross sectional radial space between the shaft (18) and process chamber (5).\",\n \"It is considered self evident from FIG.\",\n \"3 that the invention can be offered without the radial lip (29) on drive ring (21) and corresponding recess (30) in sleeve (7).\",\n \"The invention without this supporting feature is shown in FIG.\",\n \"7.If it is considered appropriate, the sleeve (7) deformation, as the result of radial force exerted by the packing (24) can be minimised by one or a combination of several factors.\",\n \"Such factors may include changing the materials of construction, reducing the squeeze hence force applied on the packing (24) or designing sufficient radial relief in the inner diameter of the sleeve (7) to accommodate the deformation.\",\n \"This principle has been illustrated in FIG.\",\n \"5 for the inboard side of the sleeve (7) but could also be applied to the outboard side of the sleeve (7) if required.\",\n \"FIG.\",\n \"7 also illustrates a design without a positive drive mechanism.\",\n \"The seal drive is transmitted through the metal to metal friction drive (54) by the screws (25) applying a compression load on sleeve (7) and drive ring (21).\",\n \"Similarly, FIG.\",\n \"8 illustrates the inboard end of sleeve (7) with a supporting end cap (38) clamping the sleeve (7) with packing (9) and the rotary seal face drive end (11) using screws (12).\",\n \"Clamp ring (38) has an axially extending lip (39) which radially locates in recess (40) of sleeve (7).\",\n \"This lip (39) supports the end of the sleeve (7) from radially deforming under the force exerted by the packing (9) when compressed.\",\n \"This is particularly important when the cartridge mechanical seal is required to fit into a thin radial cross sectional space.\",\n \"It has been surprising found that said end cap (38) could be offered in an exotic alloy material for increased chemical resistance.\",\n \"If required a non-elastomeric gasket could be added in between the end cap (38) and drive end (11) in area (41) to protect the sleeve (7) further.\",\n \"This offers a commercial advantage.\",\n \"By way of example only, FIG.\",\n \"9 shows a partial cross section of an alternate arrangement of the invention with a different outboard seal to shaft sealing arrangement.\",\n \"From FIG.\",\n \"9 the sleeve (7) terminates in the vicinity of the drive ring (42).\",\n \"The dive ring (42) is secured to the sleeve (7) using radial screws (43).\",\n \"If required an appropriate relief (63) could be included in the sleeve (7).\",\n \"The packing (44) is compressed by the clamp ring (27) using screws (28).\",\n \"When the clamp ring (27) reaches its installed axial position it is secured to the shaft (18) using radial screws (45).\",\n \"The compressed packing (44) forms a leak tight seal between the shaft (18), the sleeve (7), drive ring (42) and clamp ring (27).\",\n \"By way of example only, FIG.\",\n \"10 shows a partial cross section of an alternate arrangement of the invention with a different outboard seal to shaft sealing arrangement.\",\n \"From FIG.\",\n \"10 the sleeve (7) terminates in the vicinity of the split drive ring (46).\",\n \"The split drive ring (46) is radially supported by locking collar (47) which extends radially outwardly to the split drive ring (46).\",\n \"This arrangement ensures that the radial inwardly protruding lug (48) in the split drive ring (46) engages in the radial grove (49) in the sleeve (7) and thereby provides axial positioning.\",\n \"Spacer ring (50) radially engages into the drive ring (46) and acts to compress packing (51) when screws (52) are secured.\",\n \"Screws (52) located in clamp ring (53).\",\n \"By way of example only, FIG.\",\n \"11 illustrates the invention applied to a double cartridge mechanical seal.\",\n \"From FIG.\",\n \"11 the stationary and axially floating seal face (2) is spring biased towards an axially static rotary seal face (1).\",\n \"The rotary face (1) is allowed to slide on the stationary seal face (2).\",\n \"The interface between the rotary face (1) and stationary seal face (2) forms sealing area (3).\",\n \"This sealing area (3) is the primary seal that prevents the process media (4) from escaping from the process chamber (5).\",\n \"The second primary seal is made in the barrier media chamber (55) on the outboard side of the mechanical seal.\",\n \"The stationary and axially floating seal face (56) is spring biased towards an axially static rotary seal face (57).\",\n \"The rotary face (57) is allowed to slide on the stationary seal face (56).\",\n \"The interface between the rotary seal face (57) and stationary seal face (56) forms sealing area (58).\",\n \"This sealing area (58) is the primary seal that prevents the barrier media from escaping from the barrier media chamber (55).\",\n \"Secondary sealing members in contact with the process media include packing members (59, 60 and 61).\",\n \"As described previously, the process media is allowed to travel in the radial gap (62) between the shaft (18) and the sleeve (7).\",\n \"It then is sealed by two further secondary packing members (17 and 24) as described in detail previously.\",\n \"FIG.\",\n \"11 clearly illustrates the invention applies to yet another inboard seal configuration.\",\n \"It is therefore clear that the invention can be applied to any arrangement with a very thin radial cross sectional sleeve.\",\n \"For reference the invention could be applied to a sleeve with a radial cross section as small as 0.020″ (0.5 mm).\",\n \"By way of example only, FIG.\",\n \"12 illustrates the invention applied to the atmospheric side of a “non-mechanical” design.\",\n \"One or more packing member's (63) are radially outwardly positioned on sleeve (7).\",\n \"The packing member (63) forms a sliding contracting seal between the press chamber (5) and outer radial portion of the sleeve (7).\",\n \"Said packing mnember's (63) are compressed in position by the gland (15) and bolts (64).\",\n \"This arrangement prevents the process media (4) form escaping from the process chamber (5).\",\n \"The sleeve (7) to shaft (18) area is sealed by the invention assembly (65) as described previously.\",\n \"FIG.\",\n \"13 illustrates an alternative arrangement of the invention, The split drive ring (46) has a radially inwardly protrusion (67) that locates into a radial recess (67) of sleeve (7).\",\n \"The split drive ring (46) is radially restrained by support ring (47).\",\n \"This arrangement ensures that the protrusion (67) can not disengage from recess (68).\",\n \"The spilt drive ring (46) has an appropriate positive drive mechanism to transmit the rotational drive into the sleeve (7).\",\n \"Radial screws (66) axially connect the split drive ring (46) to clamp ring (27).\",\n \"The process media (4) is sealed when the packing member (44) is compressed by push ring (69) and axial screws (28).\",\n \"It is considered self evident to the experienced reader that the invention may be employed for both Rotary seals and Stationary seals, single, double or triple mechanical seals, whether designed in a cartridge or component seal format.\",\n \"It is also considered self evident that the invention may be used with metallic components as well as non-metallic components.\",\n \"Some types of equipment rotate the housing and have a stationary shaft.\",\n \"It is considered that the invention can be similarly applied to such designs.\",\n \"The invention may also be applied to a non-mechanical seal design, an example of which is illustrated in FIG.\",\n \"12.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471158"}}},{"rowIdx":580,"cells":{"text":{"kind":"list like","value":[["COOLING OF MILK IN AN AUTOMATIC MILKING SYSTEM","The present invention relates to a method for cooling milk in a milk storage tank of an automatic milling system comprising the steps of (i) measuring an amount of extracted milk by means of a milk flow meter, (ii) determining a cooling need for milk stored or to be stored in said milk storage tank based on said amount of milk; (iii) measuring a quantity indicative of a temperature of an inner surface area of a bottom portion of the milk storage tank; and cooling said bottom portion of said milk storage tank in consecutive periods, such that each period of cooling (τ1, τ3) is followed by a respective period of non-cooling (τ2, τ4), wherein the duration of each period of cooling and/or noncooling is based on said measured quantity indicative of said inner surface area temperature, and said cooling need.","The invention also relates to an arrangement for performing the method."],["1.A method for cooling milk in an automatic milking system, which comprises a milking robot (1) for milking animals, such as cows; a milk storage tank (5) connected to said milking robot for storing milk extracted by the milking robot; and a cooling device (7) for cooling milk stored or to be stored in said milk storage tank by cooling a bottom portion (14) of said milk storage tank, said method being characterized by the steps of: measuring an amount of milk extracted by said milking robot by means of a milk flow meter; determining a cooling need for milk stored or to be stored in said milk storage tank based on said measured amount of milk; measuring a quantity indicative of a temperature of an inner surface area (16) of the bottom portion of the milk storage tank; and cooling said bottom portion of said milk storage tank in consecutive periods, such that each period of cooling (τ1, τ3) is followed by a respective period of non-cooling (τ2, τ4), wherein the duration of each period of cooling and/or non-cooling is based on said measured quantity indicative of the inner surface temperature, and said determined cooling need.","2.The method as claimed in claim 1 wherein at least one period of cooling is started while said milk storage tank is empty of milk.","3.The method as claimed in claim 1 or 2 wherein the steps of measuring an amount of milk; determining a cooling need; measuring a quantity indicative of a temperature of an inner surface area of the bottom portion of the milk storage tank; and cooling said bottom portion of said milk storage tank in consecutive periods are preceded by the step of cooling said milk storage tank by means of cooling the bottom portion thereof.","4.The method as claimed in claim 3 wherein the step of cooling said milk storage tank by means of cooling the bottom portion thereof is performed after cleaning of said milk storage tank.","5.The method as claimed in claim 1 wherein at least one period of cooling is started while milk from at most five animals has been stored in said milk storage tank.","6.The method as claimed in any of claims 1-5 wherein said quantity indicative of a temperature of an inner surface area of the bottom portion of the milk storage tank is a temperature of the cooling device.","7.The method as claimed in claim 6 wherein said cooling device includes an evaporator (8); a compressor (9); a condenser (10); and an expansion valve (12), all of which being interconnected by a connecting piping (13), such that a cooling medium can be circulated therein; and wherein said quantity indicative of a temperature of an inner surface area of the bottom portion of the milk storage tank is a temperature of the connecting piping between the expansion valve and the evaporator, of the expansion valve or of the evaporator.","8.The method as claimed in any of claims 1-5 wherein said cooling device includes an evaporator (8); a compressor (9); a condenser (10); and an expansion valve (12), all of which being interconnected by a connecting piping (13), such that a cooling medium can be circulated therein; and wherein said quantity indicative of a temperature of an inner surface area of the bottom portion of the milk storage tank is a pressure in said evaporator or in the connecting piping between the evaporator and the compressor.","9.The method as claimed in any of claims 1-8 wherein said step of cooling said milk storage tank is performed while ensuring that the temperature of said inner surface area of the bottom portion of the milk storage tank is kept above 0° C. 10.The method as claimed in any of claims 1-9 wherein said amount of milk is measured in said milking robot.","11.The method as claimed in any of claims 1-9 wherein said amount of milk is measured in or at said milk storage tank.","12.The method as claimed in any of the preceding claims comprising the step of cooling said bottom portion of said milk storage tank in response to a temperature of milk stored in said milk storage tank if a level of milk in said milk storage tank exceeds a threshold level.","13.The method as claimed in any of the preceding claims wherein the step of determining a cooling need for milk stored or to be stored in said milk storage tank includes the establishment of an expected temperature of the milk stored or to be stored in the milk store tank at the end of the step of cooling said bottom portion of said milk storage tank in consecutive periods; the temperature of the milk is established at the end of the step of cooling said bottom portion of said milk storage tank in consecutive periods; and the method for cooling milk as claimed in any of the preceding claims is repeated, wherein the duration of each period of cooling and/or non-cooling is based also on any difference between said expected and established temperatures.","14.An arrangement for cooling milk in an automatic milking system, said milking system comprising a milking robot (1) for milking animals, such as cows; a milk flow meter (2) for measuring an amount of milk extracted by said milking robot; a milk storage tank (5) connected to the milking robot for storing milk extracted by the milking robot; and a cooling device (7) for cooling milk stored or to be stored in said milk storage tank by cooling a bottom portion (14) of said milk storage tank, characterized in that said arrangement comprises: a sensor (15) for measuring a quantity indicative of a temperature of an inner surface area (16) of the bottom portion of the milk storage tank; and a controller (17) for determining a cooling need for milk stored or to be stored in said milk storage tank based on said measured amount of milk; and for controlling said cooling device to cool said bottom portion of said milk storage tank in consecutive periods, such that each period of cooling (τ1, τ3) is followed by a respective period of non-cooling (τ2, τ4), wherein the duration of each period of cooling and/or non-cooling is based on said measured quantity indicative of the inner surface area temperature, and said determined cooling need.","15.The arrangement as claimed in claim 14 wherein the controller is adapted to control said cooling device to start at least one period of cooling while said milk storage tank is empty of milk.","16.The arrangement as claimed in claim 14 wherein the controller is adapted to control said cooling device to start at least one period of cooling while milk from at most five animals has been stored in said milk storage tank.","17.The arrangement as claimed in any of claims 14-16 wherein said quantity indicative of a temperature of an inner surface area of the bottom portion of the milk storage tank is a temperature of the cooling device.","18.The arrangement as claimed in claim 17 wherein said cooling device includes an evaporator (8); a compressor (9); a condenser (10); and an expansion valve (12), all of which being interconnected by a connecting piping (13), such that a cooling medium can be circulated therein; and wherein said sensor for measuring the quantity indicative of a temperature of an inner surface area of the bottom portion of the milk storage tank is a temperature sensor (15) arranged at the connecting piping between the expansion valve and the evaporator, at the expansion valve or at the evaporator.","19.The arrangement as claimed in any of claims 14-16 wherein said cooling device includes an evaporator (8); a compressor (9); a condenser (10); and an expansion valve (12), all of which being interconnected by a connecting piping (13), such that a cooling medium can be circulated therein; and wherein said sensor for measuring said quantity indicative of a temperature of an inner surface area of the bottom portion of the milk storage tank is a pressure sensor arranged to measure the pressure in said evaporator, or in the connecting piping between the evaporator and the compressor.","20.The arrangement as claimed in any of claims 14-19 wherein said controller is adapted to control said cooling device to cool said milk storage tank while ensuring that the temperature of said inner surface area of the bottom portion of the milk storage tank is kept above 0° C. 21.The arrangement as claimed in any of claims 14-20 wherein said milk flow meter for measuring said amount of milk extracted by said milking robot is arranged in said milking robot.","22.The arrangement as claimed in any of claims 14-20 wherein said milk flow meter for measuring said amount of milk extracted by said milking robot is arranged in or at said milk storage tank.","23.The arrangement as claimed in any of claims 14-22 wherein the controller is adapted to control said cooling device to cool said bottom portion of said milk storage tank in response to a temperature of milk stored in said milk storage tank depending on a level of milk in said milk storage tank exceeding a threshold level."],["<SOH> BACKGROUND <EOH>In dairy farming animals are milked and their milk is thereafter stored in a milk storage tank for collection on a regular time basis, e.g.","every second day.","In order to maintain the quality of the milk, it is cooled to approximately 4° C. as quickly as possible.","It is necessary to be careful during cooling of the milk since the quality is deteriorated if the milk freezes.","At a dairy farm provided with an automatic milking system, the milk usually enters the milk storage tank in small amounts spread during the day and night, compared to a dairy farm without an automatic milking system, where all animals are milked together two or three times a day.","The milk storage tank is usually equipped with a cooling device, which lowers the temperature of the milk to about 4° C. and maintains this temperature in a filled milk storage tank.","When the milk storage tank only contains small amounts of milk, there is a considerable risk of cooling the milk too much since the cooling device, when cooling, operates at full capacity.","Usually the cooling of milk in a milk storage tank is controlled in response to the temperature of the milk in the tank.","The milk temperature is usually measured on the outside of the tank due to hygienic requirements, and this results in a rather slow response when the temperature of the milk within the tank is changed.","Such a sluggish temperature response gives rise to problems, in particular when the volume of stored milk is small.","The temperature of a small milk volume is lowered rapidly at the risk of freezing the milk.","Furthermore, the milk storage tank is typically provided with a stirrer, which stirs the milk in the tank to obtain a uniform temperature of the milk.","Such a stirrer is usually not able to stir the milk in the tank when the tank contains only small amounts of milk, which thus involves a further risk of freezing milk locally at the beginning of the filling of the tank with milk.","If cooling, on the other hand, is omitted while there is only a small volume of milk in the milk storage tank, this milk is not cooled at once, and thus the quality of the milk is deteriorated."],["<SOH> SUMMARY OF THE INVENTION <EOH>An object of the present invention is to provide a method and an arrangement, respectively for cooling milk rapidly in an automatic milking system without risk of freezing the milk, particularly when only small amounts of milk are present in the milk storage tank.","This object, among others, is attained by methods and arrangements, respectively, as claimed in the appended patent claims.","By cooling in consecutive periods according to the present invention, milk is cooled without risk of being frozen, and thus a high milk quality can be safeguarded.","In accordance with one preferred embodiment of the present invention the milk storage tank itself is pre-cooled, i.e.","cooled before milk is entered therein.","In such manner, milk transported to the milk storage tank can be cooled faster, and thus an enhanced milk quality is obtained.","In accordance with another preferred embodiment of the present invention the cooling of milk may be performed firstly, when there is only a small volume of milk in the milk storage tank, as set out in the independent claims, and then, when a level of milk in said milk storage tank exceeds a threshold level, the cooling is controlled in response to the temperature of the milk in a conventional fashion.","Further features and advantages of the present invention will be evident from the following description."],["FIELD OF INVENTION The present invention relates generally to dairy farming and particularly to a method and an arrangement, respectively, for cooling of milk in an automatic milking system.","BACKGROUND In dairy farming animals are milked and their milk is thereafter stored in a milk storage tank for collection on a regular time basis, e.g.","every second day.","In order to maintain the quality of the milk, it is cooled to approximately 4° C. as quickly as possible.","It is necessary to be careful during cooling of the milk since the quality is deteriorated if the milk freezes.","At a dairy farm provided with an automatic milking system, the milk usually enters the milk storage tank in small amounts spread during the day and night, compared to a dairy farm without an automatic milking system, where all animals are milked together two or three times a day.","The milk storage tank is usually equipped with a cooling device, which lowers the temperature of the milk to about 4° C. and maintains this temperature in a filled milk storage tank.","When the milk storage tank only contains small amounts of milk, there is a considerable risk of cooling the milk too much since the cooling device, when cooling, operates at full capacity.","Usually the cooling of milk in a milk storage tank is controlled in response to the temperature of the milk in the tank.","The milk temperature is usually measured on the outside of the tank due to hygienic requirements, and this results in a rather slow response when the temperature of the milk within the tank is changed.","Such a sluggish temperature response gives rise to problems, in particular when the volume of stored milk is small.","The temperature of a small milk volume is lowered rapidly at the risk of freezing the milk.","Furthermore, the milk storage tank is typically provided with a stirrer, which stirs the milk in the tank to obtain a uniform temperature of the milk.","Such a stirrer is usually not able to stir the milk in the tank when the tank contains only small amounts of milk, which thus involves a further risk of freezing milk locally at the beginning of the filling of the tank with milk.","If cooling, on the other hand, is omitted while there is only a small volume of milk in the milk storage tank, this milk is not cooled at once, and thus the quality of the milk is deteriorated.","SUMMARY OF THE INVENTION An object of the present invention is to provide a method and an arrangement, respectively for cooling milk rapidly in an automatic milking system without risk of freezing the milk, particularly when only small amounts of milk are present in the milk storage tank.","This object, among others, is attained by methods and arrangements, respectively, as claimed in the appended patent claims.","By cooling in consecutive periods according to the present invention, milk is cooled without risk of being frozen, and thus a high milk quality can be safeguarded.","In accordance with one preferred embodiment of the present invention the milk storage tank itself is pre-cooled, i.e.","cooled before milk is entered therein.","In such manner, milk transported to the milk storage tank can be cooled faster, and thus an enhanced milk quality is obtained.","In accordance with another preferred embodiment of the present invention the cooling of milk may be performed firstly, when there is only a small volume of milk in the milk storage tank, as set out in the independent claims, and then, when a level of milk in said milk storage tank exceeds a threshold level, the cooling is controlled in response to the temperature of the milk in a conventional fashion.","Further features and advantages of the present invention will be evident from the following description.","BRIEF DESCRIPTION OF THE DRAWINGS The present invention will become better understood from the detailed description of embodiments given below and the accompanying FIGS.","1-3, which are given by way of illustration only, and thus, are not limitative of the present invention, wherein: FIG.","1 is a schematic representation of an automatic milking system including a milk cooling arrangement according to a preferred embodiment of the present invention; FIG.","2 is an exemplary diagram illustrating the cooling of the inventive milk cooling arrangement of FIG.","1 as a function of time; and FIG.","3 is another exemplary diagram illustrating the cooling of the inventive milk cooling arrangement of FIG.","1 as a function of time.","DETAILED DESCRIPTION OF EMBODIMENTS In the following description, for purpose of explanation and not limitation, specific details are set forth, such as particular techniques and applications in order to provide a thorough understanding of the present invention.","However, it will be apparent for a person skilled in the art that the present invention may be practiced in other embodiments that depart from these specific details.","An automatic milking system, as shown in FIG.","1, comprises a milking robot 1 for milking animals, such as cows, including a milk flow meter 2 for measuring the milk flow during milking; a milk storage tank 5 which stores milk for collection on a regular time basis; a milk line 4 connecting the milking robot 1 with the milk storage tank 5; a milk pump (not shown) for pumping milk from the milking robot 1 to the milk storage tank 5; and a pre-cooling device 3 for cooling milk, which is pumped over to the milk storage tank 5.The milk storage tank 5 is provided with a stirrer 6 for stirring milk in the milk storage tank 5; and a cooling device 7 for cooling milk in the milk storage tank 5.The cooling device 7 includes typically an evaporator 8; a compressor 9; a condenser 10; and an expansion valve 12, all of which being interconnected by a connecting piping 13, such that a cooling medium can be circulated therein.","The evaporator is suitably integrated in, or in contact with, a bottom portion 14 of the wall of the milk storage tank 5 and the compressor 9 is typically driven by an electrical motor (not shown).","Furthermore, the cooling device 7 comprises a sensor 15 for sensing a quantity, which is indicative of a temperature of an inner surface area 16 of the bottom portion 14 of the milk storage tank 5.The sensor is preferably a temperature sensor 15 for measuring the temperature of the connecting piping 13 between the expansion valve 12 and the evaporator 8 of the cooling device 7.Alternatively, the temperature sensor 15 may be arranged to measure the temperature of the expansion valve 12, or of the evaporator 8.As a further alternative, the sensor may be a pressure sensor (not illustrated) for sensing the pressure in the evaporator 8 or preferably in the connecting piping 13 downstream from the evaporator 8.However, a temperature sensor is preferred since it is easier to arrange a temperature sensor 15 at the outside of the connecting piping 13, the expansion valve 12, or the evaporator 8 than to introduce a pressure sensor within the connecting piping 13.The milking system further comprises a controller 17, such as a microcomputer provided with suitable software, for controlling and managing the milking robot 1, including keeping records of animals to be milked and their respective characteristics, such as milk quality, amount of milk per milking, and time lapsed since last milking.","Further, the controller 17 is adapted for controlling the cooling device 7, which includes receiving measured values from the milk flow meter 2 and the sensor 15.Alternatively, one controller is provided for the control of the milking robot and another separate controller is provided for the control of the cooling device (not illustrated), in which case the two controllers are connected with each other to provide for one-way or two-way communication between them.","The milk flow in the milking system is measured by the milk flow meter 2 and may be registered by the controller 17 to obtain an accumulated milk flow and optionally to determine the level of milk in the milk storage tank 5 by prior knowledge of the design of the milk storage tank and the obtained accumulated milk flow.","Alternatively, the level of milk in the milk storage tank may be measured by means of a level-measuring device (not illustrated) located in the milk storage tank 5.As a further alternative, the level of milk in the milk storage tank can be determined by measuring a static pressure or change in static pressure of the milk at the bottom of the milk storage tank 5, e.g.","detected by a sensor (not illustrated) at the bottom of the milk storage tank.","Instead of measuring the milk flow in the robot 1 it would be possible to provide a milk flow meter (not illustrated) in the milk line 4, e.g.","adjacent to the milk storage tank 5, to measure the milk flow there.","The cooling of the cooling device 7 is controlled, in accordance with the present invention, by operating the cooling device 7 in periods of cooling, each of which being followed by a respective period of non-cooling, where the duration of each period of cooling and/or non-cooling is determined and controlled by the controller 17.More precisely, the controller 17 is adapted to receive a value of the flow of milk from the milk flow meter 2; to determine, i.e.","calculate, a cooling need for milk to be stored in said milk storage tank based on the received value of the flow of milk; and to receive a value of the quantity sensed by the sensor 15.The duration of each period of cooling and/or period of non-cooling is then controlled based on the determined cooling need and the value of the quantity sensed by sensor 15.It is particularly important to control the cooling such that the temperature of the inner surface area 16 of the bottom portion 14 of the milk storage tank 5 does not fall below 0° C. By such an approach the cooling of milk in the milk storage tank may be started when only small amounts of milk have been stored therein, and a quick cooling of the first amounts of stored milk is obtained without any risk of freezing the same.","In FIG.","2 is shown a diagram illustrating an example of a the cooling by the cooling device 7 as a function of time, wherein two periods of cooling τ1 and τ3 and two periods of non-cooling τ2 and τ4 are shown.","The duration of two periods of cooling and two periods of non-cooling, at four different times are illustrated in FIG.","2.Such a cooling may occur as a result of the following exemplary scenario.","At t1 a sensor, e.g.","the milk flow meter 2, indicates an increased milk flow to the controller, which calculates a new increased cooling need and in response thereto the controller starts the cooling.","At t2 the temperature sensor 15 indicates a low temperature, e.g.","close to 0° C., and in response thereto the controller stops the cooling.","At t3 the temperature sensor 15 indicates a higher temperature and in response thereto the controller starts the cooling again.","Then, at t4 the temperature sensor 15 indicates a low temperature and in response thereto the controller stops the cooling.","The cooling need of the milking system depends on the amount of milk to be stored in the milk storage tank 5, which in turn is determined by the flow of milk in the milking system.","The cooling by the cooling device 7, which is required for cooling a specific volume of milk, is divided into periods of cooling and non-cooling to avoid freezing of the milk, and these periods are continued until the cooling need is fulfilled as indicated by the milk flow meter 2 and/or the sensor 15.The accumulated milk flow may be utilized to calculate how much of a heat transfer area, i.e.","the area where the cooling device efficiently draws heat from the milk storage tank, in the milk storage tank that is covered by milk.","If the milk does not cover the whole heat transfer area, the risk of freezing the milk is particularly large, which may be taken into account by the controller 17 when determining the duration of the periods of cooling and/or the periods of non-cooling.","The cooling need further depends on the design of the milk storage tank 5, and of the temperature surrounding the milk storage tank 5.The influence of the surrounding temperature will be less if the milk storage tank is insulated.","Such insulation is usually implemented as a layer of insulation material between the wall of the milk storage tank and an outer shell.","If the cooling device 7 has a minimum period of rest due to the electrical motor of the cooling device having a need to be cool down after a period of cooling, or else the motor might be damaged, such a minimum period of rest defines a minimum duration of a period of non-cooling as controlled by the controller 17.The flow pattern of milk in the milk storage tank 5, i.e.","the flow pattern of the milk obtained by inflowing milk and/or obtained by an auxiliary device, such as the stirrer 6, affects how fast temperature differences of milk in different parts of the milk storage tank are evened out, which might be a factor to consider by the controller 17.A flow pattern obtained by the stirrer 6 has only to be considered when the milk level in milk storage tank has reached the lowermost portion of the stirrer such that stirring really takes place.","Furthermore, there are a number of more operation conditions, which, more or less, may affect the cooling need, the cooling capacity, or which may be utilized to correct the cooling as performed by the cooling device 7 and controlled by the controller 17.The cooling may be controlled based on the time lapsed since the cooling device 7 ended a period of cooling, the temperature of the milk in the milk storage tank 5, and the amount of the milk in the milk storage tank 5.If the controller 17 takes these dependencies into account when determining the lengths of the periods of cooling and non-cooling, a better cooling control may be obtained.","In some designs of a milking system the first amounts of milk pumped from the milking robot after cleaning the tank may not reach the milk storage tank until several cows have been milked, due to that the milk only fills the inner volume of the milk line 4.This affects also the cooling need and accordingly the controller 17 may take this into account when controlling the operation of the cooling device 7.The controller may be arranged to initiate a period of cooling when the milk pump of the milking system, at the milking robot, is started, and to control the cooling device based on the number of times the milk pump at the milking robot has been started during a predetermined period of time.","Alternatively, or as complement, the controller 17 may be adapted to control the cooling of the cooling device based on the number of animals that have been milked during the predetermined period of time.","As the control device 8 keeps record of the cows being milked and identifies each cow prior to milking, such identification may affect the control of the cooling device 7 since a period of cooling may begin earlier or last longer if the cow identified prior to milking is known to have a high milk yield.","The efficiency of a cooling process using e.g.","an air-cooled condenser may change significantly if the temperature surrounding the condenser changes, e.g.","depending on the time of year or between daytime and nighttime.","That is, a certain amount of electrical effect fed into the cooling device results in one level of cooling during daytime and another level of cooling during nighttime.","Such environment-dependent cooling capacity could be compensated for by the controller if a temperature sensor is provided for detection of the temperature close to the condenser and using a correction factor depending on this temperature for adjusting the cooling.","Alternatively, a timer may be used to adjust the correction factor if the temperature variations during the day and night are predictable.","Further, if the pre-cooling device is a heat exchanger, e.g.","a plate heat exchanger, it may not be capable to pre-cool a large flow of milk as efficient as a lower flow of milk and thus a larger flow of milk will be warmer when reaching the milk storage tank 5.Thus, the controller 17 preferably takes into account the efficiency of the pre-cooling device 3 by means of prior knowledge of its cooling capacity or by means of measuring the temperature of the milk in milk line 4.In a particular embodiment of the present invention the milk storage tank itself is pre-cooled, i.e.","cooled before milk is entered therein.","Preferably, the controller initiates a period of cooling of the milk storage tank 5 itself directly after the cleaning of the same is finished.","In such manner, milk transported to the milk storage tank can be cooled faster, and thus an enhanced milk quality can be obtained.","FIG.","3 illustrates a diagram indicating periods of cooling followed by periods of non-cooling during such pre-cooling of the milk storage tank 5 itself.","Firstly five periods of cooling lower the temperature of the milk storage tank to approximately 4° C. by cooling the heat transfer area during each period of cooling to approximately 0° C. During the periods of non-cooling the temperature differences in the milk storage tank wall is equalized.","The sixth cooling period illustrates the cooling need of a small amount of milk entering, or about to enter, the milk storage tank.","The seventh cooling period illustrates the cooling need of a large amount or several consecutive small amounts of milk entering, or about to enter, the milk storage tank.","Next an example of cooling of a 3500-liter milk storage tank having a wall made of 300 kg stainless steel will be described.","Subsequent to cleaning, the tank has a temperature of about 20° C. and then 2400 kJ of cooling capacity is needed to bring the tank temperature down to 4° C. according to the formula E=cmΔt, where c is the heat capacity, m is the mass of the tank and Δt is the temperature drop caused by cooling (c=500 J/kg° C. for stainless steel, m=300 kg, and Δt=16° C.).","Any heat losses to the air surrounding the tank are assumed to be negligible in this calculation.","Such a milk storage tank contains about 4 kg of air, which needs 65 kJ of cooling capacity to bring the air temperature down to 4° C., since cp=1000 J/kg° C. for air.","Further, 0.06 kg of moisture in the air will be reduced to 0.03 kg during the cooling process, which consumes 74 kJ of condensation energy.","Thus, it can be seen that the cooling need of the tank in this case is 2539 kJ.","A 3 kW cooling compressor having a cooling factor of ε=2.4 needs a running time of about six minutes to produce the cooling capacity needed to bring the temperature of the milk storage tank down to 4° C. (according to running time τ=E/Pε, where the energy E is 2539 kJ, the power P is 3 kW, and the cooling factor ε is 2.4).","Since the heat transfer between the bottom wall of the empty milk storage tank and the side walls thereof takes place during a period of time, it may be advisable to start the cooling compressor subsequent to the cleaning and let it run for two minutes to get an initial lowering of the temperature at the bottom of the tank.","Thereafter a period of rest of about two minutes is used (which may be given by the temperature sensed by sensor 15) to equalize the temperature in the tank.","Two minutes running period and two minutes rest period can then be repeated (given by the temperature sensed by sensor 15) until a complete running time of the six minutes is achieved.","Milk leaving a cow has a temperature of about 37° C., but may have been reduced to about 35° C. during transport to the milk storage tank.","An amount of 15 l of milk needs an effective compressor running time of about 270 s, as m≈15 kg, Δt=31° C., c=4190 J/kg° C. for milk, P=3000 W, and ε=2.4.As the milk from the first cow only covers a part of the cooling surface in the milk storage tank, the cooling device is run in sequences of about one minute followed by a rest period of two minutes (which may be given by the temperature sensed by sensor 15), to avoid freezing the milk.","The controller keeps track of the accumulated milk flow as more cows are being milked and increases the running time when the volume of milk in the tank increases, e.g.","to two minutes running time.","It shall be appreciated that the duration of each of the periods of cooling and non-cooling is determined according to an algorithm, which uses at least the determined cooling need and the quantity sensed as inputs.","Further inputs may be employed to obtain a refined cooling process.","It shall particularly be noted that the duration of each period of non-cooling may not only be determined in response to the quantity sensed, but other measured values or prior knowledge may be used such as e.g.","an estimated average number of cows to be milked per time unit.","In yet a particular embodiment of the present invention the cooling of milk may be performed firstly, when there is only a small volume of milk in the milk storage tank 5, as described with reference to the first embodiment of the invention, and then, when a level of milk in said milk storage tank exceeds a threshold level, the cooling is controlled in a conventional fashion, i.e.","in response to a temperature of milk in the milk storage tank 5.The threshold value is preferably determined to be a level, which is sufficiently high so the stirrer 6 can effectively stir the milk in the milk storage tank 5 and even out any temperature gradients in the milk.","Thus the risk of freezing the milk is very low.","In still a particular embodiment of the present invention the controller 17 may use a feedback process for adjusting the periods of cooling and/or periods of non-cooling.","Thus, if a temperature of the milk at the end of a cooling process (e.g.","a cooling process, which ends when the milk in the milk storage tank 5 is collected, or alternatively when the milk level in the milk storage tank exceeds the above-mentioned threshold value) is detected to be different than expected (e.g.","different than an end temperature used in the calculation of the cooling need), the controller may adjust the cooling capacity of the cooling device 7 accordingly for a following cooling process.","Such an adjustment may be made by introducing a correction factor to correct for the discrepancy between the detected and the expected temperatures.","The present invention being thus described, it will be obvious that the same may be varied in a plurality of ways.","All such variations as would be obvious for a person skilled in the art are intended to be included within the scope of the present invention."]],"string":"[\n [\n \"COOLING OF MILK IN AN AUTOMATIC MILKING SYSTEM\",\n \"The present invention relates to a method for cooling milk in a milk storage tank of an automatic milling system comprising the steps of (i) measuring an amount of extracted milk by means of a milk flow meter, (ii) determining a cooling need for milk stored or to be stored in said milk storage tank based on said amount of milk; (iii) measuring a quantity indicative of a temperature of an inner surface area of a bottom portion of the milk storage tank; and cooling said bottom portion of said milk storage tank in consecutive periods, such that each period of cooling (τ1, τ3) is followed by a respective period of non-cooling (τ2, τ4), wherein the duration of each period of cooling and/or noncooling is based on said measured quantity indicative of said inner surface area temperature, and said cooling need.\",\n \"The invention also relates to an arrangement for performing the method.\"\n ],\n [\n \"1.A method for cooling milk in an automatic milking system, which comprises a milking robot (1) for milking animals, such as cows; a milk storage tank (5) connected to said milking robot for storing milk extracted by the milking robot; and a cooling device (7) for cooling milk stored or to be stored in said milk storage tank by cooling a bottom portion (14) of said milk storage tank, said method being characterized by the steps of: measuring an amount of milk extracted by said milking robot by means of a milk flow meter; determining a cooling need for milk stored or to be stored in said milk storage tank based on said measured amount of milk; measuring a quantity indicative of a temperature of an inner surface area (16) of the bottom portion of the milk storage tank; and cooling said bottom portion of said milk storage tank in consecutive periods, such that each period of cooling (τ1, τ3) is followed by a respective period of non-cooling (τ2, τ4), wherein the duration of each period of cooling and/or non-cooling is based on said measured quantity indicative of the inner surface temperature, and said determined cooling need.\",\n \"2.The method as claimed in claim 1 wherein at least one period of cooling is started while said milk storage tank is empty of milk.\",\n \"3.The method as claimed in claim 1 or 2 wherein the steps of measuring an amount of milk; determining a cooling need; measuring a quantity indicative of a temperature of an inner surface area of the bottom portion of the milk storage tank; and cooling said bottom portion of said milk storage tank in consecutive periods are preceded by the step of cooling said milk storage tank by means of cooling the bottom portion thereof.\",\n \"4.The method as claimed in claim 3 wherein the step of cooling said milk storage tank by means of cooling the bottom portion thereof is performed after cleaning of said milk storage tank.\",\n \"5.The method as claimed in claim 1 wherein at least one period of cooling is started while milk from at most five animals has been stored in said milk storage tank.\",\n \"6.The method as claimed in any of claims 1-5 wherein said quantity indicative of a temperature of an inner surface area of the bottom portion of the milk storage tank is a temperature of the cooling device.\",\n \"7.The method as claimed in claim 6 wherein said cooling device includes an evaporator (8); a compressor (9); a condenser (10); and an expansion valve (12), all of which being interconnected by a connecting piping (13), such that a cooling medium can be circulated therein; and wherein said quantity indicative of a temperature of an inner surface area of the bottom portion of the milk storage tank is a temperature of the connecting piping between the expansion valve and the evaporator, of the expansion valve or of the evaporator.\",\n \"8.The method as claimed in any of claims 1-5 wherein said cooling device includes an evaporator (8); a compressor (9); a condenser (10); and an expansion valve (12), all of which being interconnected by a connecting piping (13), such that a cooling medium can be circulated therein; and wherein said quantity indicative of a temperature of an inner surface area of the bottom portion of the milk storage tank is a pressure in said evaporator or in the connecting piping between the evaporator and the compressor.\",\n \"9.The method as claimed in any of claims 1-8 wherein said step of cooling said milk storage tank is performed while ensuring that the temperature of said inner surface area of the bottom portion of the milk storage tank is kept above 0° C. 10.The method as claimed in any of claims 1-9 wherein said amount of milk is measured in said milking robot.\",\n \"11.The method as claimed in any of claims 1-9 wherein said amount of milk is measured in or at said milk storage tank.\",\n \"12.The method as claimed in any of the preceding claims comprising the step of cooling said bottom portion of said milk storage tank in response to a temperature of milk stored in said milk storage tank if a level of milk in said milk storage tank exceeds a threshold level.\",\n \"13.The method as claimed in any of the preceding claims wherein the step of determining a cooling need for milk stored or to be stored in said milk storage tank includes the establishment of an expected temperature of the milk stored or to be stored in the milk store tank at the end of the step of cooling said bottom portion of said milk storage tank in consecutive periods; the temperature of the milk is established at the end of the step of cooling said bottom portion of said milk storage tank in consecutive periods; and the method for cooling milk as claimed in any of the preceding claims is repeated, wherein the duration of each period of cooling and/or non-cooling is based also on any difference between said expected and established temperatures.\",\n \"14.An arrangement for cooling milk in an automatic milking system, said milking system comprising a milking robot (1) for milking animals, such as cows; a milk flow meter (2) for measuring an amount of milk extracted by said milking robot; a milk storage tank (5) connected to the milking robot for storing milk extracted by the milking robot; and a cooling device (7) for cooling milk stored or to be stored in said milk storage tank by cooling a bottom portion (14) of said milk storage tank, characterized in that said arrangement comprises: a sensor (15) for measuring a quantity indicative of a temperature of an inner surface area (16) of the bottom portion of the milk storage tank; and a controller (17) for determining a cooling need for milk stored or to be stored in said milk storage tank based on said measured amount of milk; and for controlling said cooling device to cool said bottom portion of said milk storage tank in consecutive periods, such that each period of cooling (τ1, τ3) is followed by a respective period of non-cooling (τ2, τ4), wherein the duration of each period of cooling and/or non-cooling is based on said measured quantity indicative of the inner surface area temperature, and said determined cooling need.\",\n \"15.The arrangement as claimed in claim 14 wherein the controller is adapted to control said cooling device to start at least one period of cooling while said milk storage tank is empty of milk.\",\n \"16.The arrangement as claimed in claim 14 wherein the controller is adapted to control said cooling device to start at least one period of cooling while milk from at most five animals has been stored in said milk storage tank.\",\n \"17.The arrangement as claimed in any of claims 14-16 wherein said quantity indicative of a temperature of an inner surface area of the bottom portion of the milk storage tank is a temperature of the cooling device.\",\n \"18.The arrangement as claimed in claim 17 wherein said cooling device includes an evaporator (8); a compressor (9); a condenser (10); and an expansion valve (12), all of which being interconnected by a connecting piping (13), such that a cooling medium can be circulated therein; and wherein said sensor for measuring the quantity indicative of a temperature of an inner surface area of the bottom portion of the milk storage tank is a temperature sensor (15) arranged at the connecting piping between the expansion valve and the evaporator, at the expansion valve or at the evaporator.\",\n \"19.The arrangement as claimed in any of claims 14-16 wherein said cooling device includes an evaporator (8); a compressor (9); a condenser (10); and an expansion valve (12), all of which being interconnected by a connecting piping (13), such that a cooling medium can be circulated therein; and wherein said sensor for measuring said quantity indicative of a temperature of an inner surface area of the bottom portion of the milk storage tank is a pressure sensor arranged to measure the pressure in said evaporator, or in the connecting piping between the evaporator and the compressor.\",\n \"20.The arrangement as claimed in any of claims 14-19 wherein said controller is adapted to control said cooling device to cool said milk storage tank while ensuring that the temperature of said inner surface area of the bottom portion of the milk storage tank is kept above 0° C. 21.The arrangement as claimed in any of claims 14-20 wherein said milk flow meter for measuring said amount of milk extracted by said milking robot is arranged in said milking robot.\",\n \"22.The arrangement as claimed in any of claims 14-20 wherein said milk flow meter for measuring said amount of milk extracted by said milking robot is arranged in or at said milk storage tank.\",\n \"23.The arrangement as claimed in any of claims 14-22 wherein the controller is adapted to control said cooling device to cool said bottom portion of said milk storage tank in response to a temperature of milk stored in said milk storage tank depending on a level of milk in said milk storage tank exceeding a threshold level.\"\n ],\n [\n \"<SOH> BACKGROUND <EOH>In dairy farming animals are milked and their milk is thereafter stored in a milk storage tank for collection on a regular time basis, e.g.\",\n \"every second day.\",\n \"In order to maintain the quality of the milk, it is cooled to approximately 4° C. as quickly as possible.\",\n \"It is necessary to be careful during cooling of the milk since the quality is deteriorated if the milk freezes.\",\n \"At a dairy farm provided with an automatic milking system, the milk usually enters the milk storage tank in small amounts spread during the day and night, compared to a dairy farm without an automatic milking system, where all animals are milked together two or three times a day.\",\n \"The milk storage tank is usually equipped with a cooling device, which lowers the temperature of the milk to about 4° C. and maintains this temperature in a filled milk storage tank.\",\n \"When the milk storage tank only contains small amounts of milk, there is a considerable risk of cooling the milk too much since the cooling device, when cooling, operates at full capacity.\",\n \"Usually the cooling of milk in a milk storage tank is controlled in response to the temperature of the milk in the tank.\",\n \"The milk temperature is usually measured on the outside of the tank due to hygienic requirements, and this results in a rather slow response when the temperature of the milk within the tank is changed.\",\n \"Such a sluggish temperature response gives rise to problems, in particular when the volume of stored milk is small.\",\n \"The temperature of a small milk volume is lowered rapidly at the risk of freezing the milk.\",\n \"Furthermore, the milk storage tank is typically provided with a stirrer, which stirs the milk in the tank to obtain a uniform temperature of the milk.\",\n \"Such a stirrer is usually not able to stir the milk in the tank when the tank contains only small amounts of milk, which thus involves a further risk of freezing milk locally at the beginning of the filling of the tank with milk.\",\n \"If cooling, on the other hand, is omitted while there is only a small volume of milk in the milk storage tank, this milk is not cooled at once, and thus the quality of the milk is deteriorated.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>An object of the present invention is to provide a method and an arrangement, respectively for cooling milk rapidly in an automatic milking system without risk of freezing the milk, particularly when only small amounts of milk are present in the milk storage tank.\",\n \"This object, among others, is attained by methods and arrangements, respectively, as claimed in the appended patent claims.\",\n \"By cooling in consecutive periods according to the present invention, milk is cooled without risk of being frozen, and thus a high milk quality can be safeguarded.\",\n \"In accordance with one preferred embodiment of the present invention the milk storage tank itself is pre-cooled, i.e.\",\n \"cooled before milk is entered therein.\",\n \"In such manner, milk transported to the milk storage tank can be cooled faster, and thus an enhanced milk quality is obtained.\",\n \"In accordance with another preferred embodiment of the present invention the cooling of milk may be performed firstly, when there is only a small volume of milk in the milk storage tank, as set out in the independent claims, and then, when a level of milk in said milk storage tank exceeds a threshold level, the cooling is controlled in response to the temperature of the milk in a conventional fashion.\",\n \"Further features and advantages of the present invention will be evident from the following description.\"\n ],\n [\n \"FIELD OF INVENTION The present invention relates generally to dairy farming and particularly to a method and an arrangement, respectively, for cooling of milk in an automatic milking system.\",\n \"BACKGROUND In dairy farming animals are milked and their milk is thereafter stored in a milk storage tank for collection on a regular time basis, e.g.\",\n \"every second day.\",\n \"In order to maintain the quality of the milk, it is cooled to approximately 4° C. as quickly as possible.\",\n \"It is necessary to be careful during cooling of the milk since the quality is deteriorated if the milk freezes.\",\n \"At a dairy farm provided with an automatic milking system, the milk usually enters the milk storage tank in small amounts spread during the day and night, compared to a dairy farm without an automatic milking system, where all animals are milked together two or three times a day.\",\n \"The milk storage tank is usually equipped with a cooling device, which lowers the temperature of the milk to about 4° C. and maintains this temperature in a filled milk storage tank.\",\n \"When the milk storage tank only contains small amounts of milk, there is a considerable risk of cooling the milk too much since the cooling device, when cooling, operates at full capacity.\",\n \"Usually the cooling of milk in a milk storage tank is controlled in response to the temperature of the milk in the tank.\",\n \"The milk temperature is usually measured on the outside of the tank due to hygienic requirements, and this results in a rather slow response when the temperature of the milk within the tank is changed.\",\n \"Such a sluggish temperature response gives rise to problems, in particular when the volume of stored milk is small.\",\n \"The temperature of a small milk volume is lowered rapidly at the risk of freezing the milk.\",\n \"Furthermore, the milk storage tank is typically provided with a stirrer, which stirs the milk in the tank to obtain a uniform temperature of the milk.\",\n \"Such a stirrer is usually not able to stir the milk in the tank when the tank contains only small amounts of milk, which thus involves a further risk of freezing milk locally at the beginning of the filling of the tank with milk.\",\n \"If cooling, on the other hand, is omitted while there is only a small volume of milk in the milk storage tank, this milk is not cooled at once, and thus the quality of the milk is deteriorated.\",\n \"SUMMARY OF THE INVENTION An object of the present invention is to provide a method and an arrangement, respectively for cooling milk rapidly in an automatic milking system without risk of freezing the milk, particularly when only small amounts of milk are present in the milk storage tank.\",\n \"This object, among others, is attained by methods and arrangements, respectively, as claimed in the appended patent claims.\",\n \"By cooling in consecutive periods according to the present invention, milk is cooled without risk of being frozen, and thus a high milk quality can be safeguarded.\",\n \"In accordance with one preferred embodiment of the present invention the milk storage tank itself is pre-cooled, i.e.\",\n \"cooled before milk is entered therein.\",\n \"In such manner, milk transported to the milk storage tank can be cooled faster, and thus an enhanced milk quality is obtained.\",\n \"In accordance with another preferred embodiment of the present invention the cooling of milk may be performed firstly, when there is only a small volume of milk in the milk storage tank, as set out in the independent claims, and then, when a level of milk in said milk storage tank exceeds a threshold level, the cooling is controlled in response to the temperature of the milk in a conventional fashion.\",\n \"Further features and advantages of the present invention will be evident from the following description.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS The present invention will become better understood from the detailed description of embodiments given below and the accompanying FIGS.\",\n \"1-3, which are given by way of illustration only, and thus, are not limitative of the present invention, wherein: FIG.\",\n \"1 is a schematic representation of an automatic milking system including a milk cooling arrangement according to a preferred embodiment of the present invention; FIG.\",\n \"2 is an exemplary diagram illustrating the cooling of the inventive milk cooling arrangement of FIG.\",\n \"1 as a function of time; and FIG.\",\n \"3 is another exemplary diagram illustrating the cooling of the inventive milk cooling arrangement of FIG.\",\n \"1 as a function of time.\",\n \"DETAILED DESCRIPTION OF EMBODIMENTS In the following description, for purpose of explanation and not limitation, specific details are set forth, such as particular techniques and applications in order to provide a thorough understanding of the present invention.\",\n \"However, it will be apparent for a person skilled in the art that the present invention may be practiced in other embodiments that depart from these specific details.\",\n \"An automatic milking system, as shown in FIG.\",\n \"1, comprises a milking robot 1 for milking animals, such as cows, including a milk flow meter 2 for measuring the milk flow during milking; a milk storage tank 5 which stores milk for collection on a regular time basis; a milk line 4 connecting the milking robot 1 with the milk storage tank 5; a milk pump (not shown) for pumping milk from the milking robot 1 to the milk storage tank 5; and a pre-cooling device 3 for cooling milk, which is pumped over to the milk storage tank 5.The milk storage tank 5 is provided with a stirrer 6 for stirring milk in the milk storage tank 5; and a cooling device 7 for cooling milk in the milk storage tank 5.The cooling device 7 includes typically an evaporator 8; a compressor 9; a condenser 10; and an expansion valve 12, all of which being interconnected by a connecting piping 13, such that a cooling medium can be circulated therein.\",\n \"The evaporator is suitably integrated in, or in contact with, a bottom portion 14 of the wall of the milk storage tank 5 and the compressor 9 is typically driven by an electrical motor (not shown).\",\n \"Furthermore, the cooling device 7 comprises a sensor 15 for sensing a quantity, which is indicative of a temperature of an inner surface area 16 of the bottom portion 14 of the milk storage tank 5.The sensor is preferably a temperature sensor 15 for measuring the temperature of the connecting piping 13 between the expansion valve 12 and the evaporator 8 of the cooling device 7.Alternatively, the temperature sensor 15 may be arranged to measure the temperature of the expansion valve 12, or of the evaporator 8.As a further alternative, the sensor may be a pressure sensor (not illustrated) for sensing the pressure in the evaporator 8 or preferably in the connecting piping 13 downstream from the evaporator 8.However, a temperature sensor is preferred since it is easier to arrange a temperature sensor 15 at the outside of the connecting piping 13, the expansion valve 12, or the evaporator 8 than to introduce a pressure sensor within the connecting piping 13.The milking system further comprises a controller 17, such as a microcomputer provided with suitable software, for controlling and managing the milking robot 1, including keeping records of animals to be milked and their respective characteristics, such as milk quality, amount of milk per milking, and time lapsed since last milking.\",\n \"Further, the controller 17 is adapted for controlling the cooling device 7, which includes receiving measured values from the milk flow meter 2 and the sensor 15.Alternatively, one controller is provided for the control of the milking robot and another separate controller is provided for the control of the cooling device (not illustrated), in which case the two controllers are connected with each other to provide for one-way or two-way communication between them.\",\n \"The milk flow in the milking system is measured by the milk flow meter 2 and may be registered by the controller 17 to obtain an accumulated milk flow and optionally to determine the level of milk in the milk storage tank 5 by prior knowledge of the design of the milk storage tank and the obtained accumulated milk flow.\",\n \"Alternatively, the level of milk in the milk storage tank may be measured by means of a level-measuring device (not illustrated) located in the milk storage tank 5.As a further alternative, the level of milk in the milk storage tank can be determined by measuring a static pressure or change in static pressure of the milk at the bottom of the milk storage tank 5, e.g.\",\n \"detected by a sensor (not illustrated) at the bottom of the milk storage tank.\",\n \"Instead of measuring the milk flow in the robot 1 it would be possible to provide a milk flow meter (not illustrated) in the milk line 4, e.g.\",\n \"adjacent to the milk storage tank 5, to measure the milk flow there.\",\n \"The cooling of the cooling device 7 is controlled, in accordance with the present invention, by operating the cooling device 7 in periods of cooling, each of which being followed by a respective period of non-cooling, where the duration of each period of cooling and/or non-cooling is determined and controlled by the controller 17.More precisely, the controller 17 is adapted to receive a value of the flow of milk from the milk flow meter 2; to determine, i.e.\",\n \"calculate, a cooling need for milk to be stored in said milk storage tank based on the received value of the flow of milk; and to receive a value of the quantity sensed by the sensor 15.The duration of each period of cooling and/or period of non-cooling is then controlled based on the determined cooling need and the value of the quantity sensed by sensor 15.It is particularly important to control the cooling such that the temperature of the inner surface area 16 of the bottom portion 14 of the milk storage tank 5 does not fall below 0° C. By such an approach the cooling of milk in the milk storage tank may be started when only small amounts of milk have been stored therein, and a quick cooling of the first amounts of stored milk is obtained without any risk of freezing the same.\",\n \"In FIG.\",\n \"2 is shown a diagram illustrating an example of a the cooling by the cooling device 7 as a function of time, wherein two periods of cooling τ1 and τ3 and two periods of non-cooling τ2 and τ4 are shown.\",\n \"The duration of two periods of cooling and two periods of non-cooling, at four different times are illustrated in FIG.\",\n \"2.Such a cooling may occur as a result of the following exemplary scenario.\",\n \"At t1 a sensor, e.g.\",\n \"the milk flow meter 2, indicates an increased milk flow to the controller, which calculates a new increased cooling need and in response thereto the controller starts the cooling.\",\n \"At t2 the temperature sensor 15 indicates a low temperature, e.g.\",\n \"close to 0° C., and in response thereto the controller stops the cooling.\",\n \"At t3 the temperature sensor 15 indicates a higher temperature and in response thereto the controller starts the cooling again.\",\n \"Then, at t4 the temperature sensor 15 indicates a low temperature and in response thereto the controller stops the cooling.\",\n \"The cooling need of the milking system depends on the amount of milk to be stored in the milk storage tank 5, which in turn is determined by the flow of milk in the milking system.\",\n \"The cooling by the cooling device 7, which is required for cooling a specific volume of milk, is divided into periods of cooling and non-cooling to avoid freezing of the milk, and these periods are continued until the cooling need is fulfilled as indicated by the milk flow meter 2 and/or the sensor 15.The accumulated milk flow may be utilized to calculate how much of a heat transfer area, i.e.\",\n \"the area where the cooling device efficiently draws heat from the milk storage tank, in the milk storage tank that is covered by milk.\",\n \"If the milk does not cover the whole heat transfer area, the risk of freezing the milk is particularly large, which may be taken into account by the controller 17 when determining the duration of the periods of cooling and/or the periods of non-cooling.\",\n \"The cooling need further depends on the design of the milk storage tank 5, and of the temperature surrounding the milk storage tank 5.The influence of the surrounding temperature will be less if the milk storage tank is insulated.\",\n \"Such insulation is usually implemented as a layer of insulation material between the wall of the milk storage tank and an outer shell.\",\n \"If the cooling device 7 has a minimum period of rest due to the electrical motor of the cooling device having a need to be cool down after a period of cooling, or else the motor might be damaged, such a minimum period of rest defines a minimum duration of a period of non-cooling as controlled by the controller 17.The flow pattern of milk in the milk storage tank 5, i.e.\",\n \"the flow pattern of the milk obtained by inflowing milk and/or obtained by an auxiliary device, such as the stirrer 6, affects how fast temperature differences of milk in different parts of the milk storage tank are evened out, which might be a factor to consider by the controller 17.A flow pattern obtained by the stirrer 6 has only to be considered when the milk level in milk storage tank has reached the lowermost portion of the stirrer such that stirring really takes place.\",\n \"Furthermore, there are a number of more operation conditions, which, more or less, may affect the cooling need, the cooling capacity, or which may be utilized to correct the cooling as performed by the cooling device 7 and controlled by the controller 17.The cooling may be controlled based on the time lapsed since the cooling device 7 ended a period of cooling, the temperature of the milk in the milk storage tank 5, and the amount of the milk in the milk storage tank 5.If the controller 17 takes these dependencies into account when determining the lengths of the periods of cooling and non-cooling, a better cooling control may be obtained.\",\n \"In some designs of a milking system the first amounts of milk pumped from the milking robot after cleaning the tank may not reach the milk storage tank until several cows have been milked, due to that the milk only fills the inner volume of the milk line 4.This affects also the cooling need and accordingly the controller 17 may take this into account when controlling the operation of the cooling device 7.The controller may be arranged to initiate a period of cooling when the milk pump of the milking system, at the milking robot, is started, and to control the cooling device based on the number of times the milk pump at the milking robot has been started during a predetermined period of time.\",\n \"Alternatively, or as complement, the controller 17 may be adapted to control the cooling of the cooling device based on the number of animals that have been milked during the predetermined period of time.\",\n \"As the control device 8 keeps record of the cows being milked and identifies each cow prior to milking, such identification may affect the control of the cooling device 7 since a period of cooling may begin earlier or last longer if the cow identified prior to milking is known to have a high milk yield.\",\n \"The efficiency of a cooling process using e.g.\",\n \"an air-cooled condenser may change significantly if the temperature surrounding the condenser changes, e.g.\",\n \"depending on the time of year or between daytime and nighttime.\",\n \"That is, a certain amount of electrical effect fed into the cooling device results in one level of cooling during daytime and another level of cooling during nighttime.\",\n \"Such environment-dependent cooling capacity could be compensated for by the controller if a temperature sensor is provided for detection of the temperature close to the condenser and using a correction factor depending on this temperature for adjusting the cooling.\",\n \"Alternatively, a timer may be used to adjust the correction factor if the temperature variations during the day and night are predictable.\",\n \"Further, if the pre-cooling device is a heat exchanger, e.g.\",\n \"a plate heat exchanger, it may not be capable to pre-cool a large flow of milk as efficient as a lower flow of milk and thus a larger flow of milk will be warmer when reaching the milk storage tank 5.Thus, the controller 17 preferably takes into account the efficiency of the pre-cooling device 3 by means of prior knowledge of its cooling capacity or by means of measuring the temperature of the milk in milk line 4.In a particular embodiment of the present invention the milk storage tank itself is pre-cooled, i.e.\",\n \"cooled before milk is entered therein.\",\n \"Preferably, the controller initiates a period of cooling of the milk storage tank 5 itself directly after the cleaning of the same is finished.\",\n \"In such manner, milk transported to the milk storage tank can be cooled faster, and thus an enhanced milk quality can be obtained.\",\n \"FIG.\",\n \"3 illustrates a diagram indicating periods of cooling followed by periods of non-cooling during such pre-cooling of the milk storage tank 5 itself.\",\n \"Firstly five periods of cooling lower the temperature of the milk storage tank to approximately 4° C. by cooling the heat transfer area during each period of cooling to approximately 0° C. During the periods of non-cooling the temperature differences in the milk storage tank wall is equalized.\",\n \"The sixth cooling period illustrates the cooling need of a small amount of milk entering, or about to enter, the milk storage tank.\",\n \"The seventh cooling period illustrates the cooling need of a large amount or several consecutive small amounts of milk entering, or about to enter, the milk storage tank.\",\n \"Next an example of cooling of a 3500-liter milk storage tank having a wall made of 300 kg stainless steel will be described.\",\n \"Subsequent to cleaning, the tank has a temperature of about 20° C. and then 2400 kJ of cooling capacity is needed to bring the tank temperature down to 4° C. according to the formula E=cmΔt, where c is the heat capacity, m is the mass of the tank and Δt is the temperature drop caused by cooling (c=500 J/kg° C. for stainless steel, m=300 kg, and Δt=16° C.).\",\n \"Any heat losses to the air surrounding the tank are assumed to be negligible in this calculation.\",\n \"Such a milk storage tank contains about 4 kg of air, which needs 65 kJ of cooling capacity to bring the air temperature down to 4° C., since cp=1000 J/kg° C. for air.\",\n \"Further, 0.06 kg of moisture in the air will be reduced to 0.03 kg during the cooling process, which consumes 74 kJ of condensation energy.\",\n \"Thus, it can be seen that the cooling need of the tank in this case is 2539 kJ.\",\n \"A 3 kW cooling compressor having a cooling factor of ε=2.4 needs a running time of about six minutes to produce the cooling capacity needed to bring the temperature of the milk storage tank down to 4° C. (according to running time τ=E/Pε, where the energy E is 2539 kJ, the power P is 3 kW, and the cooling factor ε is 2.4).\",\n \"Since the heat transfer between the bottom wall of the empty milk storage tank and the side walls thereof takes place during a period of time, it may be advisable to start the cooling compressor subsequent to the cleaning and let it run for two minutes to get an initial lowering of the temperature at the bottom of the tank.\",\n \"Thereafter a period of rest of about two minutes is used (which may be given by the temperature sensed by sensor 15) to equalize the temperature in the tank.\",\n \"Two minutes running period and two minutes rest period can then be repeated (given by the temperature sensed by sensor 15) until a complete running time of the six minutes is achieved.\",\n \"Milk leaving a cow has a temperature of about 37° C., but may have been reduced to about 35° C. during transport to the milk storage tank.\",\n \"An amount of 15 l of milk needs an effective compressor running time of about 270 s, as m≈15 kg, Δt=31° C., c=4190 J/kg° C. for milk, P=3000 W, and ε=2.4.As the milk from the first cow only covers a part of the cooling surface in the milk storage tank, the cooling device is run in sequences of about one minute followed by a rest period of two minutes (which may be given by the temperature sensed by sensor 15), to avoid freezing the milk.\",\n \"The controller keeps track of the accumulated milk flow as more cows are being milked and increases the running time when the volume of milk in the tank increases, e.g.\",\n \"to two minutes running time.\",\n \"It shall be appreciated that the duration of each of the periods of cooling and non-cooling is determined according to an algorithm, which uses at least the determined cooling need and the quantity sensed as inputs.\",\n \"Further inputs may be employed to obtain a refined cooling process.\",\n \"It shall particularly be noted that the duration of each period of non-cooling may not only be determined in response to the quantity sensed, but other measured values or prior knowledge may be used such as e.g.\",\n \"an estimated average number of cows to be milked per time unit.\",\n \"In yet a particular embodiment of the present invention the cooling of milk may be performed firstly, when there is only a small volume of milk in the milk storage tank 5, as described with reference to the first embodiment of the invention, and then, when a level of milk in said milk storage tank exceeds a threshold level, the cooling is controlled in a conventional fashion, i.e.\",\n \"in response to a temperature of milk in the milk storage tank 5.The threshold value is preferably determined to be a level, which is sufficiently high so the stirrer 6 can effectively stir the milk in the milk storage tank 5 and even out any temperature gradients in the milk.\",\n \"Thus the risk of freezing the milk is very low.\",\n \"In still a particular embodiment of the present invention the controller 17 may use a feedback process for adjusting the periods of cooling and/or periods of non-cooling.\",\n \"Thus, if a temperature of the milk at the end of a cooling process (e.g.\",\n \"a cooling process, which ends when the milk in the milk storage tank 5 is collected, or alternatively when the milk level in the milk storage tank exceeds the above-mentioned threshold value) is detected to be different than expected (e.g.\",\n \"different than an end temperature used in the calculation of the cooling need), the controller may adjust the cooling capacity of the cooling device 7 accordingly for a following cooling process.\",\n \"Such an adjustment may be made by introducing a correction factor to correct for the discrepancy between the detected and the expected temperatures.\",\n \"The present invention being thus described, it will be obvious that the same may be varied in a plurality of ways.\",\n \"All such variations as would be obvious for a person skilled in the art are intended to be included within the scope of the present invention.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471172"}}},{"rowIdx":581,"cells":{"text":{"kind":"list like","value":[["Drainage and irrigation approach and structure as well as its implementation","This Invention relates to a drainage and irrigation approach, which primarily includes the initial stage of green belt irrigation and the later stage of rainwater drainage.","The drainage and irrigation structures employed in this approach consist of curb stones, edge stones, border stones, and boundary stones, which may have combinations of drainage troughs [3], rainwater ports [6] and drainage holes [4], or drainage troughs [3] and rainwater ports [6], or drainage troughs [3] and drainage holes [4].","Depending on the conditions of planted areas, the drainage and irrigation structures discussed above may be deployed along roads, landscaping on top of architectural structures, planted areas, seeded strips or barren hill slopes."],["1.A drainage and irrigation approach suitable for both rainwater and road cleaning water; the said approach consists of two stages: the initial stage of green belt irrigation and the later stage of rainwater drainage; firstly, rainwater at the beginning of a rainfall or in the event of road cleaning with debris flows through drainage holes in the drainage and irrigation structure into the planted areas, thereby providing the nutrients and water necessary for plant growth; secondly, if the rain continues falling and the rainwater rises to a certain level, clear water flows through the rainwater ports in the drainage and irrigation structure into the underground pipes and corresponding connected pipelines; the clear rainwater is then stored in underground reservoirs for storage or flows into rivers and lakes; from there it evaporates into the ecological cycle of nature.","2.The drainage and irrigation approach as described in claim 1, wherein, for the initial green belt irrigation stage, flat curb stones are laid horizontally along one side or both sides of the road, their top surface flush with the pavement surface; the curb barrier stones are laid perpendicularly to and tightly against the flat curb stones along the outer edge of the road, their top surface higher than the road pavement; on either side of the road, there is a green belt [A] on the outer side of the curb barrier stones; the surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole in each of the said curb barrier stones so that there is an elevation drop; at the beginning of a rainfall, the rainwater flows along a drainage channel consisting of a trough in each flat curb stone and a drainage hole in each curb barrier stone.","It then flows through the exit opening of the drainage hole onto the green belt [A].","3.The drainage and irrigation approach as described in claim 1, wherein, for the later drainage stage, rainwater ports are installed at a certain interval in the row of curb barrier stones; the rainwater ports are connected to the underground drainage pipeline; the highest position of the said drainage hole in each curb barrier stone should be lower than the bottom edge [b] of the said rainwater port; when the rainwater accumulates in the green belt [A] and the water level rises to a point [a] above the rainwater port, it flows through the outlet into the underground pipeline; the drainage holes in curb barrier stones can also effectively prevent floating debris in the green belt from entering the rainwater ports so that the water flowing into the outlets is virtually filtered.","4.The drainage and irrigation approach as described in claim 1, wherein the following engineering requirements must be met: the drainage initiation point of the pavement is at Elevation b, the drainage initiation point of the rainwater port is at Elevation a, the drainage or irrigation initiation point of the curb is at Elevation C, the blocking point of the floating debris in the green belt is at Elevation d, and the water accumulation initiation point in the green belt and the seeded strips is at Elevation e. 5.The drainage and irrigation approach as described in claim 4, wherein the elevation between b and C is 6 cm; the elevation between b and d is about 12 cm; and the elevation between d and e is about 5 cm.","6.The curb stones used in a type of irrigation and drainage structure, which curb stones consist of flat curb stones and curb barrier stones having the following features: each of the said flat curb stones has a longitudinal drainage trough in its top surface and each of the said curb barrier stones has a half-circular cut-out at either end so that a drainage hole is formed when two curb barrier stones are laid end-to-end; the said longitudinal trough on the flat curb stones and the said drainage holes on the curb barrier stones are interconnected to form an unobstructed channel for water flows.","7.The curb stones as described in claim 6, wherein the said curb barrier stones have rainwater ports installed at a certain interval and the rainwater ports are connected to underground pipelines; the highest point of the drainage hole on each curb barrier stone is lower than the bottom edge of the said rainwater port.","8.The curb stones as described in claim 6, wherein the said longitudinal trough stretches on the top surface of the said flat curb stone and along the upper edge close to the said curb barrier stone; it is a trough cut along the said upper edge of the flat curb stone.","9.The curb stones as described in claim 6, wherein the said drainage hole on the curb barrier stone goes through either end of the curb barrier stone; its opening at the flat curb stone side is the water entry and on the outer side of the curb barrier stone is the water exit; the bottom edge of the entry opening is flush with the bottom of the longitudinal trough on the flat curb stone; the exit opening is somewhat lower than the entry opening to produce a horizontal elevation drop; the entry opening and the exit opening may be set level.","10.The curb stones as described in claim 8, wherein the said longitudinal trough is a half-circular trough cut along the edge of the flat curb stone close to the said curb barrier stone; additional cut-outs may or may not be made at proper positions along the trough in correspondence to the drainage holes on the curb barrier stones.","11.The curb stones as described in claim 6, wherein the half-circular drainage hole cut-out in the curb barrier stone may be a horizontal or a slanting trough; in other words, its entry and its exit may be at the same elevation or may have a proper elevation drop.","12.The edge stones used in a second type of irrigation and drainage structure, which edge stones consist of flat stones and barrier stones having the following features: Each of the said flat stones has a longitudinal trough on the side of its top surface close to the barrier stones, and the longitudinal trough connects with a drainage hole at either end of each barrier stone and several evenly spaced drainage holes in the middle section of the barrier stone to form an unobstructed channel for water flows.","13.The edge stones as described in claim 12, wherein the bottom edge of the multiple holes in the said barrier stone is flush with the bottom of the longitudinal trough in the flat stone; the drainage hole at either end of the barrier stone is flush with and connects to the bottom of the longitudinal trough; the hole is a half-circular trough at the end of each barrier stone; when two barrier stones are set end-to-end, a circular drainage hole is formed.","14.The edge stones as described in claim 12, wherein the said flat stone and the said barrier stone may form an “L”-shaped or “--|”-shaped structure.","15.The edge stones as described in claim 14, wherein the said flat stone and the said barrier stone may be a single-piece unit or two separate pieces joined together.","16.The edge stones as described in claim 15, wherein, as separate pieces to be joined together, the said flat stones are laid horizontally on one side or both sides of the road with their top surface flush with the pavement surface while the barrier stones are set perpendicularly to and tightly against the flat stones with their top surface higher than the pavement; the green belt lies on the outer side of the barrier stones; the surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole in each of the said barrier stones so that there is an elevation drop.","17.The edge stones as described in claim 15, wherein, when the flat stones and the barrier stones form single-piece units, the edge stones may be directly laid on one side or both sides of the road; the top surface of the barrier stones is higher than the pavement surface and the top surface of the flat stones is flush with the pavement surface; the green belt lies on the outer side of the barrier stones; the surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole in each of the said barrier stones so that there is an elevation drop.","18.The border stones used in a third type of irrigation and drainage structure, which border stones consist of flat stones and barrier stones having the following features: This structure also includes supplemental barrier stones; each of the flat stones has a stopper at either end and a trough in the middle section near one side; the trough is connected with the multiple drainage holes in the barrier stones; the top surface of the barrier stones is higher than the supplemental barrier stones, whose top surface is higher than the top surface of the flat stones; the top surface of the flat stones is flush with the upper edge of the trough and higher than the drainage holes in the barrier stones; the barrier stones and the supplemental barrier stones form an overflow relief trough between themselves.","19.The border stones as described in claim 18, wherein the flat stone, the barrier stone and the supplemental barrier stone may form a “|_|”-shaped structure.","20.The border stones as described in claim 18, wherein the said flat stone and the said supplemental barrier stone may be a single-piece “L”-shaped structure, which may then join the barrier stone to form a “|_|”-shaped structure.","21.The border stones as described in claim 18, wherein the said flat stone, barrier stone, and supplemental barrier stone may be a single-piece unit or three separate pieces joined together.","22.The border stones as described in claim 18, wherein the said supplemental barrier stones may have overflow outlets and overflow pipelines connected to those overflow outlets; they constitute the border stones used in a fourth type of drainage and irrigation structure.","23.The border stones as described in claim 18, wherein, in actual application, the said border stones are laid on one side of a green belt, which lies beyond the barrier stones; the surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole in each of the said barrier stones so that there is an elevation drop; the rainwater flows into the trough in the flat stones and then through the drainage holes in the barrier stones onto the green belt; the supplemental barrier stones prevent the rainwater from flowing randomly.","24.The border stones as described in claim 22, wherein, in actual application, the border stones, consisting of the said flat stones, the said barrier stones and the said supplemental barrier stones, may be laid on one side of a sloping planted area, which lies outside the barrier stones; the surface elevation [e] of the planted area is lower than the bottom edge of the drainage hole in each of the said barrier stones so that there is an elevation drop; the water in the planted area flows through the drainage holes in the barrier stones into the overflow outlets in the supplemental barrier stones, and then through the connected overflow pipelines into drainage pipelines.","25.The boundary stones used in a fifth type of drainage and irrigation structure, wherein the boundary stones are U-shaped tubular structures; the tubular wall on one side has multiple drainage holes, which are connected with the water trough in the stones; the tubular stones have internal stoppers on both sides, or have no stopper.","26.The boundary stones as described in claim 25, wherein the drainage holes on the tubular wall are positioned lower than the top of the stoppers, which is a level surface and functions as the drainage initiation point.","27.The boundary stones as described in claim 25, wherein, in actual application, the U-shaped tubular boundary stones may be laid on one side of a planted area with the drainage holes on their walls opening towards the planted area; excessive rainwater in the planted area may flow through the drainage holes into the trough in the tubular boundary stones, which function like a dike to hold water around the tiered fields on the hill slopes; the boundary stones may also be used together with the border stones discussed earlier on barren hill slopes.","28.The curb stones as described in claim 6, wherein, in actual application, the said curb barrier stones may be set perpendicularly along one side of the road foundation with their rainwater ports below the top surface of the foundation; the top surface of the curb barrier stones is higher than the pavement surface; another row of curb barrier stones and flat curb stones is laid on the other side of the road; outside the curb barrier stones are planted areas; the rainwater ports in the curb barrier stones on both sides of the road are connected to underground drainage pipelines, which in turn lead into municipal sewage pipelines.","29.The drainage and irrigation structures as described in one of claims 6, 12, 18, and 25 wherein, depending on the conditions of the planted areas and the types of terrain, the said curb stones, edge stones, border stones and boundary stones may be set at one side of the planted areas, on one side or both sides of the roads, or at appropriate locations on barren hills; they may be connected to underground pipelines which lead to main drainage pipes, which in turn may be connected to drainage ditches."],["<SOH> BACKGROUND TECHNOLOGY <EOH>Roads, drainage pipeline networks, and landscaping are important urban infrastructures, which have a direct impact on the quality of the urban environment.","The conventional road curb generally consists of curb barrier stones and flat curb stones.","The flat curb stones are laid horizontally along one side or both sides of the road, with their upper surface flush with the road pavement.","The curb barrier stones are laid perpendicularly to and tightly against the flat curb stones along the outer edge of the road, with their upper surface higher than the road pavement.","To facilitate the drainage of rainwater, a curb barrier stone with a rainwater port is placed at a certain interval (generally 30-50 meters) along the road, or a special grated rainwater port is placed in its place.","The said rainwater port is connected through a pipe to an underground drainage pipeline.","To improve the rainwater drainage speed and efficiency, rainwater ports should be placed at the lowest positions on the road.","Therefore, when necessary, longitudinal slopes are built on the road, that is, the pavement is laid with a specific sloping grade in the longitudinal direction of the road.","Then, rainwater ports or special rainwater drainage devices are placed at the lowest positions on the pavement slopes.","As is disclosed in a Japanese patent document (Publication No.","6-248610) and shown in FIG.","15 , a row of curb barrier stones are laid on either side of the seeded median strip of the road pavement.","Each of the curb barrier stones has two evenly embedded pre-fabricated blocks, which are water pervious.","The curb barrier stones are laid in such a manner that the bottoms of the pre-fabricated pervious blocks are flush with the surface of the pavement.","In the event of a rainfall or road cleaning, the rainwater or the cleaning water is allowed to flow through the pores in the pre-fabricated pervious blocks into the soil in the seeded median strip (in the direction indicated by the arrow in FIG.","15 ) for irrigation purposes.","Sands and other debris carried by the water are stopped and stranded between the curb barrier stones and the pavement.","Consequently, regular maintenance work such as removing the debris is necessitated.","Such maintenance work requires a lot of labor and expenses.","Also, as is disclosed in a German patent document (DE 295 08 680 U1) and shown in FIG.","4 , a row of curb barrier stones are laid on either side of the sloping road pavement.","A drainage outlet is formed between each pair of adjacent curb barrier stones.","On the outer side of either row of the curb barrier stones are green barrier slopes or seeded areas.","In the event of a rainfall or road cleaning, the rainwater or the cleaning water flows down the sloping pavement and through the drainage outlets in the curb barrier stones directly into the green barrier slopes or seeded areas for irrigation purposes.","In a heavy rainfall, the vegetation may easily be damaged, the soil be washed away, and the water resources be wasted.","Again, as is disclosed in a French patent document (N° 1.368.655) and shown in FIG.","5 , a row of curb barrier stones with pre-installed slanting pipe joints is laid along either side of the road.","The pipe joints are connected to an underground pipeline.","The cross section of the curb barrier stones assumes an “L” shape.","The curb barrier stones are laid in such a manner that their top surface is even with the pavement surface.","The bottom part of the curb barrier stone tilts upward and slants inward.","The lower part of the opening of the pre-installed pipe joint is flush with the inner edge of the tilting section of the bottom part of the curb barrier stone.","In the event of a rainfall or road cleaning, the rainwater or cleaning water flows through the opening of the pipe joint directly into the underground sewage pipeline.","Sludge and other debris carried by the water may contaminate and clog the pipes.","Debris removal will require a lot of labor and expenses.","In addition, the water resources are wasted.","The conventional technology as evidenced in the examples presented in the previous paragraphs has created a lot of problems in road debris removing, which has been costly and labor intensive.","Road maintenance often generates a lot of dust, thus continually causing pollution.","In addition, rainfalls or irrigation of the green belts and seeded strips along the roads cause soil loss, underground pipe clogging and contaminating, and contamination of downstream water resources.","In short, in the event of rainfalls, the existing curb structures cause debris to be washed from green belts and surfaces of structures to the road pavements and then to underground pipelines and rivers and lakes.","We are confronted not only with the issue of soil and water loss, but also with the risk of on-going contamination."],["TECHNICAL AREA This Invention relates to a drainage and irrigation approach and structure as well as its implementation.","In particular, it relates to a drainage and irrigation approach and structure as well as its implementation in connection with roads, landscaping on top of architectural structures, green belts, planted borders, and barren hill slopes.","BACKGROUND TECHNOLOGY Roads, drainage pipeline networks, and landscaping are important urban infrastructures, which have a direct impact on the quality of the urban environment.","The conventional road curb generally consists of curb barrier stones and flat curb stones.","The flat curb stones are laid horizontally along one side or both sides of the road, with their upper surface flush with the road pavement.","The curb barrier stones are laid perpendicularly to and tightly against the flat curb stones along the outer edge of the road, with their upper surface higher than the road pavement.","To facilitate the drainage of rainwater, a curb barrier stone with a rainwater port is placed at a certain interval (generally 30-50 meters) along the road, or a special grated rainwater port is placed in its place.","The said rainwater port is connected through a pipe to an underground drainage pipeline.","To improve the rainwater drainage speed and efficiency, rainwater ports should be placed at the lowest positions on the road.","Therefore, when necessary, longitudinal slopes are built on the road, that is, the pavement is laid with a specific sloping grade in the longitudinal direction of the road.","Then, rainwater ports or special rainwater drainage devices are placed at the lowest positions on the pavement slopes.","As is disclosed in a Japanese patent document (Publication No.","6-248610) and shown in FIG.","15, a row of curb barrier stones are laid on either side of the seeded median strip of the road pavement.","Each of the curb barrier stones has two evenly embedded pre-fabricated blocks, which are water pervious.","The curb barrier stones are laid in such a manner that the bottoms of the pre-fabricated pervious blocks are flush with the surface of the pavement.","In the event of a rainfall or road cleaning, the rainwater or the cleaning water is allowed to flow through the pores in the pre-fabricated pervious blocks into the soil in the seeded median strip (in the direction indicated by the arrow in FIG.","15) for irrigation purposes.","Sands and other debris carried by the water are stopped and stranded between the curb barrier stones and the pavement.","Consequently, regular maintenance work such as removing the debris is necessitated.","Such maintenance work requires a lot of labor and expenses.","Also, as is disclosed in a German patent document (DE 295 08 680 U1) and shown in FIG.","4, a row of curb barrier stones are laid on either side of the sloping road pavement.","A drainage outlet is formed between each pair of adjacent curb barrier stones.","On the outer side of either row of the curb barrier stones are green barrier slopes or seeded areas.","In the event of a rainfall or road cleaning, the rainwater or the cleaning water flows down the sloping pavement and through the drainage outlets in the curb barrier stones directly into the green barrier slopes or seeded areas for irrigation purposes.","In a heavy rainfall, the vegetation may easily be damaged, the soil be washed away, and the water resources be wasted.","Again, as is disclosed in a French patent document (N° 1.368.655) and shown in FIG.","5, a row of curb barrier stones with pre-installed slanting pipe joints is laid along either side of the road.","The pipe joints are connected to an underground pipeline.","The cross section of the curb barrier stones assumes an “L” shape.","The curb barrier stones are laid in such a manner that their top surface is even with the pavement surface.","The bottom part of the curb barrier stone tilts upward and slants inward.","The lower part of the opening of the pre-installed pipe joint is flush with the inner edge of the tilting section of the bottom part of the curb barrier stone.","In the event of a rainfall or road cleaning, the rainwater or cleaning water flows through the opening of the pipe joint directly into the underground sewage pipeline.","Sludge and other debris carried by the water may contaminate and clog the pipes.","Debris removal will require a lot of labor and expenses.","In addition, the water resources are wasted.","The conventional technology as evidenced in the examples presented in the previous paragraphs has created a lot of problems in road debris removing, which has been costly and labor intensive.","Road maintenance often generates a lot of dust, thus continually causing pollution.","In addition, rainfalls or irrigation of the green belts and seeded strips along the roads cause soil loss, underground pipe clogging and contaminating, and contamination of downstream water resources.","In short, in the event of rainfalls, the existing curb structures cause debris to be washed from green belts and surfaces of structures to the road pavements and then to underground pipelines and rivers and lakes.","We are confronted not only with the issue of soil and water loss, but also with the risk of on-going contamination.","SCOPE OF THE INVENTION The purpose of this Invention is to provide a drainage and irrigation approach and structure, which, in the event of rainfalls and road cleaning, may function simultaneously in two ways: retaining dirt and debris in the green belts and seeded strips to avoid drainage pipeline blockage, downstream water body contamination, and soil loss on the one hand, and properly allocating water consumption and making better use of external water resources in the green belts and seeded strips to improve the environmental conditions on the roads and green belts as well as seeded strips, promote the water and soil conservation in the green belts and planting area, and cultivate the natural conditions for plant growth.","Another purpose of this Invention is to provide a drainage and irrigation approach and structure, which may offer curb stones of various structural forms to suit the infrastructural needs of hard soil drainage, roadway drainage, as well as preservation of landscaping on top of architectural structures, green belts, seeded strips, planted plots and barren hills.","The technical solution of this Invention involves a drainage and irrigation approach suitable for both rainwater and road cleaning water.","The said approach consists of two stages: the initial stage of irrigating green belts and the later stage of rainwater drainage.","In the initial irrigation stage rainwater, at the beginning of a rainfall or foul water in the event of road cleaning with debris flows through drainage holes in the drainage and irrigation structure into the planted areas, thereby providing the nutrients and water necessary for plant growth.","If the rain continues falling and the rainwater rises to a certain level, clear water flows through rainwater ports in the drainage and irrigation structure into the underground pipes and corresponding connected pipelines.","The clear rainwater is then stored in underground reservoirs for storage or flows into rivers and lakes.","From there it evaporates into the ecological cycle of nature.","The said drainage and irrigation approach has the following engineering requirements: The drainage initiation point of the pavement is at Elevation b, the drainage initiation point of the rainwater port is at Elevation a, the drainage or irrigation initiation point of the curb is at Elevation C, the blocking point of the floating debris in the green belt is at Elevation d, and the water accumulation initiation point in the green belt and the seeded strips is at Elevation e, where the said point b is higher than point C and point d, which in turn is higher than point e. This Invention relates to the curb stones used in a type of irrigation and drainage structure, which curb stones consist of flat curb stones and curb barrier stones having the following features: Each of the said flat curb stones has a longitudinal drainage trough in its top surface and each of the said curb barrier stones has a half-circular cut-out at either end so that a drainage hole is formed when two curb barrier stones are laid end-to-end.","The said longitudinal trough on the flat curb stones and the said drainage holes on the curb barrier stones are interconnected to form an unobstructed channel for water flows.","The said curb barrier stones have rainwater ports installed at a certain interval and the rainwater ports are connected to underground pipelines.","The highest point of the drainage hole in each curb barrier stone is lower than the bottom edge of the said rainwater port.","The half-circular cut-out in the curb barrier stone may be a horizontal or a slanting trough.","In other words, its entry and its exit may be at the same elevation or may have a proper elevation drop.","This Invention relates to the edge stones used in a second type of irrigation and drainage structure, which edge stones consist of flat stones and barrier stones having the following features: Each of the said flat stones has a longitudinal trough on the side of its top surface close to the barrier stones, and the longitudinal trough connects with a drainage hole at either end of each barrier stone and several evenly spaced drainage holes in the middle section of the barrier stone to form an unobstructed channel for water flows.","The said flat stone and the said barrier stone may form an “L”-shaped or “--|”-shaped structure.","The said flat stone and the said barrier stone may be a single-piece unit or two separate pieces joined together.","As separate pieces to be joined together, the said flat stones are laid horizontally on one side or both sides of the road with their top surface flush with the pavement surface while the barrier stones are set perpendicularly to and tightly against the flat stones with their top surface higher than the pavement.","The green belt [A] lies on the outer side of the barrier stones.","The surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole in each of the said barrier stones so that there is an elevation drop.","This Invention relates to the border stones used in a third type of irrigation and drainage structure, which border stones consist of flat stones and barrier stones having the following features: This structure also includes supplemental barrier stones.","Each of the flat stones has a stopper at either end and a trough in the middle section near one side.","The trough is connected with the multiple drainage holes in the barrier stones.","The top surface of the barrier stones is higher than the supplemental barrier stones, whose top surface is higher than the top surface of the flat stones.","The top surface of the flat stones is flush with the upper edge of the trough and higher than the drainage holes in the barrier stones.","The barrier stones and the supplemental barrier stones form an overflow relief trough between themselves.","The flat stone, the barrier stone and the supplemental barrier stone may form a “|_|”-shaped structure.","The said flat stone and the said supplemental barrier stone may be a single-piece “L”-shaped structure, which may then join the barrier stone to form a “|_|”-shaped structure.","The said flat stone, barrier stone, and supplemental barrier stone may be a single-piece unit or three separate pieces joined together.","The said supplemental barrier stones may have overflow outlets and overflow pipelines connected to those overflow outlets.","They constitute the border stones used in a fourth type of drainage and irrigation structure.","This Invention also relates to the boundary stones used in a fifth type of drainage and irrigation structure, wherein the boundary stones are U-shaped tubular structures.","The tubular wall on one side has multiple drainage holes, which are connected with the water trough in the stones.","The tubular stones may or may not have internal stoppers on both sides.","The said curb barrier stones may be set perpendicularly along one side of the road foundation with their rainwater ports below the top surface of the foundation.","The top surface of the curb barrier stones is higher than the pavement surface.","Another row of curb barrier stones and flat curb stones is laid on the other side of the road.","Outside the curb barrier stones are planted areas.","The rainwater ports in the curb barrier stones on both sides of the road are connected to underground drainage pipelines respectively, which in turn lead into municipal sewage pipes.","Depending on the conditions of the areas and the types of terrain, the said curb stones, edge stones, border stones and boundary stones may be set at one side of the planted areas, on one side or both sides of the roads, or at appropriate locations on barren hills.","They may be connected to underground pipelines which lead to main drainage pipes, which in turn may be connected to drainage ditches.","DESCRIPTION OF THE DRAWINGS FIG.","1 is an operation flow chart of this Invention.","FIG.","2 is a structural diagram of the curb stones used in Application Sample 1 of this Invention.","FIG.","3 is a diagram of the operational principles in the initial green belt irrigation stage in Application Sample 1 of this Invention.","FIG.","4 is a diagram of the operational principles in the later drainage stage in Application Sample 1 of this Invention.","FIG.","5 is an engineering diagram of the curb drainage structure in Application Sample 1 of this Invention.","FIG.","6 is a structural diagram of the edge stones used in Application Sample 2 of this Invention.","FIG.","7 is an operational diagram of Application Sample 2 of this Invention.","FIG.","8 is a structural diagram of the border stones used in Application Sample 3 of this Invention.","FIG.","9 is an operational diagram of Application Sample 3 of this invention.","FIG.","10 is a structural diagram of the border stones used in Application Sample 4 of this Invention.","FIG.","11 is an operational diagram of Application Sample 4 of this invention.","FIG.","12 is a structural diagram of the boundary stones used in Application Sample 5 of this Invention.","FIG.","13 is an operational diagram of Application Sample 6 of this invention.","FIG.","14 is an operational diagram of Application Sample 7 of this invention.","FIG.","15 is a structural diagram of the conventional technology (part 1).","FIG.","16 is a structural diagram of the conventional technology (part 2).","FIG.","17 is a 3-D diagram of the objects in FIG.","16.FIG.","18 is a structural diagram of the conventional technology (part 3).","FIG.","19 is a top view of the objects in FIG.","18.IMPLEMENTATION OF THE INVENTION The application samples presented in the following paragraphs are intended to illustrate in detail the implementation and benefits of this Invention so that readers may see the creativity embodied in this Invention.","The samples should be studied together with the drawings.","See FIG.","1, which is an operation flow chart of this Invention.","This Invention involves a drainage and irrigation approach suitable for both rainwater and road cleaning water.","The said approach consists of two stages: the initial stage of irrigating green belts and seeded strips and the later stage of rainwater drainage.","In the initial irrigation stage, rainwater at the beginning of a rainfall or foul water in the event of road cleaning with debris flow through drainage holes in the drainage and irrigation structure onto the planted areas, thereby providing the nutrients and water necessary for plant growth.","If the rain continues falling and the rainwater rises to a certain level, it flows through the rainwater ports in the drainage and irrigation structure into the underground pipes and connected pipelines.","The clear rainwater is then stored in underground reservoirs for storage or flows into rivers and lakes.","From there it evaporates into the ecological cycle of nature.","The concept of this Invention is as follows: At the beginning of a rainfall or in the event of road cleaning, the water with debris flows through drainage holes in the drainage and irrigation structure onto the planted areas, thereby providing the nutrients and water necessary for plant growth.","If the rain continues falling and the rainwater rises to a certain level, it flows through the rainwater ports in the drainage and irrigation structure into the underground pipes and connected pipelines.","The clear rainwater is then stored in underground reservoirs for storage or flows into rivers and lakes.","From there it evaporates into the ecological cycle of nature.","Application Sample 1 See FIG.","2, which shows the curb stones used in a type of irrigation and drainage structure.","The curb stones consist of flat curb stones [1] and curb barrier stones [2].","Each of the said flat curb stones [1] has a longitudinal drainage trough [3] in its top surface and each of the said curb barrier stones [2] has a half-circular cut-out at either end so that a drainage hole [4] is formed when two curb barrier stones [2] are laid end-to-end.","The said longitudinal trough [3] on the flat curb stones and the said drainage holes [4] on the curb barrier stones are interconnected to form an unobstructed channel for water flows.","The said curb barrier stones [2] have rainwater ports [6] installed at a certain interval and connected to underground pipelines [8].","The rainwater port [6], assuming a “” shape, can prevent the dirty water in the green belts [A] from entering into the rainwater ports [6].","The highest point of the drainage hole [4] on the curb barrier stone is lower than the bottom edge of the said rainwater port [6].","The said longitudinal trough stretches on the top surface of the said flat curb stone [1] and along the upper edge close to the said curb barrier stone [2].","It is a trough cut along the said upper edge of the flat curb stone [1].","The said drainage hole [4] on the curb barrier stone [2] goes through either end of the curb barrier stone.","Its opening at the flat curb stone [2] side is the water entry and on the outer side of the curb barrier stone [2] is the water exit.","The bottom edge of the entry opening is flush with the bottom of the longitudinal trough [3] on the flat curb stone.","The exit opening is somewhat lower than the entry opening to produce a horizontal elevation drop.","However, the entry opening and the exit opening may be set level.","The said longitudinal trough [3] is a half-circular trough cut along the edge of the flat curb stone [1] close to the said curb barrier stone [2].","Additional cut-outs may or may not be made at proper positions along the trough in correspondence to the drainage holes [4] on the curb barrier stones (cut-outs not shown in the drawing).","The half-circular drainage hole cut-out in the curb barrier stone may be a horizontal or a slanting trough.","In other words, its entry and its exit may be at the same elevation or may have a proper elevation drop.","In view of the structural features of the said curb stones, the relevant drainage and irrigation approach is as follows (see FIGS.","3-4): 1.For the initial green belt irrigation stage, flat curb stones [1] are laid horizontally along one side or both sides of the road, their top surface flush with the pavement surface [7].","The curb barrier stones [2] are laid perpendicularly to and tightly against the flat curb stones [1] along the outer edge of the road, their bottom part set in a holding frame [21] and their top surface higher than the road pavement.","On either side of the road, there is a green belt [A] on the outer side of the curb barrier stones [2].","The surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole [4] in each of the said curb barrier stones so that there is an elevation drop.","At the beginning of a rainfall, the rainwater [9] flows along a drainage channel consisting of a trough [3] in each flat curb stone and a drainage hole [4] in each curb barrier stone.","It then flows through the exit opening of the drainage hole [4] onto the green belt [A].","2.For the later drainage stage, a rainwater port [6] is installed at a certain interval in the row of curb barrier stones.","The rainwater ports [6] are connected to the underground drainage pipeline [8].","The highest position of the said drainage hole [4] in each curb barrier stone should be lower than the bottom edge [b] of the said rainwater port [6].","When the rainwater accumulates in the green belt [A] and the water level rises to a point [a] above the rainwater port, it flows through the outlet into the underground pipeline [8].","The rainwater port [6] in each curb barrier stone can also effectively prevent floating debris in the green belt from entering the rainwater port [6] so that the water flowing into the outlet is virtually filtered, thus avoiding clogging of underground pipelines [8] and rivers as well as reducing the maintenance work and labor required to remove the blockage.","The said drainage and irrigation approach has the following engineering requirements (see FIG.","5): The drainage initiation point of the pavement is at Elevation b, the drainage initiation point of the rainwater port is at Elevation a, the drainage or irrigation initiation point of the curb is at Elevation C, the blocking point of the floating debris in the green belt is at Elevation d, and the water accumulation initiation point in the green belt and the seeded strips is at Elevation e, where the elevations of point a and point b shall be calculated on the basis of the pavement width and grade while the elevations of other points should also be properly set depending on the environmental conditions.","The elevation between b and C is 6 cm; the elevation between b and d is about 12 cm; and the elevation between d and e is about 5 cm.","Application Sample 2 See FIGS.","6-7, which show the edge stones used in a second type of irrigation and drainage structure.","The edge stones consist of flat stones [10] and barrier stones [20].","Each of the said flat stones [10] has a longitudinal trough [30] on the side of its top surface close to the barrier stones [20], and the longitudinal trough connects with a drainage hole [40] at either end of the barrier stone and several evenly spaced drainage holes [40] in the middle section of the barrier stone to form an unobstructed channel for water flows.","The bottom edge of the multiple holes [40] in the said barrier stone [20] is flush with the bottom of the longitudinal trough [30] in the flat stone.","The drainage hole [40] at either end of the barrier stone [20] is flush with and connects to the bottom of the longitudinal trough [30].","The hole is a half-circular trough at the end of each barrier stone.","When two barrier stones are set end-to-end, a circular drainage hole [40] is formed.","The said flat stone [20] and the said barrier stone [30] may form an “L”-shaped or “--|”-shaped structure.","The said flat stone [20] and the said barrier stone [30] may be a single-piece unit or two separate pieces joined together.","See FIG.","7.When the flat stone 20 and the barrier stone 30 are as separate pieces to be joined together, the said flat stones [20] are laid horizontally on one side or both sides of the road with their top surface flush with the pavement surface [7] while the barrier stones [30] are set perpendicularly to and tightly against the flat stones [20] with their top surface higher than the pavement.","The green belt [A] lies on the outer side of the barrier stones [20].","The surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole [40] in each of the said barrier stones so that there is an elevation drop.","When the flat stones and the barrier stones form single-piece units, the edge stones may be directly laid on one side or both sides of the road.","The top surface of the barrier stones [20] is higher than the pavement surface [7] and the top surface of the flat stones [10] is flush with the pavement surface.","The green belt [A] lies on the outer side of the barrier stones [20].","The surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole [40] in each of the said barrier stones so that there is an elevation drop.","Application Sample 3 See FIGS.","8-9, which show the border stones used in a third type of irrigation and drainage structure.","The border stones consist of flat stones [10], barrier stones [20] and supplemental barrier stones [20′].","Each of the flat stones [10] has a stopper at either end and a trough [30′] in the middle section near one side.","The trough [30′] is connected with the multiple drainage holes [40] in the barrier stones.","The top surface of the barrier stones [20] is higher than the supplemental barrier stones [20′], whose top surface is higher than the top surface of the flat stones [10].","The top surface of the flat stones is flush with the upper edge of the trough [30′] and higher than the drainage holes [40] in the barrier stones [20].","The barrier stones [20] and the supplemental barrier stones [20′] form an overflow relief trough [12] between themselves.","The flat stone [10], the barrier stone [20] and the supplemental barrier stone [20′] may form a “|_|”-shaped structure.","The said flat stone [10] and the said supplemental barrier stone [20′] may be a single-piece “L”-shaped structure, which may then join the barrier stone [20] to form a “|_|”-shaped structure.","The said flat stone [10], barrier stone [20], and supplemental barrier stone [20′] may be a single-piece unit or three separate pieces joined together.","See FIG.","9.In actual application, the said border stones are laid on one side of a green belt [A], which lies beyond the barrier stones [20].","The surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole [40] in each of the said barrier stones so that there is an elevation drop.","The rainwater flows into the trough [30′] in the flat stones and then through the drainage holes [40] in the barrier stones onto the green belt.","The supplemental barrier stones [20′] prevent the rainwater from flowing randomly.","Application Sample 4 See FIG.","10, which shows the border stones used in a fourth type of drainage and irrigation structure.","The said structure is essentially identical to that in Application Sample 3 except that the said supplemental barrier stones [20′] may have overflow outlets [23] and overflow pipelines [24] connected to those overflow outlets.","See FIG.","11.The border stones, consisting of the said flat stones [10], the said barrier stones [20] and the said supplemental barrier stones [20′], may be laid on one side of a sloping planted area, which lies inside the barrier stones [20].","The surface elevation [e] of the planted area is lower than the bottom edge of the drainage hole [40] in each of the said barrier stones so that there is an elevation drop.","The water in the planted area flows through the drainage holes [40] in the barrier stones into the overflow outlets [23] in the supplemental barrier stones [20′], and then through the connected overflow pipelines [24] onto the lower tiers of fields.","Application Sample 5 See FIG.","12, which shows the boundary stones used in a fifth type of drainage and irrigation structure.","The boundary stones [50] are U-shaped tubular structures.","The tubular wall on one side has multiple drainage holes [51], which are connected with the water trough [52] in the stones.","The tubular stones have internal stoppers [53] on both sides, but there are no stoppers between adjacent boundary stones.","The water trough goes all the way through all the joined boundary stones.","The drainage holes [51] on the tubular wall are positioned higher than the top of the stoppers [53].","The stopper top is a level surface and functions as the drainage initiation point [a].","See FIG.","11.In actual application, the U-shaped tubular boundary stones may be laid on one side of a planted area with the drainage holes [51] on their walls opening towards the planted area.","Excessive rainwater or water from other sources may flow through the drainage holes [51] into the trough [52] in the tubular boundary stones, which function like a dike to hold water around the tiered fields on the hill slopes.","The boundary stones [50] may also be used together with the border stones discussed in previous application samples.","In addition to the applications discussed so far, this Invention may serve the following purposes: Application Sample 6 See FIGS.","13, 3 and 4.The said curb barrier stones [2] may be set perpendicularly along one side of the road foundation [19] with their rainwater ports [6] below the top surface of the foundation [19].","The top surface of the curb barrier stones [2] is higher than the pavement surface [7].","Another row of curb barrier stones [2] and flat curb stones [1] is laid on the other side of the road.","Outside the curb barrier stones [2] are planted areas [A].","The rainwater ports [6] in the curb barrier stones [2] on both sides of the road are connected to underground drainage pipelines [8], which in turn lead into municipal sewage pipes [14].","Application Sample 7 See FIG.","14.Depending on the conditions of the planted areas [A] and the types of terrain, the said curb stones, edge stones, border stones and boundary stones used in the drainage and irrigation structures of this Invention may be set at one side of the planted areas [A], on one side or both sides of the roads, or at appropriate locations on barren hills.","They may be connected through individual pipes to main drainage pipes [15], which in turn may be connected to drainage ditches [16].","This approach makes full use of the water resources, improves drainage efficiency, eliminates the need to have sloping grades in the road pavement, enhances the utilization of water resources, creates better environmental conditions for plant growth, and prevents soil loss.","This Invention has the following benefits: 1.Environmental Protection: Separate channels are designed for dirty water to flow into green belts and clear water to flow into underground pipelines.","The dirty water carries a lot of organic matter, which is nutritious to the soil and helpful to plant growth.","With only clear water flowing into underground pipelines, the amount of work required in removing sewage slurry and reducing waterway contamination is greatly reduced.","2.Improvement of Drainage Efficiency: The possibility of pavement flooding is eliminated.","There is no need to build longitudinal grades into the pavement.","Drainage troughs may be used to replace pavement grades, thus reducing the cost.","3.Improvement of Road Cleaning Efficiency: Washing the pavement is much faster and produces a cleaner result than sweeping it.","In addition, there is much less dust contamination.","The road cleaning water flows into green belts.","Thus, two purposes are served simultaneously: The pavement gets cleaned and the green belts get the water they need.","4.Enhanced Utilization of Natural Resources: In the event of a rainfall, as long as there is water on the pavement, the green belts are likely to get the water.","Thus, the rainwater is effectively utilized.","Any excessive amount of water may be stored for future use.","5.Prevention of Soil Loss: As the pavement is higher than the planted areas, the possibility of any debris being washed onto the pavement is eliminated, thus reducing the risk of consecutive contamination.","An appropriate water accumulation level may be set in tiered fields on hill slopes so that any excessive water may be drained through pipes or ditches, thus preventing any soil loss or mud streams.","The application samples presented in the foregoing paragraphs have illustrated the benefits of this Invention.","However, those samples are not intended to limit the applications of this Invention.","Instead, anyone familiar with the technology embodied in this Invention is encouraged to come up with new designs and modifications that conform to the principles of this Invention and are within the technical area of this Invention.","The inventor of this Invention requests that any such designs be included in the claims of this Invention."]],"string":"[\n [\n \"Drainage and irrigation approach and structure as well as its implementation\",\n \"This Invention relates to a drainage and irrigation approach, which primarily includes the initial stage of green belt irrigation and the later stage of rainwater drainage.\",\n \"The drainage and irrigation structures employed in this approach consist of curb stones, edge stones, border stones, and boundary stones, which may have combinations of drainage troughs [3], rainwater ports [6] and drainage holes [4], or drainage troughs [3] and rainwater ports [6], or drainage troughs [3] and drainage holes [4].\",\n \"Depending on the conditions of planted areas, the drainage and irrigation structures discussed above may be deployed along roads, landscaping on top of architectural structures, planted areas, seeded strips or barren hill slopes.\"\n ],\n [\n \"1.A drainage and irrigation approach suitable for both rainwater and road cleaning water; the said approach consists of two stages: the initial stage of green belt irrigation and the later stage of rainwater drainage; firstly, rainwater at the beginning of a rainfall or in the event of road cleaning with debris flows through drainage holes in the drainage and irrigation structure into the planted areas, thereby providing the nutrients and water necessary for plant growth; secondly, if the rain continues falling and the rainwater rises to a certain level, clear water flows through the rainwater ports in the drainage and irrigation structure into the underground pipes and corresponding connected pipelines; the clear rainwater is then stored in underground reservoirs for storage or flows into rivers and lakes; from there it evaporates into the ecological cycle of nature.\",\n \"2.The drainage and irrigation approach as described in claim 1, wherein, for the initial green belt irrigation stage, flat curb stones are laid horizontally along one side or both sides of the road, their top surface flush with the pavement surface; the curb barrier stones are laid perpendicularly to and tightly against the flat curb stones along the outer edge of the road, their top surface higher than the road pavement; on either side of the road, there is a green belt [A] on the outer side of the curb barrier stones; the surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole in each of the said curb barrier stones so that there is an elevation drop; at the beginning of a rainfall, the rainwater flows along a drainage channel consisting of a trough in each flat curb stone and a drainage hole in each curb barrier stone.\",\n \"It then flows through the exit opening of the drainage hole onto the green belt [A].\",\n \"3.The drainage and irrigation approach as described in claim 1, wherein, for the later drainage stage, rainwater ports are installed at a certain interval in the row of curb barrier stones; the rainwater ports are connected to the underground drainage pipeline; the highest position of the said drainage hole in each curb barrier stone should be lower than the bottom edge [b] of the said rainwater port; when the rainwater accumulates in the green belt [A] and the water level rises to a point [a] above the rainwater port, it flows through the outlet into the underground pipeline; the drainage holes in curb barrier stones can also effectively prevent floating debris in the green belt from entering the rainwater ports so that the water flowing into the outlets is virtually filtered.\",\n \"4.The drainage and irrigation approach as described in claim 1, wherein the following engineering requirements must be met: the drainage initiation point of the pavement is at Elevation b, the drainage initiation point of the rainwater port is at Elevation a, the drainage or irrigation initiation point of the curb is at Elevation C, the blocking point of the floating debris in the green belt is at Elevation d, and the water accumulation initiation point in the green belt and the seeded strips is at Elevation e. 5.The drainage and irrigation approach as described in claim 4, wherein the elevation between b and C is 6 cm; the elevation between b and d is about 12 cm; and the elevation between d and e is about 5 cm.\",\n \"6.The curb stones used in a type of irrigation and drainage structure, which curb stones consist of flat curb stones and curb barrier stones having the following features: each of the said flat curb stones has a longitudinal drainage trough in its top surface and each of the said curb barrier stones has a half-circular cut-out at either end so that a drainage hole is formed when two curb barrier stones are laid end-to-end; the said longitudinal trough on the flat curb stones and the said drainage holes on the curb barrier stones are interconnected to form an unobstructed channel for water flows.\",\n \"7.The curb stones as described in claim 6, wherein the said curb barrier stones have rainwater ports installed at a certain interval and the rainwater ports are connected to underground pipelines; the highest point of the drainage hole on each curb barrier stone is lower than the bottom edge of the said rainwater port.\",\n \"8.The curb stones as described in claim 6, wherein the said longitudinal trough stretches on the top surface of the said flat curb stone and along the upper edge close to the said curb barrier stone; it is a trough cut along the said upper edge of the flat curb stone.\",\n \"9.The curb stones as described in claim 6, wherein the said drainage hole on the curb barrier stone goes through either end of the curb barrier stone; its opening at the flat curb stone side is the water entry and on the outer side of the curb barrier stone is the water exit; the bottom edge of the entry opening is flush with the bottom of the longitudinal trough on the flat curb stone; the exit opening is somewhat lower than the entry opening to produce a horizontal elevation drop; the entry opening and the exit opening may be set level.\",\n \"10.The curb stones as described in claim 8, wherein the said longitudinal trough is a half-circular trough cut along the edge of the flat curb stone close to the said curb barrier stone; additional cut-outs may or may not be made at proper positions along the trough in correspondence to the drainage holes on the curb barrier stones.\",\n \"11.The curb stones as described in claim 6, wherein the half-circular drainage hole cut-out in the curb barrier stone may be a horizontal or a slanting trough; in other words, its entry and its exit may be at the same elevation or may have a proper elevation drop.\",\n \"12.The edge stones used in a second type of irrigation and drainage structure, which edge stones consist of flat stones and barrier stones having the following features: Each of the said flat stones has a longitudinal trough on the side of its top surface close to the barrier stones, and the longitudinal trough connects with a drainage hole at either end of each barrier stone and several evenly spaced drainage holes in the middle section of the barrier stone to form an unobstructed channel for water flows.\",\n \"13.The edge stones as described in claim 12, wherein the bottom edge of the multiple holes in the said barrier stone is flush with the bottom of the longitudinal trough in the flat stone; the drainage hole at either end of the barrier stone is flush with and connects to the bottom of the longitudinal trough; the hole is a half-circular trough at the end of each barrier stone; when two barrier stones are set end-to-end, a circular drainage hole is formed.\",\n \"14.The edge stones as described in claim 12, wherein the said flat stone and the said barrier stone may form an “L”-shaped or “--|”-shaped structure.\",\n \"15.The edge stones as described in claim 14, wherein the said flat stone and the said barrier stone may be a single-piece unit or two separate pieces joined together.\",\n \"16.The edge stones as described in claim 15, wherein, as separate pieces to be joined together, the said flat stones are laid horizontally on one side or both sides of the road with their top surface flush with the pavement surface while the barrier stones are set perpendicularly to and tightly against the flat stones with their top surface higher than the pavement; the green belt lies on the outer side of the barrier stones; the surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole in each of the said barrier stones so that there is an elevation drop.\",\n \"17.The edge stones as described in claim 15, wherein, when the flat stones and the barrier stones form single-piece units, the edge stones may be directly laid on one side or both sides of the road; the top surface of the barrier stones is higher than the pavement surface and the top surface of the flat stones is flush with the pavement surface; the green belt lies on the outer side of the barrier stones; the surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole in each of the said barrier stones so that there is an elevation drop.\",\n \"18.The border stones used in a third type of irrigation and drainage structure, which border stones consist of flat stones and barrier stones having the following features: This structure also includes supplemental barrier stones; each of the flat stones has a stopper at either end and a trough in the middle section near one side; the trough is connected with the multiple drainage holes in the barrier stones; the top surface of the barrier stones is higher than the supplemental barrier stones, whose top surface is higher than the top surface of the flat stones; the top surface of the flat stones is flush with the upper edge of the trough and higher than the drainage holes in the barrier stones; the barrier stones and the supplemental barrier stones form an overflow relief trough between themselves.\",\n \"19.The border stones as described in claim 18, wherein the flat stone, the barrier stone and the supplemental barrier stone may form a “|_|”-shaped structure.\",\n \"20.The border stones as described in claim 18, wherein the said flat stone and the said supplemental barrier stone may be a single-piece “L”-shaped structure, which may then join the barrier stone to form a “|_|”-shaped structure.\",\n \"21.The border stones as described in claim 18, wherein the said flat stone, barrier stone, and supplemental barrier stone may be a single-piece unit or three separate pieces joined together.\",\n \"22.The border stones as described in claim 18, wherein the said supplemental barrier stones may have overflow outlets and overflow pipelines connected to those overflow outlets; they constitute the border stones used in a fourth type of drainage and irrigation structure.\",\n \"23.The border stones as described in claim 18, wherein, in actual application, the said border stones are laid on one side of a green belt, which lies beyond the barrier stones; the surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole in each of the said barrier stones so that there is an elevation drop; the rainwater flows into the trough in the flat stones and then through the drainage holes in the barrier stones onto the green belt; the supplemental barrier stones prevent the rainwater from flowing randomly.\",\n \"24.The border stones as described in claim 22, wherein, in actual application, the border stones, consisting of the said flat stones, the said barrier stones and the said supplemental barrier stones, may be laid on one side of a sloping planted area, which lies outside the barrier stones; the surface elevation [e] of the planted area is lower than the bottom edge of the drainage hole in each of the said barrier stones so that there is an elevation drop; the water in the planted area flows through the drainage holes in the barrier stones into the overflow outlets in the supplemental barrier stones, and then through the connected overflow pipelines into drainage pipelines.\",\n \"25.The boundary stones used in a fifth type of drainage and irrigation structure, wherein the boundary stones are U-shaped tubular structures; the tubular wall on one side has multiple drainage holes, which are connected with the water trough in the stones; the tubular stones have internal stoppers on both sides, or have no stopper.\",\n \"26.The boundary stones as described in claim 25, wherein the drainage holes on the tubular wall are positioned lower than the top of the stoppers, which is a level surface and functions as the drainage initiation point.\",\n \"27.The boundary stones as described in claim 25, wherein, in actual application, the U-shaped tubular boundary stones may be laid on one side of a planted area with the drainage holes on their walls opening towards the planted area; excessive rainwater in the planted area may flow through the drainage holes into the trough in the tubular boundary stones, which function like a dike to hold water around the tiered fields on the hill slopes; the boundary stones may also be used together with the border stones discussed earlier on barren hill slopes.\",\n \"28.The curb stones as described in claim 6, wherein, in actual application, the said curb barrier stones may be set perpendicularly along one side of the road foundation with their rainwater ports below the top surface of the foundation; the top surface of the curb barrier stones is higher than the pavement surface; another row of curb barrier stones and flat curb stones is laid on the other side of the road; outside the curb barrier stones are planted areas; the rainwater ports in the curb barrier stones on both sides of the road are connected to underground drainage pipelines, which in turn lead into municipal sewage pipelines.\",\n \"29.The drainage and irrigation structures as described in one of claims 6, 12, 18, and 25 wherein, depending on the conditions of the planted areas and the types of terrain, the said curb stones, edge stones, border stones and boundary stones may be set at one side of the planted areas, on one side or both sides of the roads, or at appropriate locations on barren hills; they may be connected to underground pipelines which lead to main drainage pipes, which in turn may be connected to drainage ditches.\"\n ],\n [\n \"<SOH> BACKGROUND TECHNOLOGY <EOH>Roads, drainage pipeline networks, and landscaping are important urban infrastructures, which have a direct impact on the quality of the urban environment.\",\n \"The conventional road curb generally consists of curb barrier stones and flat curb stones.\",\n \"The flat curb stones are laid horizontally along one side or both sides of the road, with their upper surface flush with the road pavement.\",\n \"The curb barrier stones are laid perpendicularly to and tightly against the flat curb stones along the outer edge of the road, with their upper surface higher than the road pavement.\",\n \"To facilitate the drainage of rainwater, a curb barrier stone with a rainwater port is placed at a certain interval (generally 30-50 meters) along the road, or a special grated rainwater port is placed in its place.\",\n \"The said rainwater port is connected through a pipe to an underground drainage pipeline.\",\n \"To improve the rainwater drainage speed and efficiency, rainwater ports should be placed at the lowest positions on the road.\",\n \"Therefore, when necessary, longitudinal slopes are built on the road, that is, the pavement is laid with a specific sloping grade in the longitudinal direction of the road.\",\n \"Then, rainwater ports or special rainwater drainage devices are placed at the lowest positions on the pavement slopes.\",\n \"As is disclosed in a Japanese patent document (Publication No.\",\n \"6-248610) and shown in FIG.\",\n \"15 , a row of curb barrier stones are laid on either side of the seeded median strip of the road pavement.\",\n \"Each of the curb barrier stones has two evenly embedded pre-fabricated blocks, which are water pervious.\",\n \"The curb barrier stones are laid in such a manner that the bottoms of the pre-fabricated pervious blocks are flush with the surface of the pavement.\",\n \"In the event of a rainfall or road cleaning, the rainwater or the cleaning water is allowed to flow through the pores in the pre-fabricated pervious blocks into the soil in the seeded median strip (in the direction indicated by the arrow in FIG.\",\n \"15 ) for irrigation purposes.\",\n \"Sands and other debris carried by the water are stopped and stranded between the curb barrier stones and the pavement.\",\n \"Consequently, regular maintenance work such as removing the debris is necessitated.\",\n \"Such maintenance work requires a lot of labor and expenses.\",\n \"Also, as is disclosed in a German patent document (DE 295 08 680 U1) and shown in FIG.\",\n \"4 , a row of curb barrier stones are laid on either side of the sloping road pavement.\",\n \"A drainage outlet is formed between each pair of adjacent curb barrier stones.\",\n \"On the outer side of either row of the curb barrier stones are green barrier slopes or seeded areas.\",\n \"In the event of a rainfall or road cleaning, the rainwater or the cleaning water flows down the sloping pavement and through the drainage outlets in the curb barrier stones directly into the green barrier slopes or seeded areas for irrigation purposes.\",\n \"In a heavy rainfall, the vegetation may easily be damaged, the soil be washed away, and the water resources be wasted.\",\n \"Again, as is disclosed in a French patent document (N° 1.368.655) and shown in FIG.\",\n \"5 , a row of curb barrier stones with pre-installed slanting pipe joints is laid along either side of the road.\",\n \"The pipe joints are connected to an underground pipeline.\",\n \"The cross section of the curb barrier stones assumes an “L” shape.\",\n \"The curb barrier stones are laid in such a manner that their top surface is even with the pavement surface.\",\n \"The bottom part of the curb barrier stone tilts upward and slants inward.\",\n \"The lower part of the opening of the pre-installed pipe joint is flush with the inner edge of the tilting section of the bottom part of the curb barrier stone.\",\n \"In the event of a rainfall or road cleaning, the rainwater or cleaning water flows through the opening of the pipe joint directly into the underground sewage pipeline.\",\n \"Sludge and other debris carried by the water may contaminate and clog the pipes.\",\n \"Debris removal will require a lot of labor and expenses.\",\n \"In addition, the water resources are wasted.\",\n \"The conventional technology as evidenced in the examples presented in the previous paragraphs has created a lot of problems in road debris removing, which has been costly and labor intensive.\",\n \"Road maintenance often generates a lot of dust, thus continually causing pollution.\",\n \"In addition, rainfalls or irrigation of the green belts and seeded strips along the roads cause soil loss, underground pipe clogging and contaminating, and contamination of downstream water resources.\",\n \"In short, in the event of rainfalls, the existing curb structures cause debris to be washed from green belts and surfaces of structures to the road pavements and then to underground pipelines and rivers and lakes.\",\n \"We are confronted not only with the issue of soil and water loss, but also with the risk of on-going contamination.\"\n ],\n [\n \"TECHNICAL AREA This Invention relates to a drainage and irrigation approach and structure as well as its implementation.\",\n \"In particular, it relates to a drainage and irrigation approach and structure as well as its implementation in connection with roads, landscaping on top of architectural structures, green belts, planted borders, and barren hill slopes.\",\n \"BACKGROUND TECHNOLOGY Roads, drainage pipeline networks, and landscaping are important urban infrastructures, which have a direct impact on the quality of the urban environment.\",\n \"The conventional road curb generally consists of curb barrier stones and flat curb stones.\",\n \"The flat curb stones are laid horizontally along one side or both sides of the road, with their upper surface flush with the road pavement.\",\n \"The curb barrier stones are laid perpendicularly to and tightly against the flat curb stones along the outer edge of the road, with their upper surface higher than the road pavement.\",\n \"To facilitate the drainage of rainwater, a curb barrier stone with a rainwater port is placed at a certain interval (generally 30-50 meters) along the road, or a special grated rainwater port is placed in its place.\",\n \"The said rainwater port is connected through a pipe to an underground drainage pipeline.\",\n \"To improve the rainwater drainage speed and efficiency, rainwater ports should be placed at the lowest positions on the road.\",\n \"Therefore, when necessary, longitudinal slopes are built on the road, that is, the pavement is laid with a specific sloping grade in the longitudinal direction of the road.\",\n \"Then, rainwater ports or special rainwater drainage devices are placed at the lowest positions on the pavement slopes.\",\n \"As is disclosed in a Japanese patent document (Publication No.\",\n \"6-248610) and shown in FIG.\",\n \"15, a row of curb barrier stones are laid on either side of the seeded median strip of the road pavement.\",\n \"Each of the curb barrier stones has two evenly embedded pre-fabricated blocks, which are water pervious.\",\n \"The curb barrier stones are laid in such a manner that the bottoms of the pre-fabricated pervious blocks are flush with the surface of the pavement.\",\n \"In the event of a rainfall or road cleaning, the rainwater or the cleaning water is allowed to flow through the pores in the pre-fabricated pervious blocks into the soil in the seeded median strip (in the direction indicated by the arrow in FIG.\",\n \"15) for irrigation purposes.\",\n \"Sands and other debris carried by the water are stopped and stranded between the curb barrier stones and the pavement.\",\n \"Consequently, regular maintenance work such as removing the debris is necessitated.\",\n \"Such maintenance work requires a lot of labor and expenses.\",\n \"Also, as is disclosed in a German patent document (DE 295 08 680 U1) and shown in FIG.\",\n \"4, a row of curb barrier stones are laid on either side of the sloping road pavement.\",\n \"A drainage outlet is formed between each pair of adjacent curb barrier stones.\",\n \"On the outer side of either row of the curb barrier stones are green barrier slopes or seeded areas.\",\n \"In the event of a rainfall or road cleaning, the rainwater or the cleaning water flows down the sloping pavement and through the drainage outlets in the curb barrier stones directly into the green barrier slopes or seeded areas for irrigation purposes.\",\n \"In a heavy rainfall, the vegetation may easily be damaged, the soil be washed away, and the water resources be wasted.\",\n \"Again, as is disclosed in a French patent document (N° 1.368.655) and shown in FIG.\",\n \"5, a row of curb barrier stones with pre-installed slanting pipe joints is laid along either side of the road.\",\n \"The pipe joints are connected to an underground pipeline.\",\n \"The cross section of the curb barrier stones assumes an “L” shape.\",\n \"The curb barrier stones are laid in such a manner that their top surface is even with the pavement surface.\",\n \"The bottom part of the curb barrier stone tilts upward and slants inward.\",\n \"The lower part of the opening of the pre-installed pipe joint is flush with the inner edge of the tilting section of the bottom part of the curb barrier stone.\",\n \"In the event of a rainfall or road cleaning, the rainwater or cleaning water flows through the opening of the pipe joint directly into the underground sewage pipeline.\",\n \"Sludge and other debris carried by the water may contaminate and clog the pipes.\",\n \"Debris removal will require a lot of labor and expenses.\",\n \"In addition, the water resources are wasted.\",\n \"The conventional technology as evidenced in the examples presented in the previous paragraphs has created a lot of problems in road debris removing, which has been costly and labor intensive.\",\n \"Road maintenance often generates a lot of dust, thus continually causing pollution.\",\n \"In addition, rainfalls or irrigation of the green belts and seeded strips along the roads cause soil loss, underground pipe clogging and contaminating, and contamination of downstream water resources.\",\n \"In short, in the event of rainfalls, the existing curb structures cause debris to be washed from green belts and surfaces of structures to the road pavements and then to underground pipelines and rivers and lakes.\",\n \"We are confronted not only with the issue of soil and water loss, but also with the risk of on-going contamination.\",\n \"SCOPE OF THE INVENTION The purpose of this Invention is to provide a drainage and irrigation approach and structure, which, in the event of rainfalls and road cleaning, may function simultaneously in two ways: retaining dirt and debris in the green belts and seeded strips to avoid drainage pipeline blockage, downstream water body contamination, and soil loss on the one hand, and properly allocating water consumption and making better use of external water resources in the green belts and seeded strips to improve the environmental conditions on the roads and green belts as well as seeded strips, promote the water and soil conservation in the green belts and planting area, and cultivate the natural conditions for plant growth.\",\n \"Another purpose of this Invention is to provide a drainage and irrigation approach and structure, which may offer curb stones of various structural forms to suit the infrastructural needs of hard soil drainage, roadway drainage, as well as preservation of landscaping on top of architectural structures, green belts, seeded strips, planted plots and barren hills.\",\n \"The technical solution of this Invention involves a drainage and irrigation approach suitable for both rainwater and road cleaning water.\",\n \"The said approach consists of two stages: the initial stage of irrigating green belts and the later stage of rainwater drainage.\",\n \"In the initial irrigation stage rainwater, at the beginning of a rainfall or foul water in the event of road cleaning with debris flows through drainage holes in the drainage and irrigation structure into the planted areas, thereby providing the nutrients and water necessary for plant growth.\",\n \"If the rain continues falling and the rainwater rises to a certain level, clear water flows through rainwater ports in the drainage and irrigation structure into the underground pipes and corresponding connected pipelines.\",\n \"The clear rainwater is then stored in underground reservoirs for storage or flows into rivers and lakes.\",\n \"From there it evaporates into the ecological cycle of nature.\",\n \"The said drainage and irrigation approach has the following engineering requirements: The drainage initiation point of the pavement is at Elevation b, the drainage initiation point of the rainwater port is at Elevation a, the drainage or irrigation initiation point of the curb is at Elevation C, the blocking point of the floating debris in the green belt is at Elevation d, and the water accumulation initiation point in the green belt and the seeded strips is at Elevation e, where the said point b is higher than point C and point d, which in turn is higher than point e. This Invention relates to the curb stones used in a type of irrigation and drainage structure, which curb stones consist of flat curb stones and curb barrier stones having the following features: Each of the said flat curb stones has a longitudinal drainage trough in its top surface and each of the said curb barrier stones has a half-circular cut-out at either end so that a drainage hole is formed when two curb barrier stones are laid end-to-end.\",\n \"The said longitudinal trough on the flat curb stones and the said drainage holes on the curb barrier stones are interconnected to form an unobstructed channel for water flows.\",\n \"The said curb barrier stones have rainwater ports installed at a certain interval and the rainwater ports are connected to underground pipelines.\",\n \"The highest point of the drainage hole in each curb barrier stone is lower than the bottom edge of the said rainwater port.\",\n \"The half-circular cut-out in the curb barrier stone may be a horizontal or a slanting trough.\",\n \"In other words, its entry and its exit may be at the same elevation or may have a proper elevation drop.\",\n \"This Invention relates to the edge stones used in a second type of irrigation and drainage structure, which edge stones consist of flat stones and barrier stones having the following features: Each of the said flat stones has a longitudinal trough on the side of its top surface close to the barrier stones, and the longitudinal trough connects with a drainage hole at either end of each barrier stone and several evenly spaced drainage holes in the middle section of the barrier stone to form an unobstructed channel for water flows.\",\n \"The said flat stone and the said barrier stone may form an “L”-shaped or “--|”-shaped structure.\",\n \"The said flat stone and the said barrier stone may be a single-piece unit or two separate pieces joined together.\",\n \"As separate pieces to be joined together, the said flat stones are laid horizontally on one side or both sides of the road with their top surface flush with the pavement surface while the barrier stones are set perpendicularly to and tightly against the flat stones with their top surface higher than the pavement.\",\n \"The green belt [A] lies on the outer side of the barrier stones.\",\n \"The surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole in each of the said barrier stones so that there is an elevation drop.\",\n \"This Invention relates to the border stones used in a third type of irrigation and drainage structure, which border stones consist of flat stones and barrier stones having the following features: This structure also includes supplemental barrier stones.\",\n \"Each of the flat stones has a stopper at either end and a trough in the middle section near one side.\",\n \"The trough is connected with the multiple drainage holes in the barrier stones.\",\n \"The top surface of the barrier stones is higher than the supplemental barrier stones, whose top surface is higher than the top surface of the flat stones.\",\n \"The top surface of the flat stones is flush with the upper edge of the trough and higher than the drainage holes in the barrier stones.\",\n \"The barrier stones and the supplemental barrier stones form an overflow relief trough between themselves.\",\n \"The flat stone, the barrier stone and the supplemental barrier stone may form a “|_|”-shaped structure.\",\n \"The said flat stone and the said supplemental barrier stone may be a single-piece “L”-shaped structure, which may then join the barrier stone to form a “|_|”-shaped structure.\",\n \"The said flat stone, barrier stone, and supplemental barrier stone may be a single-piece unit or three separate pieces joined together.\",\n \"The said supplemental barrier stones may have overflow outlets and overflow pipelines connected to those overflow outlets.\",\n \"They constitute the border stones used in a fourth type of drainage and irrigation structure.\",\n \"This Invention also relates to the boundary stones used in a fifth type of drainage and irrigation structure, wherein the boundary stones are U-shaped tubular structures.\",\n \"The tubular wall on one side has multiple drainage holes, which are connected with the water trough in the stones.\",\n \"The tubular stones may or may not have internal stoppers on both sides.\",\n \"The said curb barrier stones may be set perpendicularly along one side of the road foundation with their rainwater ports below the top surface of the foundation.\",\n \"The top surface of the curb barrier stones is higher than the pavement surface.\",\n \"Another row of curb barrier stones and flat curb stones is laid on the other side of the road.\",\n \"Outside the curb barrier stones are planted areas.\",\n \"The rainwater ports in the curb barrier stones on both sides of the road are connected to underground drainage pipelines respectively, which in turn lead into municipal sewage pipes.\",\n \"Depending on the conditions of the areas and the types of terrain, the said curb stones, edge stones, border stones and boundary stones may be set at one side of the planted areas, on one side or both sides of the roads, or at appropriate locations on barren hills.\",\n \"They may be connected to underground pipelines which lead to main drainage pipes, which in turn may be connected to drainage ditches.\",\n \"DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is an operation flow chart of this Invention.\",\n \"FIG.\",\n \"2 is a structural diagram of the curb stones used in Application Sample 1 of this Invention.\",\n \"FIG.\",\n \"3 is a diagram of the operational principles in the initial green belt irrigation stage in Application Sample 1 of this Invention.\",\n \"FIG.\",\n \"4 is a diagram of the operational principles in the later drainage stage in Application Sample 1 of this Invention.\",\n \"FIG.\",\n \"5 is an engineering diagram of the curb drainage structure in Application Sample 1 of this Invention.\",\n \"FIG.\",\n \"6 is a structural diagram of the edge stones used in Application Sample 2 of this Invention.\",\n \"FIG.\",\n \"7 is an operational diagram of Application Sample 2 of this Invention.\",\n \"FIG.\",\n \"8 is a structural diagram of the border stones used in Application Sample 3 of this Invention.\",\n \"FIG.\",\n \"9 is an operational diagram of Application Sample 3 of this invention.\",\n \"FIG.\",\n \"10 is a structural diagram of the border stones used in Application Sample 4 of this Invention.\",\n \"FIG.\",\n \"11 is an operational diagram of Application Sample 4 of this invention.\",\n \"FIG.\",\n \"12 is a structural diagram of the boundary stones used in Application Sample 5 of this Invention.\",\n \"FIG.\",\n \"13 is an operational diagram of Application Sample 6 of this invention.\",\n \"FIG.\",\n \"14 is an operational diagram of Application Sample 7 of this invention.\",\n \"FIG.\",\n \"15 is a structural diagram of the conventional technology (part 1).\",\n \"FIG.\",\n \"16 is a structural diagram of the conventional technology (part 2).\",\n \"FIG.\",\n \"17 is a 3-D diagram of the objects in FIG.\",\n \"16.FIG.\",\n \"18 is a structural diagram of the conventional technology (part 3).\",\n \"FIG.\",\n \"19 is a top view of the objects in FIG.\",\n \"18.IMPLEMENTATION OF THE INVENTION The application samples presented in the following paragraphs are intended to illustrate in detail the implementation and benefits of this Invention so that readers may see the creativity embodied in this Invention.\",\n \"The samples should be studied together with the drawings.\",\n \"See FIG.\",\n \"1, which is an operation flow chart of this Invention.\",\n \"This Invention involves a drainage and irrigation approach suitable for both rainwater and road cleaning water.\",\n \"The said approach consists of two stages: the initial stage of irrigating green belts and seeded strips and the later stage of rainwater drainage.\",\n \"In the initial irrigation stage, rainwater at the beginning of a rainfall or foul water in the event of road cleaning with debris flow through drainage holes in the drainage and irrigation structure onto the planted areas, thereby providing the nutrients and water necessary for plant growth.\",\n \"If the rain continues falling and the rainwater rises to a certain level, it flows through the rainwater ports in the drainage and irrigation structure into the underground pipes and connected pipelines.\",\n \"The clear rainwater is then stored in underground reservoirs for storage or flows into rivers and lakes.\",\n \"From there it evaporates into the ecological cycle of nature.\",\n \"The concept of this Invention is as follows: At the beginning of a rainfall or in the event of road cleaning, the water with debris flows through drainage holes in the drainage and irrigation structure onto the planted areas, thereby providing the nutrients and water necessary for plant growth.\",\n \"If the rain continues falling and the rainwater rises to a certain level, it flows through the rainwater ports in the drainage and irrigation structure into the underground pipes and connected pipelines.\",\n \"The clear rainwater is then stored in underground reservoirs for storage or flows into rivers and lakes.\",\n \"From there it evaporates into the ecological cycle of nature.\",\n \"Application Sample 1 See FIG.\",\n \"2, which shows the curb stones used in a type of irrigation and drainage structure.\",\n \"The curb stones consist of flat curb stones [1] and curb barrier stones [2].\",\n \"Each of the said flat curb stones [1] has a longitudinal drainage trough [3] in its top surface and each of the said curb barrier stones [2] has a half-circular cut-out at either end so that a drainage hole [4] is formed when two curb barrier stones [2] are laid end-to-end.\",\n \"The said longitudinal trough [3] on the flat curb stones and the said drainage holes [4] on the curb barrier stones are interconnected to form an unobstructed channel for water flows.\",\n \"The said curb barrier stones [2] have rainwater ports [6] installed at a certain interval and connected to underground pipelines [8].\",\n \"The rainwater port [6], assuming a “” shape, can prevent the dirty water in the green belts [A] from entering into the rainwater ports [6].\",\n \"The highest point of the drainage hole [4] on the curb barrier stone is lower than the bottom edge of the said rainwater port [6].\",\n \"The said longitudinal trough stretches on the top surface of the said flat curb stone [1] and along the upper edge close to the said curb barrier stone [2].\",\n \"It is a trough cut along the said upper edge of the flat curb stone [1].\",\n \"The said drainage hole [4] on the curb barrier stone [2] goes through either end of the curb barrier stone.\",\n \"Its opening at the flat curb stone [2] side is the water entry and on the outer side of the curb barrier stone [2] is the water exit.\",\n \"The bottom edge of the entry opening is flush with the bottom of the longitudinal trough [3] on the flat curb stone.\",\n \"The exit opening is somewhat lower than the entry opening to produce a horizontal elevation drop.\",\n \"However, the entry opening and the exit opening may be set level.\",\n \"The said longitudinal trough [3] is a half-circular trough cut along the edge of the flat curb stone [1] close to the said curb barrier stone [2].\",\n \"Additional cut-outs may or may not be made at proper positions along the trough in correspondence to the drainage holes [4] on the curb barrier stones (cut-outs not shown in the drawing).\",\n \"The half-circular drainage hole cut-out in the curb barrier stone may be a horizontal or a slanting trough.\",\n \"In other words, its entry and its exit may be at the same elevation or may have a proper elevation drop.\",\n \"In view of the structural features of the said curb stones, the relevant drainage and irrigation approach is as follows (see FIGS.\",\n \"3-4): 1.For the initial green belt irrigation stage, flat curb stones [1] are laid horizontally along one side or both sides of the road, their top surface flush with the pavement surface [7].\",\n \"The curb barrier stones [2] are laid perpendicularly to and tightly against the flat curb stones [1] along the outer edge of the road, their bottom part set in a holding frame [21] and their top surface higher than the road pavement.\",\n \"On either side of the road, there is a green belt [A] on the outer side of the curb barrier stones [2].\",\n \"The surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole [4] in each of the said curb barrier stones so that there is an elevation drop.\",\n \"At the beginning of a rainfall, the rainwater [9] flows along a drainage channel consisting of a trough [3] in each flat curb stone and a drainage hole [4] in each curb barrier stone.\",\n \"It then flows through the exit opening of the drainage hole [4] onto the green belt [A].\",\n \"2.For the later drainage stage, a rainwater port [6] is installed at a certain interval in the row of curb barrier stones.\",\n \"The rainwater ports [6] are connected to the underground drainage pipeline [8].\",\n \"The highest position of the said drainage hole [4] in each curb barrier stone should be lower than the bottom edge [b] of the said rainwater port [6].\",\n \"When the rainwater accumulates in the green belt [A] and the water level rises to a point [a] above the rainwater port, it flows through the outlet into the underground pipeline [8].\",\n \"The rainwater port [6] in each curb barrier stone can also effectively prevent floating debris in the green belt from entering the rainwater port [6] so that the water flowing into the outlet is virtually filtered, thus avoiding clogging of underground pipelines [8] and rivers as well as reducing the maintenance work and labor required to remove the blockage.\",\n \"The said drainage and irrigation approach has the following engineering requirements (see FIG.\",\n \"5): The drainage initiation point of the pavement is at Elevation b, the drainage initiation point of the rainwater port is at Elevation a, the drainage or irrigation initiation point of the curb is at Elevation C, the blocking point of the floating debris in the green belt is at Elevation d, and the water accumulation initiation point in the green belt and the seeded strips is at Elevation e, where the elevations of point a and point b shall be calculated on the basis of the pavement width and grade while the elevations of other points should also be properly set depending on the environmental conditions.\",\n \"The elevation between b and C is 6 cm; the elevation between b and d is about 12 cm; and the elevation between d and e is about 5 cm.\",\n \"Application Sample 2 See FIGS.\",\n \"6-7, which show the edge stones used in a second type of irrigation and drainage structure.\",\n \"The edge stones consist of flat stones [10] and barrier stones [20].\",\n \"Each of the said flat stones [10] has a longitudinal trough [30] on the side of its top surface close to the barrier stones [20], and the longitudinal trough connects with a drainage hole [40] at either end of the barrier stone and several evenly spaced drainage holes [40] in the middle section of the barrier stone to form an unobstructed channel for water flows.\",\n \"The bottom edge of the multiple holes [40] in the said barrier stone [20] is flush with the bottom of the longitudinal trough [30] in the flat stone.\",\n \"The drainage hole [40] at either end of the barrier stone [20] is flush with and connects to the bottom of the longitudinal trough [30].\",\n \"The hole is a half-circular trough at the end of each barrier stone.\",\n \"When two barrier stones are set end-to-end, a circular drainage hole [40] is formed.\",\n \"The said flat stone [20] and the said barrier stone [30] may form an “L”-shaped or “--|”-shaped structure.\",\n \"The said flat stone [20] and the said barrier stone [30] may be a single-piece unit or two separate pieces joined together.\",\n \"See FIG.\",\n \"7.When the flat stone 20 and the barrier stone 30 are as separate pieces to be joined together, the said flat stones [20] are laid horizontally on one side or both sides of the road with their top surface flush with the pavement surface [7] while the barrier stones [30] are set perpendicularly to and tightly against the flat stones [20] with their top surface higher than the pavement.\",\n \"The green belt [A] lies on the outer side of the barrier stones [20].\",\n \"The surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole [40] in each of the said barrier stones so that there is an elevation drop.\",\n \"When the flat stones and the barrier stones form single-piece units, the edge stones may be directly laid on one side or both sides of the road.\",\n \"The top surface of the barrier stones [20] is higher than the pavement surface [7] and the top surface of the flat stones [10] is flush with the pavement surface.\",\n \"The green belt [A] lies on the outer side of the barrier stones [20].\",\n \"The surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole [40] in each of the said barrier stones so that there is an elevation drop.\",\n \"Application Sample 3 See FIGS.\",\n \"8-9, which show the border stones used in a third type of irrigation and drainage structure.\",\n \"The border stones consist of flat stones [10], barrier stones [20] and supplemental barrier stones [20′].\",\n \"Each of the flat stones [10] has a stopper at either end and a trough [30′] in the middle section near one side.\",\n \"The trough [30′] is connected with the multiple drainage holes [40] in the barrier stones.\",\n \"The top surface of the barrier stones [20] is higher than the supplemental barrier stones [20′], whose top surface is higher than the top surface of the flat stones [10].\",\n \"The top surface of the flat stones is flush with the upper edge of the trough [30′] and higher than the drainage holes [40] in the barrier stones [20].\",\n \"The barrier stones [20] and the supplemental barrier stones [20′] form an overflow relief trough [12] between themselves.\",\n \"The flat stone [10], the barrier stone [20] and the supplemental barrier stone [20′] may form a “|_|”-shaped structure.\",\n \"The said flat stone [10] and the said supplemental barrier stone [20′] may be a single-piece “L”-shaped structure, which may then join the barrier stone [20] to form a “|_|”-shaped structure.\",\n \"The said flat stone [10], barrier stone [20], and supplemental barrier stone [20′] may be a single-piece unit or three separate pieces joined together.\",\n \"See FIG.\",\n \"9.In actual application, the said border stones are laid on one side of a green belt [A], which lies beyond the barrier stones [20].\",\n \"The surface elevation [e] of the green belt is lower than the bottom edge of the exit opening of the drainage hole [40] in each of the said barrier stones so that there is an elevation drop.\",\n \"The rainwater flows into the trough [30′] in the flat stones and then through the drainage holes [40] in the barrier stones onto the green belt.\",\n \"The supplemental barrier stones [20′] prevent the rainwater from flowing randomly.\",\n \"Application Sample 4 See FIG.\",\n \"10, which shows the border stones used in a fourth type of drainage and irrigation structure.\",\n \"The said structure is essentially identical to that in Application Sample 3 except that the said supplemental barrier stones [20′] may have overflow outlets [23] and overflow pipelines [24] connected to those overflow outlets.\",\n \"See FIG.\",\n \"11.The border stones, consisting of the said flat stones [10], the said barrier stones [20] and the said supplemental barrier stones [20′], may be laid on one side of a sloping planted area, which lies inside the barrier stones [20].\",\n \"The surface elevation [e] of the planted area is lower than the bottom edge of the drainage hole [40] in each of the said barrier stones so that there is an elevation drop.\",\n \"The water in the planted area flows through the drainage holes [40] in the barrier stones into the overflow outlets [23] in the supplemental barrier stones [20′], and then through the connected overflow pipelines [24] onto the lower tiers of fields.\",\n \"Application Sample 5 See FIG.\",\n \"12, which shows the boundary stones used in a fifth type of drainage and irrigation structure.\",\n \"The boundary stones [50] are U-shaped tubular structures.\",\n \"The tubular wall on one side has multiple drainage holes [51], which are connected with the water trough [52] in the stones.\",\n \"The tubular stones have internal stoppers [53] on both sides, but there are no stoppers between adjacent boundary stones.\",\n \"The water trough goes all the way through all the joined boundary stones.\",\n \"The drainage holes [51] on the tubular wall are positioned higher than the top of the stoppers [53].\",\n \"The stopper top is a level surface and functions as the drainage initiation point [a].\",\n \"See FIG.\",\n \"11.In actual application, the U-shaped tubular boundary stones may be laid on one side of a planted area with the drainage holes [51] on their walls opening towards the planted area.\",\n \"Excessive rainwater or water from other sources may flow through the drainage holes [51] into the trough [52] in the tubular boundary stones, which function like a dike to hold water around the tiered fields on the hill slopes.\",\n \"The boundary stones [50] may also be used together with the border stones discussed in previous application samples.\",\n \"In addition to the applications discussed so far, this Invention may serve the following purposes: Application Sample 6 See FIGS.\",\n \"13, 3 and 4.The said curb barrier stones [2] may be set perpendicularly along one side of the road foundation [19] with their rainwater ports [6] below the top surface of the foundation [19].\",\n \"The top surface of the curb barrier stones [2] is higher than the pavement surface [7].\",\n \"Another row of curb barrier stones [2] and flat curb stones [1] is laid on the other side of the road.\",\n \"Outside the curb barrier stones [2] are planted areas [A].\",\n \"The rainwater ports [6] in the curb barrier stones [2] on both sides of the road are connected to underground drainage pipelines [8], which in turn lead into municipal sewage pipes [14].\",\n \"Application Sample 7 See FIG.\",\n \"14.Depending on the conditions of the planted areas [A] and the types of terrain, the said curb stones, edge stones, border stones and boundary stones used in the drainage and irrigation structures of this Invention may be set at one side of the planted areas [A], on one side or both sides of the roads, or at appropriate locations on barren hills.\",\n \"They may be connected through individual pipes to main drainage pipes [15], which in turn may be connected to drainage ditches [16].\",\n \"This approach makes full use of the water resources, improves drainage efficiency, eliminates the need to have sloping grades in the road pavement, enhances the utilization of water resources, creates better environmental conditions for plant growth, and prevents soil loss.\",\n \"This Invention has the following benefits: 1.Environmental Protection: Separate channels are designed for dirty water to flow into green belts and clear water to flow into underground pipelines.\",\n \"The dirty water carries a lot of organic matter, which is nutritious to the soil and helpful to plant growth.\",\n \"With only clear water flowing into underground pipelines, the amount of work required in removing sewage slurry and reducing waterway contamination is greatly reduced.\",\n \"2.Improvement of Drainage Efficiency: The possibility of pavement flooding is eliminated.\",\n \"There is no need to build longitudinal grades into the pavement.\",\n \"Drainage troughs may be used to replace pavement grades, thus reducing the cost.\",\n \"3.Improvement of Road Cleaning Efficiency: Washing the pavement is much faster and produces a cleaner result than sweeping it.\",\n \"In addition, there is much less dust contamination.\",\n \"The road cleaning water flows into green belts.\",\n \"Thus, two purposes are served simultaneously: The pavement gets cleaned and the green belts get the water they need.\",\n \"4.Enhanced Utilization of Natural Resources: In the event of a rainfall, as long as there is water on the pavement, the green belts are likely to get the water.\",\n \"Thus, the rainwater is effectively utilized.\",\n \"Any excessive amount of water may be stored for future use.\",\n \"5.Prevention of Soil Loss: As the pavement is higher than the planted areas, the possibility of any debris being washed onto the pavement is eliminated, thus reducing the risk of consecutive contamination.\",\n \"An appropriate water accumulation level may be set in tiered fields on hill slopes so that any excessive water may be drained through pipes or ditches, thus preventing any soil loss or mud streams.\",\n \"The application samples presented in the foregoing paragraphs have illustrated the benefits of this Invention.\",\n \"However, those samples are not intended to limit the applications of this Invention.\",\n \"Instead, anyone familiar with the technology embodied in this Invention is encouraged to come up with new designs and modifications that conform to the principles of this Invention and are within the technical area of this Invention.\",\n \"The inventor of this Invention requests that any such designs be included in the claims of this Invention.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471198"}}},{"rowIdx":582,"cells":{"text":{"kind":"list like","value":[["Apparatus and method for displaying virtual endoscopy display","An apparatus and method for displaying a three-dimensional virtual endoscopic image are provided.","In the method, information on a virtual endoscopic image is input in the form of volume data expressed as a function of three-dimensional position.","A two-dimensional reference image, a three-dimensional reference image, and a virtual endoscopic image are detected from the volume data.","The detected images are displayed on one screen.","Virtual cameras are respectively displayed on areas, in which the two and three-dimensional reference images are respectively displayed.","Here, a camera display sphere and a camera display circle are defined on the basis of a current position of each virtual camera.","When information regarding to one image, among the two and three-dimensional reference images and the virtual endoscopic image on one screen, is changed by a user's operation, information regarding to the other images is changed based on the information changed by the user's operation."],["1.An apparatus for displaying a three-dimensional virtual endoscopic image, the apparatus comprising: a volume data input unit, which inputs information on a virtual endoscopic image in the form of volume data expressed as a function of three-dimensional position; a two-dimensional reference image display control unit, which derives a two-dimensional reference image from the volume data and displays the two-dimensional reference image; a three-dimensional reference image display control unit, which derives a three-dimensional reference image from the volume data using parallel volume rendering and displays the three-dimensional reference image; a virtual endoscopic image display control unit, which applies position and direction information of a virtual camera used for collecting virtual endoscopic image information to a predetermined perspective ray-casting algorithm, thereby generating image information, and displays a virtual endoscopic image based on the generated image information; a user interface unit, which receives a user's operating signal; and a controller, which when the operating signal for changing image information displayed by one among the two-dimensional reference image display control unit, the three-dimensional reference image display control unit, and the virtual endoscopic image display control unit is input through the user interface unit, controls the two- and three-dimensional reference image display control units and the virtual endoscopic image display control unit to be associated with one another based on the operating signal.","2.The apparatus of claim 1, wherein the two-dimensional reference image display control unit comprises: a control signal input section, which receives a control signal for controlling a two-dimensional reference image among the operating signal input through the user interface unit; an image information receiver, which receives changed three-dimensional reference image information from the three-dimensional reference image display control unit and changed virtual endoscopic image information from the virtual endoscopic image display control unit; a painting section, which receives painting information regarding to a region of interest in the two-dimensional reference image from the control signal input section and changes two-dimensional reference image information based on the painting information; a display area control section, which changes the two-dimensional reference image information based on position change information regarding to a region of interest in the two-dimensional reference image, which is received through the control signal input section, or based on position change information regarding to a region of interest in the three-dimensional reference image or the virtual endoscopic image, which is received through the image information receiver; a camera control section, which changes virtual camera display information with respect to the two-dimensional reference image based on virtual camera display information regarding to the two-dimensional reference image, which is received through the control signal input section, or based on virtual camera display information regarding to the three-dimensional reference image and the virtual endoscopic image, which is received through the image information receiver; a display format determining section, which determines one among an axial image, a coronal image, a sagittal image, and a path MPR image as the format of the two-dimensional reference image, based on display information regarding to the two-dimensional reference image, which is received through the control signal input section; a two-dimensional image generator, which receives the changed three-dimensional reference image information and the changed virtual endoscopic image information from the image information receiver and converts the received three-dimensional reference image information and the received virtual endoscopic image information to two-dimensional image information; an image information display section, which detects a changed two-dimensional reference image based on the two-dimensional reference image information received from the painting section, the display area control section, the camera control section, the display format determining section, and the two-dimensional image generator, and displays the changed two-dimensional reference image; and an image information support section, which when the two-dimensional reference image information is changed in the painting section, the display area control section, and the camera control section in response to the control signals received from the control signal input section, transmits the changed two-dimensional reference image information to the three-dimensional reference image display control unit and the virtual endoscopic image display control unit so that the changed two-dimensional reference image information is applied to the three-dimensional reference image and the virtual endoscopic image.","3.The apparatus of claim 1, wherein the three-dimensional reference image display control unit comprises: a control signal input section, which receives a control signal for controlling a three-dimensional reference image among the operating signal input through the user interface unit; an image information receiver, which receives changed two-dimensional reference image information from the two-dimensional reference image display control unit and changed virtual endoscopic image information from the virtual endoscopic image display control unit; a painting section, which receives painting information regarding to a region of interest in the three-dimensional reference image from the control signal input section and changes three-dimensional reference image information based on the painting information; a display area control section, which changes the three-dimensional reference image information based on position change information regarding to a region of interest in the three-dimensional reference image, which is received through the control signal input section, or based on position change information regarding to a region of interest in the two-dimensional reference image or the virtual endoscopic image, which is received through the image information receiver; a camera control section, which changes virtual camera display information with respect to the three-dimensional reference image based on virtual camera display information regarding to the three-dimensional reference image, which is received through the control signal input section, or based on virtual camera display information regarding to the two-dimensional reference image and the virtual endoscopic image, which is received through the image information receiver; a three-dimensional volume rendering section, which receives the changed two-dimensional reference image information and the changed virtual endoscopic image information from the image information receiver and performs three-dimensional volume rendering based on the received two-dimensional reference image and virtual endoscopic image information; an image information display section, which detects a changed three-dimensional reference image based on the three-dimensional reference image information received from the painting section, the display area control section, the camera control section, and the three-dimensional volume rendering section, and displays the changed three-dimensional reference image; and an image information support section, which when the three-dimensional reference image information is changed in the painting section, the display area control section, and the camera control section in response to the control signals received from the control signal input section, transmits the changed three-dimensional reference image information to the two-dimensional reference image display control unit and the virtual endoscopic image display control unit so that the changed three-dimensional reference image information is applied to the two-dimensional reference image and the virtual endoscopic image.","4.The apparatus of claim 1, wherein the virtual endoscopic image display control unit comprises: a control signal input section, which receives a control signal for controlling a virtual endoscopic image among the operating signal input through the user interface unit; an image information receiver, which receives changed two-dimensional reference image information from the two-dimensional reference image display control unit and changed three-dimensional reference image information from the three-dimensional reference image display control unit; a painting section, which receives painting information regarding to a region of interest in the virtual endoscopic image from the control signal input section and changes virtual endoscopic image information based on the painting information; a perspective ray-casting section, which applies the processing result received from the painting section and the changed two- and three-dimensional reference image information received from the image information receiver to a predetermined perspective ray-casting algorithm so as to adjust the opacity of the region of interest, and then changes the virtual endoscopic image information based on the result of the adjustment; a display area control section, which changes the virtual endoscopic image information based on position change information regarding to the region of interest in the two- or three-dimensional reference image, which is received through the image information receiver; a camera control section, which changes the virtual endoscopic image information based on position and direction information of a virtual camera, which is received through the control signal input section; a focal zone generator, which designates a region of a user's interest on the virtual endoscopic image based on a control signal transmitted through the control signal input section; an image information display section, which detects a changed virtual endoscopic image based on the virtual endoscopic image information received from the perspective ray-casting section, the display area control section, the camera control section, and the focal zone generator, and displays the changed virtual endoscopic image; and an image information support section, which when the virtual endoscopic image information is changed in the painting section, the perspective ray-casting section, the camera control section, and the focal zone generator in response to the control signals received from the control signal input section, transmits the changed virtual endoscopic image information to the two-dimensional reference image display control unit and the three-dimensional reference image display control unit so that the changed virtual endoscopic image information is applied to the two-dimensional reference image and the three-dimensional reference image.","5.A method of displaying a three-dimensional virtual endoscopic image, the method comprising: inputting information on a virtual endoscopic image in the form of volume data expressed as a function of three-dimensional position; detecting a two-dimensional reference image, a three-dimensional reference image, and a virtual endoscopic image from the volume data; displaying the two-dimensional reference image, the three-dimensional reference image, and the virtual endoscopic image on one screen; displaying virtual cameras on areas, respectively, in which the two-dimensional reference image and the three-dimensional reference image are respectively displayed, wherein a camera display sphere and a camera display circle are defined on the basis of a current position of each virtual camera; and when information regarding to one image among the two-dimensional reference image, the three-dimensional reference image, and the virtual endoscopic image, which are displayed on one screen, is changed by a user's operation, changing information regarding to the other images based on the information that has been changed by the user's operation.","6.The method of claim 5, wherein the detection comprises detecting the three-dimensional reference image from the volume data by performing parallel volume rendering.","7.The method of claim 5, wherein the detection comprises detecting virtual endoscopic image information by applying position and direction information of each virtual camera to a predetermined perspective ray-casting algorithm.","8.The method of claim 5, wherein the displaying of the virtual cameras comprises: displaying a sphere, which has a predetermined radius from the current position of the virtual camera on the two-dimensional reference image, as the camera display sphere and displaying a set of intersection points between the camera display sphere and the two-dimensional reference image as the camera display circle; and displaying a sphere, which has a predetermined radius from the current position of the virtual camera on the three-dimensional reference image, as the camera display sphere and displaying a set of intersection points between the camera display sphere and a plane, which passes the current position of the virtual camera displayed on the three-dimensional reference image and is parallel with the screen, as the camera display circle.","9.The method of claim 8, wherein the displaying of the virtual cameras comprises: assuming that there is an infinite ray in the direction of each virtual camera, detecting an intersection point between the infinite ray and a corresponding camera display sphere, and representing the direction of the virtual camera with an arrow starting from the center of the virtual camera and passing the intersection point; and setting a corresponding camera display circle and the camera to have different colors when the virtual camera faces the surface of a hemisphere in front of an image and when the virtual camera faces the surface of a hemisphere in the rear of the image.","10.The method of claim 5, wherein the change of the information comprises: when information regarding to the two-dimensional reference image is changed by the user's operation, applying changed content to the two-dimensional reference image and then applying the changed content to the virtual endoscopic image using a predetermined perspective ray-casting algorithm and to the three-dimensional reference image using three-dimensional volume rendering; when information regarding to the three-dimensional reference image is changed by the user's operation, applying changed content to the three-dimensional reference image and then applying the changed content to the virtual endoscopic image using the predetermined perspective ray-casting algorithm and to the two-dimensional reference image using two-dimensional image generation; and when information regarding to the virtual endoscopic image is changed by the user's operation, applying changed content to the virtual endoscopic image using the predetermined perspective ray-casting algorithm and then applying the changed content to the two-dimensional reference image using two-dimensional image generation and to the three-dimensional reference image using three-dimensional volume rendering."],["<SOH> BACKGROUND ART <EOH>In order to avoid giving patients discomfort provoked by an endoscope used for observing the state of the patients' internal organs, virtual endoscopy has been developed and widely and usefully used with respect to a bronchus, a blood vessel, a large intestine, a joint, and so on in a human body.","In virtual endoscopy, an image similar to a real endoscopic image is generated using a series of two-dimensional images collected through CT or MRI.","In other words, a virtual endoscope is noninvasive and thus allows the inside of a patient's internal organs to be observed without giving the patient pain, so recently, it is very usefully used.","A real endoscope allows the inside of internal organs to be observed only in a direction in which a camera moves.","Unlikely, a virtual endoscope allows a user to observe the inside of internal organ in desired directions and is thus very helpful to diagnosis.","However, a conventional virtual endoscope is difficult to operate due to inefficient user interface.","For example, a conventional virtual endoscopic method is composed of a two-dimensional reference image acquired using an image input apparatus and a virtual endoscopic image generated from the two-dimensional reference image.","Accordingly, in the conventional virtual endoscopic method, a correlation between the reference image and the virtual endoscopic image is two-dimensionally shown, so it is difficult to express a three-dimensional correlation and detect the position of a part shown in the virtual endoscopic image or a relative relation with other structures.","In the conventional virtual endoscopic method, a virtual camera is controlled and expressed in a two-dimensional reference image.","Accordingly, it is difficult for a user to operate the virtual camera in a desired direction and recognize the direction of the virtual camera."],["<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.","1 is a schematic block diagram of an apparatus for displaying a three-dimensional virtual endoscopic image according to an embodiment of the present invention.","FIG.","1A is a block diagram of a two-dimensional reference image display control unit according to the embodiment of the present invention.","FIG.","1B is a block diagram of a three-dimensional reference image display control unit according to the embodiment of the present invention.","FIG.","1C is a block diagram of a virtual endoscopic image display control unit according to the embodiment of the present invention.","FIG.","2 is a flowchart of a method of displaying a three-dimensional virtual endoscopic image according to an embodiment of the present invention.","FIG.","2A is a flowchart of a procedure of displaying virtual cameras according to the embodiment of the present invention.","FIG.","2B is a flowchart of a procedure of changing a display area according to the embodiment of the present invention.","FIGS.","3A through 3E show examples of a method of displaying and operating a virtual camera according to the embodiment of the present invention.","FIGS.","4A through 4G show examples of a three-dimensional virtual endoscopic image according to the embodiment of the present invention.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["TECHNICAL FIELD The present invention relates to an apparatus and method for displaying a three-dimensional virtual endoscopic image, and more particularly, to an apparatus and method for displaying a three-dimensional virtual endoscopic image, through which a virtual endoscopic image is displayed along with two- and three-dimensional reference images, a virtual camera is intuitively displayed on the two- and three-dimensional reference images, and the images are controlled to be associated with one another.","Generally, three-dimensional image processing systems are applied to various fields such as design and construction of structures, for example, cars and ships, having a three-dimensional shapes and medical imaging.","In particular, applying three-dimensional image processing systems to the field of medical imaging is referred to as a “three-dimensional medical imaging”.","Three-dimensional medical imaging is generating a three-dimensional image from a series of two-dimensional images obtained through computed tomography (CT) or magnetic resonance imaging (MRI).","While it is difficult to feel the solidity of an entire object and is impossible to observe a cross-section of the object with only a series of two-dimensional images, three-dimensional medical imaging allows the position of an affected part to be accurately estimated and a surgical operation to be predicted similarly to the real thing.","Accordingly, three-dimensional medical imaging has been widely used.","BACKGROUND ART In order to avoid giving patients discomfort provoked by an endoscope used for observing the state of the patients' internal organs, virtual endoscopy has been developed and widely and usefully used with respect to a bronchus, a blood vessel, a large intestine, a joint, and so on in a human body.","In virtual endoscopy, an image similar to a real endoscopic image is generated using a series of two-dimensional images collected through CT or MRI.","In other words, a virtual endoscope is noninvasive and thus allows the inside of a patient's internal organs to be observed without giving the patient pain, so recently, it is very usefully used.","A real endoscope allows the inside of internal organs to be observed only in a direction in which a camera moves.","Unlikely, a virtual endoscope allows a user to observe the inside of internal organ in desired directions and is thus very helpful to diagnosis.","However, a conventional virtual endoscope is difficult to operate due to inefficient user interface.","For example, a conventional virtual endoscopic method is composed of a two-dimensional reference image acquired using an image input apparatus and a virtual endoscopic image generated from the two-dimensional reference image.","Accordingly, in the conventional virtual endoscopic method, a correlation between the reference image and the virtual endoscopic image is two-dimensionally shown, so it is difficult to express a three-dimensional correlation and detect the position of a part shown in the virtual endoscopic image or a relative relation with other structures.","In the conventional virtual endoscopic method, a virtual camera is controlled and expressed in a two-dimensional reference image.","Accordingly, it is difficult for a user to operate the virtual camera in a desired direction and recognize the direction of the virtual camera.","DISCLOSURE OF THE INVENTION The present invention provides an apparatus and method for displaying a three-dimensional virtual endoscopic image, through which a virtual endoscopic image is displayed along with two- and three-dimensional reference images in order to effectively show a correlation between the reference images and the virtual endoscopic image, and a virtual camera is intuitively displayed on the two- and three-dimensional reference images in order to facilitate the operation of the virtual camera.","According to an aspect of the present invention, there is provided an apparatus for displaying a three-dimensional virtual endoscopic image.","The apparatus includes a volume data input unit, which inputs information on a virtual endoscopic image in the form of volume data expressed as a function of three-dimensional position; a two-dimensional reference image display control unit, which derives a two-dimensional reference image from the volume data and displays the two-dimensional reference image; a three-dimensional reference image display control unit, which derives a three-dimensional reference image from the volume data using parallel volume rendering and displays the three-dimensional reference image; a virtual endoscopic image display control unit, which applies position and direction information of a virtual camera used for collecting virtual endoscopic image information to a predetermined perspective ray-casting algorithm, thereby generating image information, and displays a virtual endoscopic image based on the generated image information; a user interface unit, which receives a user's operating signal; and a controller, which when the operating signal for changing image information displayed by one among the two-dimensional reference image display control unit, the three-dimensional reference image display control unit, and the virtual endoscopic image display control unit is input through the user interface unit, controls the two- and three-dimensional reference image display control units and the virtual endoscopic image display control unit to be associated with one another based on the operating signal.","According to another aspect of the present invention, there is provided a method of displaying a three-dimensional virtual endoscopic image.","The method includes inputting information on a virtual endoscopic image in the form of volume data expressed as a function of three-dimensional position; detecting a two-dimensional reference image, a three-dimensional reference image, and a virtual endoscopic image from the volume data; displaying the two-dimensional reference image, the three-dimensional reference image, and the virtual endoscopic image on one screen; displaying virtual cameras on areas, respectively, in which the two-dimensional reference image and the three-dimensional reference image are respectively displayed, wherein a camera display sphere and a camera display circle are defined on the basis of a current position of each virtual camera; and when information regarding to one image among the two-dimensional reference image, the three-dimensional reference image, and the virtual endoscopic image, which are displayed on one screen, is changed by a user's operation, changing information regarding to the other images based on the information that has been changed by the user's operation.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 is a schematic block diagram of an apparatus for displaying a three-dimensional virtual endoscopic image according to an embodiment of the present invention.","FIG.","1A is a block diagram of a two-dimensional reference image display control unit according to the embodiment of the present invention.","FIG.","1B is a block diagram of a three-dimensional reference image display control unit according to the embodiment of the present invention.","FIG.","1C is a block diagram of a virtual endoscopic image display control unit according to the embodiment of the present invention.","FIG.","2 is a flowchart of a method of displaying a three-dimensional virtual endoscopic image according to an embodiment of the present invention.","FIG.","2A is a flowchart of a procedure of displaying virtual cameras according to the embodiment of the present invention.","FIG.","2B is a flowchart of a procedure of changing a display area according to the embodiment of the present invention.","FIGS.","3A through 3E show examples of a method of displaying and operating a virtual camera according to the embodiment of the present invention.","FIGS.","4A through 4G show examples of a three-dimensional virtual endoscopic image according to the embodiment of the present invention.","BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings.","FIG.","1 is a schematic block diagram of an apparatus for displaying a three-dimensional virtual endoscopic image according to an embodiment of the present invention.","Referring to FIG.","1, the apparatus for displaying a three-dimensional virtual endoscopic image includes volume data input unit 100, a two-dimensional reference image display control unit 200, a three-dimensional reference image display control unit 300, a virtual endoscopic image display control unit 400, a user interface unit 500, and a controller 600.The volume data input unit 100 inputs information on a virtual endoscopic image in the form of volume data expressed as a function of three-dimensional position.","For example, in a case where volume data, which is generated as the result of computed tomography (CT) scan or magnetic resonance imaging (MRI), is directly received from each device and then stored, the volume data input unit 100 can be composed of connection hardware connected to the device in order to receive volume data and a hard disc or memory device storing the received volume data.","Alternatively, in a case where data, which has already been measured and stored in other storage media (for example, a floppy disc and a CD-ROM), is read, the volume data input unit 100 can be composed of connection hardware connected to internal/external storage devices of systems for these storage media in order to receive the data and a hard disc or memory device storing the received data.","The two-dimensional reference image display control unit 200 derives a two-dimensional reference image from the volume data and displays it.","The three-dimensional reference image display control unit 300 derives a three-dimensional reference image from the volume data using parallel volume rendering and displays it.","The virtual endoscopic image display control unit 400 applies the position and direction information of a virtual camera used for collecting virtual endoscopic image information to a predetermined perspective ray-casting algorithm, thereby generating image information, and displays a virtual endoscopic image based on the generated image information.","When an operating signal for changing image information displayed by one among the two-dimensional reference image display control unit 200, the three-dimensional reference image display control unit 300, and the virtual endoscopic image display control unit 400 is input through the user interface unit 500, the controller 600 controls the three display control units 200, 300, and 400 to be associated with one another based on the operating signal.","In other words, the controller 600 controls the two- and three-dimensional reference images and the virtual endoscopic image to be associated with one another.","Referring to FIG.","1A, the two-dimensional reference image display control unit 200 includes a control signal input section 210, an image information receiver 220, a painting section 230, a display area control section 240, a camera control section 250, a display format determining section 260, a two-dimensional image generator 270, an image information support section 280, and an image information display section 290.The two-dimensional reference image display control unit 200 is largely divided into an input unit, which receives a control signal for changing a two-dimensional reference image; a main processing unit, which generates changed information with respect to the two-dimensional reference image based on the control signal; and an output unit, which outputs the changed information.","The input unit includes the control signal input section 210 and the image information receiver 220.The main processing unit includes the painting section 230, the display area control section 240, the camera control section 250, the display format determining section 260, and the two-dimensional image generator 270.The output unit includes the image information support section 280 and the image information display section 290.The image information support section 280 transmits the changed information to the other image display control units 300 and 400 so that the changed information regarding to the two-dimensional reference image can be applied to the other images, i.e., a three-dimensional reference image and a virtual endoscopic image.","The image information display section 290 displays the two-dimensional reference image based on the changed information.","The control signal input section 210 receives a control signal for controlling a two-dimensional reference image among the operating signal input through the user interface unit 500 shown in FIG.","1.The image information receiver 220 receives changed three-dimensional reference image information from the three-dimensional reference image display control unit 300 shown in FIG.","1 and changed virtual endoscopic image information from the virtual endoscopic image display control unit 400 shown in FIG.","1.Upon receiving painting information regarding to a region of interest in the two-dimensional reference image through the control signal input section 210, the painting section 230 changes two-dimensional reference image information based on the painting information and then transmits the changed information to the image information support section 280 and the image information display section 290 so that the changed content can be applied to all of the two-dimensional reference image, the three-dimensional reference image, and the virtual endoscopic image.","The display area control section 240 changes the two-dimensional reference image information based on position change information regarding to a region of interest in the two-dimensional reference image, which is received through the control signal input section 210, or position change information regarding to a region of interest in the three-dimensional reference image or the virtual endoscopic image, which is received through the image information receiver 220.For example, when a user changes a region of interest in a two-dimensional reference image through a user interface, such as a scroll bar appearing on a screen or a mouse wheel, changed information is applied to the two-dimensional reference image, and the corresponding regions of interest in both three-dimensional reference image and virtual endoscopic image are also changed based on information about the region of interest in the two-dimensional reference image.","The camera control section 250 changes virtual camera display information with respect to the two-dimensional reference image based on virtual camera display information regarding to the two-dimensional reference image, which is received through the control signal input section 210, or virtual camera display information regarding to the three-dimensional reference image and the virtual endoscopic image, which is received through the image information receiver 220.The display format determining section 260 determines one among an axial image, a coronal image, a sagittal image, and a path MPR image as the format of the two-dimensional reference image based on display information with respect to the two-dimensional reference image, which is received through the control signal input section 210.The axial image is a two-dimensional image having the same z-value.","The coronal image is a two-dimensional image having the same y-value.","The sagittal image is a two-dimensional image having the same x-value.","Upon receiving changed three-dimensional reference image information and changed virtual endoscopic image information from the image information receiver 220, the two-dimensional image generator 270 converts the received three-dimensional reference image and virtual endoscopic image information to two-dimensional image information so that the received image information is applied to the two-dimensional reference image.","The image information display section 290 detects a changed two-dimensional reference image based on the two-dimensional reference image information received from the painting section 230, the display area control section 240, the camera control section 250, the display format determining section 260, and the two-dimensional image generator 270, and displays the changed two-dimensional reference image.","When the two-dimensional reference image information is changed in the painting section 230, the display area control section 240, and the camera control section 250 in response to the control signals received from the control signal input section 210, the image information support section 280 transmits the changed two-dimensional reference image information to the three-dimensional reference image display control unit 300 and the virtual endoscopic image display control unit 400 so that the changed two-dimensional reference image information is applied to a three-dimensional reference image and a virtual endoscopic image.","Referring to FIG.","1B, the three-dimensional reference image display control unit 300 includes a control signal input section 310, an image information receiver 320, a painting section 330, a display area control section 340, a camera control section 350, a three-dimensional volume rendering section 360, an image information support section 370, and an image information display section 380.Like the two-dimensional reference image display control unit 200, the three-dimensional reference image display control unit 300 is largely divided into an input unit, which receives a control signal for changing a three-dimensional reference image; a main processing unit, which generates changed information with respect to the three-dimensional reference image based on the control signal; and an output unit, which outputs the changed information.","The input unit includes the control signal input section 310 and the image information receiver 320.The main processing unit includes the painting section 330, the display area control section 340, the camera control section 350, and the three-dimensional volume rendering section 360.The output unit includes the image information support section 370 and the image information display section 380.The image information support section 370 transmits the changed information to the other image display control units 200 and 400 so that the changed information regarding to the three-dimensional reference image can be applied to the other images, i.e., a two-dimensional reference image and a virtual endoscopic image.","The image information display section 380 displays a three-dimensional reference image based on the changed information.","The control signal input section 310 receives a control signal for controlling a three-dimensional reference image among the operating signal input through the user interface unit 500 shown in FIG.","1.The image information receiver 320 receives changed two-dimensional reference image information from the two-dimensional reference image display control unit 200 shown in FIG.","1 and changed virtual endoscopic image information from the virtual endoscopic image display control unit 400 shown in FIG.","1.Upon receiving painting information regarding to a region of interest in the three-dimensional reference image through the control signal input section 310, the painting section 330 changes three-dimensional reference image information based on the painting information and then transmits the changed information to the image information support section 370 and the image information display section 380 so that the changed content can be applied to all of the three-dimensional reference image, the two-dimensional reference image, and the virtual endoscopic image.","The display area control section 340 changes the three-dimensional reference image information based on position change information regarding to a region of interest in the three-dimensional reference image, which is received through the control signal input section 310, or position change information regarding to a region of interest in the two-dimensional reference image or the virtual endoscopic image, which is received through the image information receiver 320.For example, a new two-dimensional reference image display straight line can be displayed on the three-dimensional reference image, and a slice locator can be directly dragged on the three-dimensional reference image to change the position of a two-dimensional reference image.","The camera control section 350 changes virtual camera display information with respect to the three-dimensional reference image based on virtual camera display information regarding to the three-dimensional reference image, which is received through the control signal input section 310, or virtual camera display information regarding to the two-dimensional reference image and the virtual endoscopic image, which is received through the image information receiver 320.Upon receiving changed two-dimensional reference image information and changed virtual endoscopic image information from the image information receiver 320, the three-dimensional volume rendering section 360 performs three-dimensional volume rendering based on the received image information.","For example, the three-dimensional volume rendering section 360 receives the result of performing painting operation on a virtual endoscopic image and the result of performing painting operation on a two-dimensional reference image from the image information receiver 320 and performs three-dimensional rendering.","Three-dimensional rendering is a process of adding three-dimensional texture such as shadow or a change in color or depth of color to an object, thereby giving reality to computer graphics.","The image information display section 380 detects a changed three-dimensional reference image based on the three-dimensional reference image information received from the painting section 330, the display area control section 340, the camera control section 350, and the three-dimensional volume rendering section 360, and displays the changed three-dimensional reference image.","When the three-dimensional reference image information is changed in the painting section 330, the display area control section 340, and the camera control section 350 in response to the control signals received from the control signal input section 310, the image information support section 370 transmits the changed three-dimensional reference image information to the two-dimensional reference image display control unit 200 and the virtual endoscopic image display control unit 400 so that the changed three-dimensional reference image information is applied to a two-dimensional reference image and a virtual endoscopic image.","Referring to FIG.","1C, the virtual endoscopic image display control unit 400 includes a control signal input section 410, an image information receiver 420, a painting section 430, a perspective ray-casting section 440, a display area control section 450, a camera control section 460, a focal zone generator 470, an image information support section 480, and an image information display section 490.Like the two-dimensional reference image display control unit 200 and the three-dimensional reference image display control unit 300, the virtual endoscopic image display control unit 400 is largely divided into an input unit, which receives a control signal for changing a virtual endoscopic image; a main processing unit, which generates changed information with respect to the virtual endoscopic image based on the control signal; and an output unit, which outputs the changed information.","The input unit includes the control signal input section 410 and the image information receiver 420.The main processing unit includes the painting section 430, the perspective ray-casting section 440, the display area control section 450, the camera control section 460, and the focal zone generator 470.The output unit includes the image information support section 480 and the image information display section 490.The image information support section 480 transmits the changed information to the other image display control units 200 and 300 so that the changed information regarding to the virtual endoscopic image can be applied to the other images, i.e., a two-dimensional reference image and a three-dimensional reference image.","The image information display section 490 displays the virtual endoscopic image based on the changed information.","The control signal input section 410 receives a control signal for controlling a virtual endoscopic image among the operating signal input through the user interface unit 500 shown in FIG.","1.The image information receiver 420 receives changed two-dimensional reference image information from the two-dimensional reference image display control unit 200 shown in FIG.","1 and changed three-dimensional reference image information from the three-dimensional reference image display control unit 300 shown in FIG.","1.Upon receiving painting information regarding to a region of interest in the virtual endoscopic image through the control signal input section 410, the painting section 430 changes virtual endoscopic image information based on the painting information and then transmits the changed information to the image information support section 480 so that the changed content can be applied to the two-dimensional reference image and the three-dimensional reference image.","The painting section 430 also transmits the changed information to the perspective ray-casting section 440 so that the changed content can be applied to the virtual endoscopic image.","The perspective ray-casting section 440 applies the processing result received from the painting section 430 and the changed two- and three-dimensional reference image information received from the image information receiver 420 to a predetermined perspective ray-casting algorithm so as to adjust the opacity of a region of interest, and then changes the virtual endoscopic image information based on the result of the adjustment.","The perspective ray-casting algorithm is a process of accumulating brightness of points, where light beams diverging in different directions from the same start point meet an object, on a projection plane so as to generate an image.","Since it is widely known, a detailed description thereof will be omitted.","The display area control section 450 changes the virtual endoscopic image information based on position change information regarding to a region of interest in the two- or three-dimensional reference image, which is received through the image information receiver 420.In other words, when changed information regarding to the region of interest is received from the two-dimensional reference image display control unit 200 or the three-dimensional reference image display control unit 300, the display area control section 450 renders the two- or three-dimensional reference image in the region of interest on the virtual endoscopic image.","For example, in a case where a two-dimensional axial image is rendered on the virtual endoscopic image, when a plurality of light beams diverging from one point meet the surface of an object, if the z-value of a voxel corresponding to the surface is the same as the z-value of the two-dimensional axial image, the display area control section 450 changes the color of the voxel.","The camera control section 460 changes the virtual endoscopic image information based on the position and direction information of a virtual camera, which is received through the control signal input section 410, and displays the result of the change through the image information display section 490 or applies it to the other images through the image information support section 480.The focal zone generator 470 designates a region of a user's interest on a virtual endoscopic image based on the control signal transmitted through the control signal input section 410.In other words, if a user selects a region of interest on a virtual endoscopic image screen, the focal zone generator 470 defines the region of interest as a focal zone and transmits the result of the definition to the image information display section 490 or the image information support section 480 so that the result of the definition is displayed or applied to two- and three-dimensional reference images.","Accordingly, a correlation between each reference image and the virtual endoscopic image can be effectively shown.","The focal zone is a region defined on a screen and does not change when a new virtual endoscopic image is generated due to a change in the position or direction information of the virtual camera.","Volume rendering is performed to generate a three-dimensional reference image so that the region of volume data included in the focal zone defined on the virtual endoscopic image screen is distinguished from the remaining region.","For example, if the focal zone is a quadrilateral, four planes defined by Formula (1) are generated in three-dimensional space.","f1(x)=a1x+b1y+c1y+d1=0 f2(x)=a2x+b2y+c2y+d2=0 f3(x)=a3x+b3y+c3y+d3=0 f4(x)=a4x+b4y+c4y+d4=0 (1) Here, if the region of volume data is included in the focal zone, Formula (2) is satisfied.","f1(x)<0, f2(x)<0, f1(x)<0, f4(x)<0 (2) The focal zone can be determined with respect to other primitives, using a similar method to the above-described one.","The image information display section 490 detects a changed virtual endoscopic image based on the virtual endoscopic image information received from the perspective ray-casting section 440, the display area control section 450, the camera control section 460, and the focal zone generator 470, and displays the changed virtual endoscopic image.","When the virtual endoscopic image information is changed in the painting section 430, the perspective ray-casting section 440, the camera control section 460, and the focal zone generator 470 in response to the control signal received from the control signal input section 410, the image information support section 480 transmits the changed virtual endoscopic image information to the two-dimensional reference image display control unit 200 and the three-dimensional reference image display control unit 300 so that the changed virtual endoscopic image information is applied to a two-dimensional reference image and a three-dimensional reference image.","Referring to FIGS.","1A through 1C, the image display control units 200, 300, and 400 receive changed information regarding to different images than their own from the other image display control units using the respective image information receivers 220, 320, and 420 and transmit their own changed image information to the other image display control units using the respective image information support sections 280, 370, and 480.Accordingly, all of the different images are associated with one another.","A method of displaying a three-dimensional virtual endoscopic image according to an embodiment of the present invention will be described below with reference to FIG.","2.Information on a virtual endoscopic image is input in the form of volume data expressed in a three-dimensional position function in step s100.Here, the volume data is generated through CT scan or MRI.","A two-dimensional reference image, a three-dimensional reference image, and a virtual endoscopic image are detected from the volume data in step s200.Two-dimensional image generation is performed in order to detect the two-dimensional reference image from the volume data.","Parallel volume rendering is performed in order to detect the three-dimensional reference image from the volume data.","The position and direction information of a virtual camera is applied to a predetermined perspective ray-casting algorithm in order to detect the virtual endoscopic image from the volume data.","Thereafter, the two-dimensional reference image, the three-dimensional reference image, and the virtual endoscopic image are displayed on one screen in step s300.In other words, a display area on the screen is divided into three sub areas, and the three images are displayed on the respective three sub areas.","It is preferable that a sub area for displaying the virtual endoscopic image is largest and is located at the center of the display area.","Virtual cameras are displayed on the respective sub areas, in which the two- and three-dimensional reference images are displayed, in step s400.With respect to each of the virtual cameras, a camera display sphere and a camera display circle are provided centering around the current position of each virtual camera.","For example, in a case of a two-dimensional reference image, a sphere having a predetermined radius from the current position of a virtual camera is displayed as a camera display sphere, and a circle composed of intersection points between the camera display sphere and the current two-dimensional reference image is displayed as a camera display circle.","In a case of a three-dimensional reference image, a sphere having a predetermined radius from the current position of a virtual camera is displayed as a camera display sphere, and a circle composed of intersection points between a plane, which passes the current position of the virtual camera displayed on the three-dimensional reference image and parallel with the screen, and the camera display sphere is displayed as a camera display circle.","The direction of a virtual camera is represented with an arrow.","On the assumption that there is an infinite ray in the direction of the virtual camera, an intersection point between the infinite ray and a camera display sphere is detected, and then the direction of the virtual camera is represented with an arrow starting from the center of the virtual camera and passing the intersection point.","In order to identify whether the virtual camera faces the surface of the hemisphere in front or the rear of an image, different colors are used for the camera display circle and the virtual camera.","For example, when the virtual camera faces the surface of the hemisphere in front of the image, the camera display circle and the virtual camera are colored in red.","Conversely, when the virtual camera faces the surface of the hemisphere in the rear of the image, the camera display circle and the virtual camera are colored in blue.","Examples of a method of displaying a virtual camera will be described later more specifically with reference to FIGS.","3A through 3E.","In the meantime, when one of the images displayed on the screen is changed by a user's operation, the other images are changed based on changed image information in association with one another in step s500.FIG.","2A is a flowchart of a procedure of displaying a virtual camera according to the embodiment of the present invention.","Referring to FIG.","2A, the virtual cameras are displayed at predetermined positions on the respective two- and three-dimensional reference images in step s401.If the position and direction of the virtual camera are changed on the virtual endoscopic image in step s402, the changed position and direction information is applied to the predetermined perspective ray-casting algorithm to detect a new virtual endoscopic image and the new virtual endoscopic image is displayed in step s403.The positions and directions of the virtual cameras displayed on the respective two- and three-dimensional reference images are changed based on the changed position and direction information regarding to the virtual camera on the virtual endoscopic image in step s404.If the position and direction of the virtual camera are changed on the two-dimensional reference image in step s405, the changed position and direction information is applied to the predetermined perspective ray-casting algorithm to detect a new virtual endoscopic image and the new virtual endoscopic image is displayed in step s406.The position and direction of the virtual camera displayed on the three-dimensional reference image are changed based on the changed position and direction information regarding to the virtual camera on the two-dimensional reference image in step s407.A new three-dimensional reference image is detected based on the changed position and direction information regarding to the old three-dimensional reference image in step s408.If the position and direction of the virtual camera are changed on the three-dimensional reference image in step s409, the changed position and direction information is applied to the predetermined perspective ray-casting algorithm to detect a new virtual endoscopic image and the new virtual endoscopic image is displayed in step s410.The position and direction of the virtual camera displayed on the two-dimensional reference image are changed based on the changed position and direction information regarding to the virtual camera on the three-dimensional reference image in step s411.A new two-dimensional reference image is detected based on the changed position and direction information regarding to the old two-dimensional reference image in step s412.FIG.","2B is a flowchart of a procedure of changing an image in a display area according to the embodiment of the present invention.","Referring to FIG.","2B, when one among the two-dimensional reference image, the three-dimensional reference image, and the virtual endoscopic image, which are displayed on the same screen, is changed, the following operation is performed in order to change the other images in association with one another.","If information regarding to the two-dimensional reference image is changed in step s501, the changed information is applied to the two-dimensional reference image in step s502.Next, the changed information is applied to the virtual endoscopic image using the predetermined perspective ray-casting algorithm in step s503 and is applied to the three-dimensional reference image using three-dimensional volume rendering in step s504.If information regarding to the three-dimensional reference image is changed in step s505, the changed information is applied to the three-dimensional reference image in step s506.Next, the changed information is applied to the virtual endoscopic image using the predetermined perspective ray-casting algorithm in step s507 and is applied to the two-dimensional reference image using two-dimensional image generation in step s508.If information regarding to the virtual endoscopic image is changed by the user's operation in step s509, the changed information is applied to the virtual endoscopic image using the predetermined perspective ray-casting algorithm in step s510.Thereafter, the changed information is applied to the two-dimensional reference image using two-dimensional image generation in step s511 and is applied to the three-dimensional reference image using three-dimensional volume rendering in step s512.FIGS.","3A through 3E show examples of a method of displaying and operating a virtual camera according to the embodiment of the present invention.","FIG.","3A shows an example in which a virtual camera is displayed on a two-dimensional reference image.","Referring to FIG.","3A, with respect to a two-dimensional reference image 10, a sphere (hereinafter, a camera display sphere) 12 having a predetermined radius from the current position of a virtual camera 11 is assumed.","A set of intersection points between the camera display sphere 12 and the current two-dimensional reference image 10 is defined as a circle (hereinafter, referred to as a camera display circle) 13.Here, if it is assumed that an infinite ray exists in the direction of the virtual camera 11, the infinite ray meets the camera display sphere 12 at one point.","An arrow 14 starting from the center of the virtual camera 11 and passing the intersection point between the camera display sphere 12 and the infinite ray indicates the direction of the virtual camera 11.In a coordinate system having the center of the virtual camera 11 as an origin, the infinite ray with respect to the direction d(dx,dy,dz) of the virtual camera 11 is expressed by Formula (3).","x=dxk, y=dyk, z=dzk (where k is a constant) (3) In the meantime, the camera display sphere 12 is expressed by Formula (4), and thus the coordinates of the intersection point between the infinite ray and the camera display sphere 12 are given by Formula (5) based on Formulae (3) and (4), x2+y2+z2=r2 (4) x = d x k _ , y = d y k _ , z = d z k _ ( k _ = r 2 d x 2 + d y 2 + d z 2 ) ( 5 ) Here, the virtual camera 11 may face the surface of a hemisphere in front or the rear of the two-dimensional reference image 10.In order to identify whether the virtual camera 11 faces the surface of the hemisphere in front or the rear of the two-dimensional reference image 10, different colors are used for the camera display circle 13 and the virtual camera 11.For example, the direction of the virtual camera 11 is the same in the plane views of FIGS.","3A and 3B.","However, the virtual camera 11 in FIG.","3A faces the surface of the hemisphere in front of the two-dimensional reference image 10 while the virtual camera 11 in FIG.","3B faces the surface of the hemisphere in the rear of the two-dimensional reference image 10.Accordingly, the color of the camera display circle 13 and the virtual camera 11 shown in FIG.","3A is different from the color of the camera display circle 13 and the virtual camera 11 shown in FIG.","3B.","For example, the camera display circle 13 and the virtual camera 11 are colored in red in FIG.","3A and colored in blue in FIG.","3B so that the front and the rear of the two-dimensional reference image 10 can be discriminated from each other.","FIG.","3C shows an example, in which a virtual camera is displayed on a three-dimensional reference image.","Referring to FIG.","3C, in order to display a virtual camera 21 on a three-dimensional reference image, a sphere having a predetermined radius from the current position of a virtual camera 21 is defined as a camera display sphere 22, and a set of the intersection points between the camera display sphere 22 and a plane 20, which passes the current position of the virtual camera 21 and is parallel with a screen displaying the three-dimensional reference image, is defined as a camera display circle 23.Here, if it is assumed that an infinite ray exists in the direction of the virtual camera 21, the infinite ray intersects the camera display sphere 22 at one point.","An arrow 24 starting from the center of the virtual camera 21 and passing the intersection point between the camera display sphere 22 and the infinite ray indicates the direction of the virtual camera 21.In order to discriminate the direction of the virtual camera 21 facing the surface of a hemisphere in front of the plane 20 from the direction of the virtual camera 21 facing the surface of a hemisphere in the rear of the plane 20, different colors are used for the camera display circle 23 and the virtual camera 21.FIGS.","3D and 3E are diagrams for explaining a method of operating a virtual camera on a two-dimensional reference image, according to the present invention.","When the virtual camera 11 faces the surface of the hemisphere in front of the two-dimensional reference image 10 as shown in FIG.","3D(a), if a user clicks a predetermined position 41 within the camera display circle 13, an intersection point between the hemisphere and an infinite ray, which passes the predetermined position 41 and goes toward the front of the two-dimensional reference image 10, is calculated and the virtual camera 11 is adjusted to face the intersection point, as shown in FIG.","3D(b).","In a coordinate system having the center of the virtual camera 11 as an origin, the infinite ray, with respect to the direction d(dx,dy,dz) of the virtual camera 11 and the predetermined position 41 p(px,pypz), is expressed by Formula (6).","x=px+dxk, y=py+dyk, z=pz+dzk (where k is a constant) (6) In the meantime, the camera display sphere 12 is expressed by Formula (4), and thus the coordinates of the intersection point between the infinite ray and the camera display sphere 12 are given by Formula (7) based on Formulae (6) and (4).","x=px+dxk, y=py+dy{overscore (k)}, z=pz+dz{overscore (k)}({overscore (k)} satisfies (d2x+d2y+d2z){overscore (k)}2+2(pxdx+pydy+pzdz) {overscore (k)}+p2x+p2y+p2z−r2=0) (7) In the meantime, when the virtual camera 11 faces the surface of the hemisphere in front of the two-dimensional reference image 10 as shown in FIG.","3E(a), if a user clicks a predetermined position 42 on the camera display circle 13, the direction of the virtual camera 11 is converted to face the surface of the hemisphere in the rear of the two-dimensional reference image 10.In other words, in the case where the virtual camera 11 is colored in blue because it faces the front hemisphere, if one point on the camera display circle is clicked, the direction of the virtual camera 11 is symmetrically changed with respect to the two-dimensional reference image 10 so that the virtual camera 11 faces the rear hemisphere, as shown in FIG.","3E(b).","FIGS.","4A through 4G show examples of a three-dimensional virtual endoscopic image according to the embodiment of the present invention.","FIG.","4A shows a user interface of a virtual endoscopy system composed of a two-dimensional reference image, a three-dimensional reference image, and a virtual endoscopic image.","Referring to FIG.","4A, in the virtual endoscopy system, the three-dimensional reference image is displayed on the right, the virtual endoscopic image is displayed on the upper right, and the two-dimensional reference image is displayed on the lower right.","A two-dimensional reference image display line 51 is displayed on the three-dimensional reference image in order to indicate the position of an axial image, i.e., the current two-dimensional reference image.","Although the axial image is shown as the two-dimensional reference image, a user can view the two-dimensional reference image in a desired format using buttons, i.e., Axial, Coronal, Sagittal, and Path MPR, in an image title bar.","FIG.","4B shows an example of a sagittal image to which the axial image shown in FIG.","4A is converted by pressing the Sagittal button.","FIGS.","4C and 4D show the results of adjusting the position of the two-dimensional reference image shown in FIG.","4A.","FIG.","4C shows the result of moving a scroll bar 52 located at the lower end of the two-dimensional reference image, and FIG.","4D shows the result of moving the two-dimensional reference image display line 51 displayed on the three-dimensional reference image using a mouse.","FIG.","4E shows an example of the case where a virtual camera positioned in a particular region of a bronchus is displayed on two- and three-dimensional reference image areas.","Referring to FIG.","4E, virtual cameras 53 and 54 are displayed on the respective two- and three-dimensional reference images.","Here, in order to identify whether the virtual camera 53 on the two-dimensional reference image faces above or below the two-dimensional reference image, different colors are used for the virtual camera 53.For example, in the case where the virtual camera 53 facing above the two-dimensional reference image is set to red color while the virtual camera 53 facing below the two-dimensional reference image is set to blue color, if the virtual camera 53 is colored in red on the two-dimensional reference image, it can be inferred that the virtual camera 53 faces above the two-dimensional reference image.","Like the case of the virtual camera 53 on the two-dimensional reference image, it is identified whether the virtual camera 54 on the three-dimensional reference image faces the front or rear of the three-dimensional reference image using the color of the virtual camera 54.For example, in the case where the virtual camera 54 facing the front of the three-dimensional reference image is set to red color while the virtual camera 54 facing the rear of the three-dimensional reference image is set to blue color, if the virtual camera 54 is colored in blue on the three-dimensional reference image, it can be inferred that the virtual camera 54 faces the rear of the three-dimensional reference image.","FIG.","4F shows the result of adjusting the direction of the virtual camera 53 on the two-dimensional reference image shown in FIG.","4E.","In other words, FIG.","4F shows the result of changing the direction of the virtual camera 53 on the two-dimensional reference image by clicking a point within a camera display circle on the two-dimensional reference image shown in FIG.","4E.","Accordingly, if the virtual camera 53 is colored in red before its direction is changed, it is colored in blue after its direction is changed.","FIG.","4G shows the result of adjusting the direction of the virtual camera 54 on the three-dimensional reference image shown in FIG.","4E.","In other words, FIG.","4G shows the result of changing the direction of the virtual camera 54 on the three-dimensional reference image by clicking a point within a camera display circle on the three-dimensional reference image shown in FIG.","4E.","Here, the direction of the virtual camera 54 is symmetrically changed with respect to the three-dimensional reference image.","Accordingly, if the virtual camera 54 is colored in blue before its direction is changed, it is colored in red after its direction is changed.","The above description just concerns embodiments of the present invention.","The present invention is not restricted to the above embodiments, and various modifications can be made thereto within the scope defined by the attached claims.","For example, the shape and structure of each member specified in the embodiments can be changed without departing from the spirit of the invention.","Industrial Applicability As described above, in an apparatus and method for displaying a three-dimensional virtual endoscopic image according to the present invention, a virtual endoscopic image is displayed along with two- and three-dimensional reference images, and these images are associated with one another.","Accordingly, correlations among the reference images and the virtual endoscopic image can be effectively presented.","In addition, since a virtual camera is intuitively displayed on each of the two- and three-dimensional reference images, and the virtual camera can be operated using a normal user interface such as a mouse, the virtual camera is easily operated.","Therefore, by using the present invention, a virtual endoscopy system can be improved to integrate various images and techniques into a synthetic system."]],"string":"[\n [\n \"Apparatus and method for displaying virtual endoscopy display\",\n \"An apparatus and method for displaying a three-dimensional virtual endoscopic image are provided.\",\n \"In the method, information on a virtual endoscopic image is input in the form of volume data expressed as a function of three-dimensional position.\",\n \"A two-dimensional reference image, a three-dimensional reference image, and a virtual endoscopic image are detected from the volume data.\",\n \"The detected images are displayed on one screen.\",\n \"Virtual cameras are respectively displayed on areas, in which the two and three-dimensional reference images are respectively displayed.\",\n \"Here, a camera display sphere and a camera display circle are defined on the basis of a current position of each virtual camera.\",\n \"When information regarding to one image, among the two and three-dimensional reference images and the virtual endoscopic image on one screen, is changed by a user's operation, information regarding to the other images is changed based on the information changed by the user's operation.\"\n ],\n [\n \"1.An apparatus for displaying a three-dimensional virtual endoscopic image, the apparatus comprising: a volume data input unit, which inputs information on a virtual endoscopic image in the form of volume data expressed as a function of three-dimensional position; a two-dimensional reference image display control unit, which derives a two-dimensional reference image from the volume data and displays the two-dimensional reference image; a three-dimensional reference image display control unit, which derives a three-dimensional reference image from the volume data using parallel volume rendering and displays the three-dimensional reference image; a virtual endoscopic image display control unit, which applies position and direction information of a virtual camera used for collecting virtual endoscopic image information to a predetermined perspective ray-casting algorithm, thereby generating image information, and displays a virtual endoscopic image based on the generated image information; a user interface unit, which receives a user's operating signal; and a controller, which when the operating signal for changing image information displayed by one among the two-dimensional reference image display control unit, the three-dimensional reference image display control unit, and the virtual endoscopic image display control unit is input through the user interface unit, controls the two- and three-dimensional reference image display control units and the virtual endoscopic image display control unit to be associated with one another based on the operating signal.\",\n \"2.The apparatus of claim 1, wherein the two-dimensional reference image display control unit comprises: a control signal input section, which receives a control signal for controlling a two-dimensional reference image among the operating signal input through the user interface unit; an image information receiver, which receives changed three-dimensional reference image information from the three-dimensional reference image display control unit and changed virtual endoscopic image information from the virtual endoscopic image display control unit; a painting section, which receives painting information regarding to a region of interest in the two-dimensional reference image from the control signal input section and changes two-dimensional reference image information based on the painting information; a display area control section, which changes the two-dimensional reference image information based on position change information regarding to a region of interest in the two-dimensional reference image, which is received through the control signal input section, or based on position change information regarding to a region of interest in the three-dimensional reference image or the virtual endoscopic image, which is received through the image information receiver; a camera control section, which changes virtual camera display information with respect to the two-dimensional reference image based on virtual camera display information regarding to the two-dimensional reference image, which is received through the control signal input section, or based on virtual camera display information regarding to the three-dimensional reference image and the virtual endoscopic image, which is received through the image information receiver; a display format determining section, which determines one among an axial image, a coronal image, a sagittal image, and a path MPR image as the format of the two-dimensional reference image, based on display information regarding to the two-dimensional reference image, which is received through the control signal input section; a two-dimensional image generator, which receives the changed three-dimensional reference image information and the changed virtual endoscopic image information from the image information receiver and converts the received three-dimensional reference image information and the received virtual endoscopic image information to two-dimensional image information; an image information display section, which detects a changed two-dimensional reference image based on the two-dimensional reference image information received from the painting section, the display area control section, the camera control section, the display format determining section, and the two-dimensional image generator, and displays the changed two-dimensional reference image; and an image information support section, which when the two-dimensional reference image information is changed in the painting section, the display area control section, and the camera control section in response to the control signals received from the control signal input section, transmits the changed two-dimensional reference image information to the three-dimensional reference image display control unit and the virtual endoscopic image display control unit so that the changed two-dimensional reference image information is applied to the three-dimensional reference image and the virtual endoscopic image.\",\n \"3.The apparatus of claim 1, wherein the three-dimensional reference image display control unit comprises: a control signal input section, which receives a control signal for controlling a three-dimensional reference image among the operating signal input through the user interface unit; an image information receiver, which receives changed two-dimensional reference image information from the two-dimensional reference image display control unit and changed virtual endoscopic image information from the virtual endoscopic image display control unit; a painting section, which receives painting information regarding to a region of interest in the three-dimensional reference image from the control signal input section and changes three-dimensional reference image information based on the painting information; a display area control section, which changes the three-dimensional reference image information based on position change information regarding to a region of interest in the three-dimensional reference image, which is received through the control signal input section, or based on position change information regarding to a region of interest in the two-dimensional reference image or the virtual endoscopic image, which is received through the image information receiver; a camera control section, which changes virtual camera display information with respect to the three-dimensional reference image based on virtual camera display information regarding to the three-dimensional reference image, which is received through the control signal input section, or based on virtual camera display information regarding to the two-dimensional reference image and the virtual endoscopic image, which is received through the image information receiver; a three-dimensional volume rendering section, which receives the changed two-dimensional reference image information and the changed virtual endoscopic image information from the image information receiver and performs three-dimensional volume rendering based on the received two-dimensional reference image and virtual endoscopic image information; an image information display section, which detects a changed three-dimensional reference image based on the three-dimensional reference image information received from the painting section, the display area control section, the camera control section, and the three-dimensional volume rendering section, and displays the changed three-dimensional reference image; and an image information support section, which when the three-dimensional reference image information is changed in the painting section, the display area control section, and the camera control section in response to the control signals received from the control signal input section, transmits the changed three-dimensional reference image information to the two-dimensional reference image display control unit and the virtual endoscopic image display control unit so that the changed three-dimensional reference image information is applied to the two-dimensional reference image and the virtual endoscopic image.\",\n \"4.The apparatus of claim 1, wherein the virtual endoscopic image display control unit comprises: a control signal input section, which receives a control signal for controlling a virtual endoscopic image among the operating signal input through the user interface unit; an image information receiver, which receives changed two-dimensional reference image information from the two-dimensional reference image display control unit and changed three-dimensional reference image information from the three-dimensional reference image display control unit; a painting section, which receives painting information regarding to a region of interest in the virtual endoscopic image from the control signal input section and changes virtual endoscopic image information based on the painting information; a perspective ray-casting section, which applies the processing result received from the painting section and the changed two- and three-dimensional reference image information received from the image information receiver to a predetermined perspective ray-casting algorithm so as to adjust the opacity of the region of interest, and then changes the virtual endoscopic image information based on the result of the adjustment; a display area control section, which changes the virtual endoscopic image information based on position change information regarding to the region of interest in the two- or three-dimensional reference image, which is received through the image information receiver; a camera control section, which changes the virtual endoscopic image information based on position and direction information of a virtual camera, which is received through the control signal input section; a focal zone generator, which designates a region of a user's interest on the virtual endoscopic image based on a control signal transmitted through the control signal input section; an image information display section, which detects a changed virtual endoscopic image based on the virtual endoscopic image information received from the perspective ray-casting section, the display area control section, the camera control section, and the focal zone generator, and displays the changed virtual endoscopic image; and an image information support section, which when the virtual endoscopic image information is changed in the painting section, the perspective ray-casting section, the camera control section, and the focal zone generator in response to the control signals received from the control signal input section, transmits the changed virtual endoscopic image information to the two-dimensional reference image display control unit and the three-dimensional reference image display control unit so that the changed virtual endoscopic image information is applied to the two-dimensional reference image and the three-dimensional reference image.\",\n \"5.A method of displaying a three-dimensional virtual endoscopic image, the method comprising: inputting information on a virtual endoscopic image in the form of volume data expressed as a function of three-dimensional position; detecting a two-dimensional reference image, a three-dimensional reference image, and a virtual endoscopic image from the volume data; displaying the two-dimensional reference image, the three-dimensional reference image, and the virtual endoscopic image on one screen; displaying virtual cameras on areas, respectively, in which the two-dimensional reference image and the three-dimensional reference image are respectively displayed, wherein a camera display sphere and a camera display circle are defined on the basis of a current position of each virtual camera; and when information regarding to one image among the two-dimensional reference image, the three-dimensional reference image, and the virtual endoscopic image, which are displayed on one screen, is changed by a user's operation, changing information regarding to the other images based on the information that has been changed by the user's operation.\",\n \"6.The method of claim 5, wherein the detection comprises detecting the three-dimensional reference image from the volume data by performing parallel volume rendering.\",\n \"7.The method of claim 5, wherein the detection comprises detecting virtual endoscopic image information by applying position and direction information of each virtual camera to a predetermined perspective ray-casting algorithm.\",\n \"8.The method of claim 5, wherein the displaying of the virtual cameras comprises: displaying a sphere, which has a predetermined radius from the current position of the virtual camera on the two-dimensional reference image, as the camera display sphere and displaying a set of intersection points between the camera display sphere and the two-dimensional reference image as the camera display circle; and displaying a sphere, which has a predetermined radius from the current position of the virtual camera on the three-dimensional reference image, as the camera display sphere and displaying a set of intersection points between the camera display sphere and a plane, which passes the current position of the virtual camera displayed on the three-dimensional reference image and is parallel with the screen, as the camera display circle.\",\n \"9.The method of claim 8, wherein the displaying of the virtual cameras comprises: assuming that there is an infinite ray in the direction of each virtual camera, detecting an intersection point between the infinite ray and a corresponding camera display sphere, and representing the direction of the virtual camera with an arrow starting from the center of the virtual camera and passing the intersection point; and setting a corresponding camera display circle and the camera to have different colors when the virtual camera faces the surface of a hemisphere in front of an image and when the virtual camera faces the surface of a hemisphere in the rear of the image.\",\n \"10.The method of claim 5, wherein the change of the information comprises: when information regarding to the two-dimensional reference image is changed by the user's operation, applying changed content to the two-dimensional reference image and then applying the changed content to the virtual endoscopic image using a predetermined perspective ray-casting algorithm and to the three-dimensional reference image using three-dimensional volume rendering; when information regarding to the three-dimensional reference image is changed by the user's operation, applying changed content to the three-dimensional reference image and then applying the changed content to the virtual endoscopic image using the predetermined perspective ray-casting algorithm and to the two-dimensional reference image using two-dimensional image generation; and when information regarding to the virtual endoscopic image is changed by the user's operation, applying changed content to the virtual endoscopic image using the predetermined perspective ray-casting algorithm and then applying the changed content to the two-dimensional reference image using two-dimensional image generation and to the three-dimensional reference image using three-dimensional volume rendering.\"\n ],\n [\n \"<SOH> BACKGROUND ART <EOH>In order to avoid giving patients discomfort provoked by an endoscope used for observing the state of the patients' internal organs, virtual endoscopy has been developed and widely and usefully used with respect to a bronchus, a blood vessel, a large intestine, a joint, and so on in a human body.\",\n \"In virtual endoscopy, an image similar to a real endoscopic image is generated using a series of two-dimensional images collected through CT or MRI.\",\n \"In other words, a virtual endoscope is noninvasive and thus allows the inside of a patient's internal organs to be observed without giving the patient pain, so recently, it is very usefully used.\",\n \"A real endoscope allows the inside of internal organs to be observed only in a direction in which a camera moves.\",\n \"Unlikely, a virtual endoscope allows a user to observe the inside of internal organ in desired directions and is thus very helpful to diagnosis.\",\n \"However, a conventional virtual endoscope is difficult to operate due to inefficient user interface.\",\n \"For example, a conventional virtual endoscopic method is composed of a two-dimensional reference image acquired using an image input apparatus and a virtual endoscopic image generated from the two-dimensional reference image.\",\n \"Accordingly, in the conventional virtual endoscopic method, a correlation between the reference image and the virtual endoscopic image is two-dimensionally shown, so it is difficult to express a three-dimensional correlation and detect the position of a part shown in the virtual endoscopic image or a relative relation with other structures.\",\n \"In the conventional virtual endoscopic method, a virtual camera is controlled and expressed in a two-dimensional reference image.\",\n \"Accordingly, it is difficult for a user to operate the virtual camera in a desired direction and recognize the direction of the virtual camera.\"\n ],\n [\n \"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.\",\n \"1 is a schematic block diagram of an apparatus for displaying a three-dimensional virtual endoscopic image according to an embodiment of the present invention.\",\n \"FIG.\",\n \"1A is a block diagram of a two-dimensional reference image display control unit according to the embodiment of the present invention.\",\n \"FIG.\",\n \"1B is a block diagram of a three-dimensional reference image display control unit according to the embodiment of the present invention.\",\n \"FIG.\",\n \"1C is a block diagram of a virtual endoscopic image display control unit according to the embodiment of the present invention.\",\n \"FIG.\",\n \"2 is a flowchart of a method of displaying a three-dimensional virtual endoscopic image according to an embodiment of the present invention.\",\n \"FIG.\",\n \"2A is a flowchart of a procedure of displaying virtual cameras according to the embodiment of the present invention.\",\n \"FIG.\",\n \"2B is a flowchart of a procedure of changing a display area according to the embodiment of the present invention.\",\n \"FIGS.\",\n \"3A through 3E show examples of a method of displaying and operating a virtual camera according to the embodiment of the present invention.\",\n \"FIGS.\",\n \"4A through 4G show examples of a three-dimensional virtual endoscopic image according to the embodiment of the present invention.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"TECHNICAL FIELD The present invention relates to an apparatus and method for displaying a three-dimensional virtual endoscopic image, and more particularly, to an apparatus and method for displaying a three-dimensional virtual endoscopic image, through which a virtual endoscopic image is displayed along with two- and three-dimensional reference images, a virtual camera is intuitively displayed on the two- and three-dimensional reference images, and the images are controlled to be associated with one another.\",\n \"Generally, three-dimensional image processing systems are applied to various fields such as design and construction of structures, for example, cars and ships, having a three-dimensional shapes and medical imaging.\",\n \"In particular, applying three-dimensional image processing systems to the field of medical imaging is referred to as a “three-dimensional medical imaging”.\",\n \"Three-dimensional medical imaging is generating a three-dimensional image from a series of two-dimensional images obtained through computed tomography (CT) or magnetic resonance imaging (MRI).\",\n \"While it is difficult to feel the solidity of an entire object and is impossible to observe a cross-section of the object with only a series of two-dimensional images, three-dimensional medical imaging allows the position of an affected part to be accurately estimated and a surgical operation to be predicted similarly to the real thing.\",\n \"Accordingly, three-dimensional medical imaging has been widely used.\",\n \"BACKGROUND ART In order to avoid giving patients discomfort provoked by an endoscope used for observing the state of the patients' internal organs, virtual endoscopy has been developed and widely and usefully used with respect to a bronchus, a blood vessel, a large intestine, a joint, and so on in a human body.\",\n \"In virtual endoscopy, an image similar to a real endoscopic image is generated using a series of two-dimensional images collected through CT or MRI.\",\n \"In other words, a virtual endoscope is noninvasive and thus allows the inside of a patient's internal organs to be observed without giving the patient pain, so recently, it is very usefully used.\",\n \"A real endoscope allows the inside of internal organs to be observed only in a direction in which a camera moves.\",\n \"Unlikely, a virtual endoscope allows a user to observe the inside of internal organ in desired directions and is thus very helpful to diagnosis.\",\n \"However, a conventional virtual endoscope is difficult to operate due to inefficient user interface.\",\n \"For example, a conventional virtual endoscopic method is composed of a two-dimensional reference image acquired using an image input apparatus and a virtual endoscopic image generated from the two-dimensional reference image.\",\n \"Accordingly, in the conventional virtual endoscopic method, a correlation between the reference image and the virtual endoscopic image is two-dimensionally shown, so it is difficult to express a three-dimensional correlation and detect the position of a part shown in the virtual endoscopic image or a relative relation with other structures.\",\n \"In the conventional virtual endoscopic method, a virtual camera is controlled and expressed in a two-dimensional reference image.\",\n \"Accordingly, it is difficult for a user to operate the virtual camera in a desired direction and recognize the direction of the virtual camera.\",\n \"DISCLOSURE OF THE INVENTION The present invention provides an apparatus and method for displaying a three-dimensional virtual endoscopic image, through which a virtual endoscopic image is displayed along with two- and three-dimensional reference images in order to effectively show a correlation between the reference images and the virtual endoscopic image, and a virtual camera is intuitively displayed on the two- and three-dimensional reference images in order to facilitate the operation of the virtual camera.\",\n \"According to an aspect of the present invention, there is provided an apparatus for displaying a three-dimensional virtual endoscopic image.\",\n \"The apparatus includes a volume data input unit, which inputs information on a virtual endoscopic image in the form of volume data expressed as a function of three-dimensional position; a two-dimensional reference image display control unit, which derives a two-dimensional reference image from the volume data and displays the two-dimensional reference image; a three-dimensional reference image display control unit, which derives a three-dimensional reference image from the volume data using parallel volume rendering and displays the three-dimensional reference image; a virtual endoscopic image display control unit, which applies position and direction information of a virtual camera used for collecting virtual endoscopic image information to a predetermined perspective ray-casting algorithm, thereby generating image information, and displays a virtual endoscopic image based on the generated image information; a user interface unit, which receives a user's operating signal; and a controller, which when the operating signal for changing image information displayed by one among the two-dimensional reference image display control unit, the three-dimensional reference image display control unit, and the virtual endoscopic image display control unit is input through the user interface unit, controls the two- and three-dimensional reference image display control units and the virtual endoscopic image display control unit to be associated with one another based on the operating signal.\",\n \"According to another aspect of the present invention, there is provided a method of displaying a three-dimensional virtual endoscopic image.\",\n \"The method includes inputting information on a virtual endoscopic image in the form of volume data expressed as a function of three-dimensional position; detecting a two-dimensional reference image, a three-dimensional reference image, and a virtual endoscopic image from the volume data; displaying the two-dimensional reference image, the three-dimensional reference image, and the virtual endoscopic image on one screen; displaying virtual cameras on areas, respectively, in which the two-dimensional reference image and the three-dimensional reference image are respectively displayed, wherein a camera display sphere and a camera display circle are defined on the basis of a current position of each virtual camera; and when information regarding to one image among the two-dimensional reference image, the three-dimensional reference image, and the virtual endoscopic image, which are displayed on one screen, is changed by a user's operation, changing information regarding to the other images based on the information that has been changed by the user's operation.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is a schematic block diagram of an apparatus for displaying a three-dimensional virtual endoscopic image according to an embodiment of the present invention.\",\n \"FIG.\",\n \"1A is a block diagram of a two-dimensional reference image display control unit according to the embodiment of the present invention.\",\n \"FIG.\",\n \"1B is a block diagram of a three-dimensional reference image display control unit according to the embodiment of the present invention.\",\n \"FIG.\",\n \"1C is a block diagram of a virtual endoscopic image display control unit according to the embodiment of the present invention.\",\n \"FIG.\",\n \"2 is a flowchart of a method of displaying a three-dimensional virtual endoscopic image according to an embodiment of the present invention.\",\n \"FIG.\",\n \"2A is a flowchart of a procedure of displaying virtual cameras according to the embodiment of the present invention.\",\n \"FIG.\",\n \"2B is a flowchart of a procedure of changing a display area according to the embodiment of the present invention.\",\n \"FIGS.\",\n \"3A through 3E show examples of a method of displaying and operating a virtual camera according to the embodiment of the present invention.\",\n \"FIGS.\",\n \"4A through 4G show examples of a three-dimensional virtual endoscopic image according to the embodiment of the present invention.\",\n \"BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings.\",\n \"FIG.\",\n \"1 is a schematic block diagram of an apparatus for displaying a three-dimensional virtual endoscopic image according to an embodiment of the present invention.\",\n \"Referring to FIG.\",\n \"1, the apparatus for displaying a three-dimensional virtual endoscopic image includes volume data input unit 100, a two-dimensional reference image display control unit 200, a three-dimensional reference image display control unit 300, a virtual endoscopic image display control unit 400, a user interface unit 500, and a controller 600.The volume data input unit 100 inputs information on a virtual endoscopic image in the form of volume data expressed as a function of three-dimensional position.\",\n \"For example, in a case where volume data, which is generated as the result of computed tomography (CT) scan or magnetic resonance imaging (MRI), is directly received from each device and then stored, the volume data input unit 100 can be composed of connection hardware connected to the device in order to receive volume data and a hard disc or memory device storing the received volume data.\",\n \"Alternatively, in a case where data, which has already been measured and stored in other storage media (for example, a floppy disc and a CD-ROM), is read, the volume data input unit 100 can be composed of connection hardware connected to internal/external storage devices of systems for these storage media in order to receive the data and a hard disc or memory device storing the received data.\",\n \"The two-dimensional reference image display control unit 200 derives a two-dimensional reference image from the volume data and displays it.\",\n \"The three-dimensional reference image display control unit 300 derives a three-dimensional reference image from the volume data using parallel volume rendering and displays it.\",\n \"The virtual endoscopic image display control unit 400 applies the position and direction information of a virtual camera used for collecting virtual endoscopic image information to a predetermined perspective ray-casting algorithm, thereby generating image information, and displays a virtual endoscopic image based on the generated image information.\",\n \"When an operating signal for changing image information displayed by one among the two-dimensional reference image display control unit 200, the three-dimensional reference image display control unit 300, and the virtual endoscopic image display control unit 400 is input through the user interface unit 500, the controller 600 controls the three display control units 200, 300, and 400 to be associated with one another based on the operating signal.\",\n \"In other words, the controller 600 controls the two- and three-dimensional reference images and the virtual endoscopic image to be associated with one another.\",\n \"Referring to FIG.\",\n \"1A, the two-dimensional reference image display control unit 200 includes a control signal input section 210, an image information receiver 220, a painting section 230, a display area control section 240, a camera control section 250, a display format determining section 260, a two-dimensional image generator 270, an image information support section 280, and an image information display section 290.The two-dimensional reference image display control unit 200 is largely divided into an input unit, which receives a control signal for changing a two-dimensional reference image; a main processing unit, which generates changed information with respect to the two-dimensional reference image based on the control signal; and an output unit, which outputs the changed information.\",\n \"The input unit includes the control signal input section 210 and the image information receiver 220.The main processing unit includes the painting section 230, the display area control section 240, the camera control section 250, the display format determining section 260, and the two-dimensional image generator 270.The output unit includes the image information support section 280 and the image information display section 290.The image information support section 280 transmits the changed information to the other image display control units 300 and 400 so that the changed information regarding to the two-dimensional reference image can be applied to the other images, i.e., a three-dimensional reference image and a virtual endoscopic image.\",\n \"The image information display section 290 displays the two-dimensional reference image based on the changed information.\",\n \"The control signal input section 210 receives a control signal for controlling a two-dimensional reference image among the operating signal input through the user interface unit 500 shown in FIG.\",\n \"1.The image information receiver 220 receives changed three-dimensional reference image information from the three-dimensional reference image display control unit 300 shown in FIG.\",\n \"1 and changed virtual endoscopic image information from the virtual endoscopic image display control unit 400 shown in FIG.\",\n \"1.Upon receiving painting information regarding to a region of interest in the two-dimensional reference image through the control signal input section 210, the painting section 230 changes two-dimensional reference image information based on the painting information and then transmits the changed information to the image information support section 280 and the image information display section 290 so that the changed content can be applied to all of the two-dimensional reference image, the three-dimensional reference image, and the virtual endoscopic image.\",\n \"The display area control section 240 changes the two-dimensional reference image information based on position change information regarding to a region of interest in the two-dimensional reference image, which is received through the control signal input section 210, or position change information regarding to a region of interest in the three-dimensional reference image or the virtual endoscopic image, which is received through the image information receiver 220.For example, when a user changes a region of interest in a two-dimensional reference image through a user interface, such as a scroll bar appearing on a screen or a mouse wheel, changed information is applied to the two-dimensional reference image, and the corresponding regions of interest in both three-dimensional reference image and virtual endoscopic image are also changed based on information about the region of interest in the two-dimensional reference image.\",\n \"The camera control section 250 changes virtual camera display information with respect to the two-dimensional reference image based on virtual camera display information regarding to the two-dimensional reference image, which is received through the control signal input section 210, or virtual camera display information regarding to the three-dimensional reference image and the virtual endoscopic image, which is received through the image information receiver 220.The display format determining section 260 determines one among an axial image, a coronal image, a sagittal image, and a path MPR image as the format of the two-dimensional reference image based on display information with respect to the two-dimensional reference image, which is received through the control signal input section 210.The axial image is a two-dimensional image having the same z-value.\",\n \"The coronal image is a two-dimensional image having the same y-value.\",\n \"The sagittal image is a two-dimensional image having the same x-value.\",\n \"Upon receiving changed three-dimensional reference image information and changed virtual endoscopic image information from the image information receiver 220, the two-dimensional image generator 270 converts the received three-dimensional reference image and virtual endoscopic image information to two-dimensional image information so that the received image information is applied to the two-dimensional reference image.\",\n \"The image information display section 290 detects a changed two-dimensional reference image based on the two-dimensional reference image information received from the painting section 230, the display area control section 240, the camera control section 250, the display format determining section 260, and the two-dimensional image generator 270, and displays the changed two-dimensional reference image.\",\n \"When the two-dimensional reference image information is changed in the painting section 230, the display area control section 240, and the camera control section 250 in response to the control signals received from the control signal input section 210, the image information support section 280 transmits the changed two-dimensional reference image information to the three-dimensional reference image display control unit 300 and the virtual endoscopic image display control unit 400 so that the changed two-dimensional reference image information is applied to a three-dimensional reference image and a virtual endoscopic image.\",\n \"Referring to FIG.\",\n \"1B, the three-dimensional reference image display control unit 300 includes a control signal input section 310, an image information receiver 320, a painting section 330, a display area control section 340, a camera control section 350, a three-dimensional volume rendering section 360, an image information support section 370, and an image information display section 380.Like the two-dimensional reference image display control unit 200, the three-dimensional reference image display control unit 300 is largely divided into an input unit, which receives a control signal for changing a three-dimensional reference image; a main processing unit, which generates changed information with respect to the three-dimensional reference image based on the control signal; and an output unit, which outputs the changed information.\",\n \"The input unit includes the control signal input section 310 and the image information receiver 320.The main processing unit includes the painting section 330, the display area control section 340, the camera control section 350, and the three-dimensional volume rendering section 360.The output unit includes the image information support section 370 and the image information display section 380.The image information support section 370 transmits the changed information to the other image display control units 200 and 400 so that the changed information regarding to the three-dimensional reference image can be applied to the other images, i.e., a two-dimensional reference image and a virtual endoscopic image.\",\n \"The image information display section 380 displays a three-dimensional reference image based on the changed information.\",\n \"The control signal input section 310 receives a control signal for controlling a three-dimensional reference image among the operating signal input through the user interface unit 500 shown in FIG.\",\n \"1.The image information receiver 320 receives changed two-dimensional reference image information from the two-dimensional reference image display control unit 200 shown in FIG.\",\n \"1 and changed virtual endoscopic image information from the virtual endoscopic image display control unit 400 shown in FIG.\",\n \"1.Upon receiving painting information regarding to a region of interest in the three-dimensional reference image through the control signal input section 310, the painting section 330 changes three-dimensional reference image information based on the painting information and then transmits the changed information to the image information support section 370 and the image information display section 380 so that the changed content can be applied to all of the three-dimensional reference image, the two-dimensional reference image, and the virtual endoscopic image.\",\n \"The display area control section 340 changes the three-dimensional reference image information based on position change information regarding to a region of interest in the three-dimensional reference image, which is received through the control signal input section 310, or position change information regarding to a region of interest in the two-dimensional reference image or the virtual endoscopic image, which is received through the image information receiver 320.For example, a new two-dimensional reference image display straight line can be displayed on the three-dimensional reference image, and a slice locator can be directly dragged on the three-dimensional reference image to change the position of a two-dimensional reference image.\",\n \"The camera control section 350 changes virtual camera display information with respect to the three-dimensional reference image based on virtual camera display information regarding to the three-dimensional reference image, which is received through the control signal input section 310, or virtual camera display information regarding to the two-dimensional reference image and the virtual endoscopic image, which is received through the image information receiver 320.Upon receiving changed two-dimensional reference image information and changed virtual endoscopic image information from the image information receiver 320, the three-dimensional volume rendering section 360 performs three-dimensional volume rendering based on the received image information.\",\n \"For example, the three-dimensional volume rendering section 360 receives the result of performing painting operation on a virtual endoscopic image and the result of performing painting operation on a two-dimensional reference image from the image information receiver 320 and performs three-dimensional rendering.\",\n \"Three-dimensional rendering is a process of adding three-dimensional texture such as shadow or a change in color or depth of color to an object, thereby giving reality to computer graphics.\",\n \"The image information display section 380 detects a changed three-dimensional reference image based on the three-dimensional reference image information received from the painting section 330, the display area control section 340, the camera control section 350, and the three-dimensional volume rendering section 360, and displays the changed three-dimensional reference image.\",\n \"When the three-dimensional reference image information is changed in the painting section 330, the display area control section 340, and the camera control section 350 in response to the control signals received from the control signal input section 310, the image information support section 370 transmits the changed three-dimensional reference image information to the two-dimensional reference image display control unit 200 and the virtual endoscopic image display control unit 400 so that the changed three-dimensional reference image information is applied to a two-dimensional reference image and a virtual endoscopic image.\",\n \"Referring to FIG.\",\n \"1C, the virtual endoscopic image display control unit 400 includes a control signal input section 410, an image information receiver 420, a painting section 430, a perspective ray-casting section 440, a display area control section 450, a camera control section 460, a focal zone generator 470, an image information support section 480, and an image information display section 490.Like the two-dimensional reference image display control unit 200 and the three-dimensional reference image display control unit 300, the virtual endoscopic image display control unit 400 is largely divided into an input unit, which receives a control signal for changing a virtual endoscopic image; a main processing unit, which generates changed information with respect to the virtual endoscopic image based on the control signal; and an output unit, which outputs the changed information.\",\n \"The input unit includes the control signal input section 410 and the image information receiver 420.The main processing unit includes the painting section 430, the perspective ray-casting section 440, the display area control section 450, the camera control section 460, and the focal zone generator 470.The output unit includes the image information support section 480 and the image information display section 490.The image information support section 480 transmits the changed information to the other image display control units 200 and 300 so that the changed information regarding to the virtual endoscopic image can be applied to the other images, i.e., a two-dimensional reference image and a three-dimensional reference image.\",\n \"The image information display section 490 displays the virtual endoscopic image based on the changed information.\",\n \"The control signal input section 410 receives a control signal for controlling a virtual endoscopic image among the operating signal input through the user interface unit 500 shown in FIG.\",\n \"1.The image information receiver 420 receives changed two-dimensional reference image information from the two-dimensional reference image display control unit 200 shown in FIG.\",\n \"1 and changed three-dimensional reference image information from the three-dimensional reference image display control unit 300 shown in FIG.\",\n \"1.Upon receiving painting information regarding to a region of interest in the virtual endoscopic image through the control signal input section 410, the painting section 430 changes virtual endoscopic image information based on the painting information and then transmits the changed information to the image information support section 480 so that the changed content can be applied to the two-dimensional reference image and the three-dimensional reference image.\",\n \"The painting section 430 also transmits the changed information to the perspective ray-casting section 440 so that the changed content can be applied to the virtual endoscopic image.\",\n \"The perspective ray-casting section 440 applies the processing result received from the painting section 430 and the changed two- and three-dimensional reference image information received from the image information receiver 420 to a predetermined perspective ray-casting algorithm so as to adjust the opacity of a region of interest, and then changes the virtual endoscopic image information based on the result of the adjustment.\",\n \"The perspective ray-casting algorithm is a process of accumulating brightness of points, where light beams diverging in different directions from the same start point meet an object, on a projection plane so as to generate an image.\",\n \"Since it is widely known, a detailed description thereof will be omitted.\",\n \"The display area control section 450 changes the virtual endoscopic image information based on position change information regarding to a region of interest in the two- or three-dimensional reference image, which is received through the image information receiver 420.In other words, when changed information regarding to the region of interest is received from the two-dimensional reference image display control unit 200 or the three-dimensional reference image display control unit 300, the display area control section 450 renders the two- or three-dimensional reference image in the region of interest on the virtual endoscopic image.\",\n \"For example, in a case where a two-dimensional axial image is rendered on the virtual endoscopic image, when a plurality of light beams diverging from one point meet the surface of an object, if the z-value of a voxel corresponding to the surface is the same as the z-value of the two-dimensional axial image, the display area control section 450 changes the color of the voxel.\",\n \"The camera control section 460 changes the virtual endoscopic image information based on the position and direction information of a virtual camera, which is received through the control signal input section 410, and displays the result of the change through the image information display section 490 or applies it to the other images through the image information support section 480.The focal zone generator 470 designates a region of a user's interest on a virtual endoscopic image based on the control signal transmitted through the control signal input section 410.In other words, if a user selects a region of interest on a virtual endoscopic image screen, the focal zone generator 470 defines the region of interest as a focal zone and transmits the result of the definition to the image information display section 490 or the image information support section 480 so that the result of the definition is displayed or applied to two- and three-dimensional reference images.\",\n \"Accordingly, a correlation between each reference image and the virtual endoscopic image can be effectively shown.\",\n \"The focal zone is a region defined on a screen and does not change when a new virtual endoscopic image is generated due to a change in the position or direction information of the virtual camera.\",\n \"Volume rendering is performed to generate a three-dimensional reference image so that the region of volume data included in the focal zone defined on the virtual endoscopic image screen is distinguished from the remaining region.\",\n \"For example, if the focal zone is a quadrilateral, four planes defined by Formula (1) are generated in three-dimensional space.\",\n \"f1(x)=a1x+b1y+c1y+d1=0 f2(x)=a2x+b2y+c2y+d2=0 f3(x)=a3x+b3y+c3y+d3=0 f4(x)=a4x+b4y+c4y+d4=0 (1) Here, if the region of volume data is included in the focal zone, Formula (2) is satisfied.\",\n \"f1(x)<0, f2(x)<0, f1(x)<0, f4(x)<0 (2) The focal zone can be determined with respect to other primitives, using a similar method to the above-described one.\",\n \"The image information display section 490 detects a changed virtual endoscopic image based on the virtual endoscopic image information received from the perspective ray-casting section 440, the display area control section 450, the camera control section 460, and the focal zone generator 470, and displays the changed virtual endoscopic image.\",\n \"When the virtual endoscopic image information is changed in the painting section 430, the perspective ray-casting section 440, the camera control section 460, and the focal zone generator 470 in response to the control signal received from the control signal input section 410, the image information support section 480 transmits the changed virtual endoscopic image information to the two-dimensional reference image display control unit 200 and the three-dimensional reference image display control unit 300 so that the changed virtual endoscopic image information is applied to a two-dimensional reference image and a three-dimensional reference image.\",\n \"Referring to FIGS.\",\n \"1A through 1C, the image display control units 200, 300, and 400 receive changed information regarding to different images than their own from the other image display control units using the respective image information receivers 220, 320, and 420 and transmit their own changed image information to the other image display control units using the respective image information support sections 280, 370, and 480.Accordingly, all of the different images are associated with one another.\",\n \"A method of displaying a three-dimensional virtual endoscopic image according to an embodiment of the present invention will be described below with reference to FIG.\",\n \"2.Information on a virtual endoscopic image is input in the form of volume data expressed in a three-dimensional position function in step s100.Here, the volume data is generated through CT scan or MRI.\",\n \"A two-dimensional reference image, a three-dimensional reference image, and a virtual endoscopic image are detected from the volume data in step s200.Two-dimensional image generation is performed in order to detect the two-dimensional reference image from the volume data.\",\n \"Parallel volume rendering is performed in order to detect the three-dimensional reference image from the volume data.\",\n \"The position and direction information of a virtual camera is applied to a predetermined perspective ray-casting algorithm in order to detect the virtual endoscopic image from the volume data.\",\n \"Thereafter, the two-dimensional reference image, the three-dimensional reference image, and the virtual endoscopic image are displayed on one screen in step s300.In other words, a display area on the screen is divided into three sub areas, and the three images are displayed on the respective three sub areas.\",\n \"It is preferable that a sub area for displaying the virtual endoscopic image is largest and is located at the center of the display area.\",\n \"Virtual cameras are displayed on the respective sub areas, in which the two- and three-dimensional reference images are displayed, in step s400.With respect to each of the virtual cameras, a camera display sphere and a camera display circle are provided centering around the current position of each virtual camera.\",\n \"For example, in a case of a two-dimensional reference image, a sphere having a predetermined radius from the current position of a virtual camera is displayed as a camera display sphere, and a circle composed of intersection points between the camera display sphere and the current two-dimensional reference image is displayed as a camera display circle.\",\n \"In a case of a three-dimensional reference image, a sphere having a predetermined radius from the current position of a virtual camera is displayed as a camera display sphere, and a circle composed of intersection points between a plane, which passes the current position of the virtual camera displayed on the three-dimensional reference image and parallel with the screen, and the camera display sphere is displayed as a camera display circle.\",\n \"The direction of a virtual camera is represented with an arrow.\",\n \"On the assumption that there is an infinite ray in the direction of the virtual camera, an intersection point between the infinite ray and a camera display sphere is detected, and then the direction of the virtual camera is represented with an arrow starting from the center of the virtual camera and passing the intersection point.\",\n \"In order to identify whether the virtual camera faces the surface of the hemisphere in front or the rear of an image, different colors are used for the camera display circle and the virtual camera.\",\n \"For example, when the virtual camera faces the surface of the hemisphere in front of the image, the camera display circle and the virtual camera are colored in red.\",\n \"Conversely, when the virtual camera faces the surface of the hemisphere in the rear of the image, the camera display circle and the virtual camera are colored in blue.\",\n \"Examples of a method of displaying a virtual camera will be described later more specifically with reference to FIGS.\",\n \"3A through 3E.\",\n \"In the meantime, when one of the images displayed on the screen is changed by a user's operation, the other images are changed based on changed image information in association with one another in step s500.FIG.\",\n \"2A is a flowchart of a procedure of displaying a virtual camera according to the embodiment of the present invention.\",\n \"Referring to FIG.\",\n \"2A, the virtual cameras are displayed at predetermined positions on the respective two- and three-dimensional reference images in step s401.If the position and direction of the virtual camera are changed on the virtual endoscopic image in step s402, the changed position and direction information is applied to the predetermined perspective ray-casting algorithm to detect a new virtual endoscopic image and the new virtual endoscopic image is displayed in step s403.The positions and directions of the virtual cameras displayed on the respective two- and three-dimensional reference images are changed based on the changed position and direction information regarding to the virtual camera on the virtual endoscopic image in step s404.If the position and direction of the virtual camera are changed on the two-dimensional reference image in step s405, the changed position and direction information is applied to the predetermined perspective ray-casting algorithm to detect a new virtual endoscopic image and the new virtual endoscopic image is displayed in step s406.The position and direction of the virtual camera displayed on the three-dimensional reference image are changed based on the changed position and direction information regarding to the virtual camera on the two-dimensional reference image in step s407.A new three-dimensional reference image is detected based on the changed position and direction information regarding to the old three-dimensional reference image in step s408.If the position and direction of the virtual camera are changed on the three-dimensional reference image in step s409, the changed position and direction information is applied to the predetermined perspective ray-casting algorithm to detect a new virtual endoscopic image and the new virtual endoscopic image is displayed in step s410.The position and direction of the virtual camera displayed on the two-dimensional reference image are changed based on the changed position and direction information regarding to the virtual camera on the three-dimensional reference image in step s411.A new two-dimensional reference image is detected based on the changed position and direction information regarding to the old two-dimensional reference image in step s412.FIG.\",\n \"2B is a flowchart of a procedure of changing an image in a display area according to the embodiment of the present invention.\",\n \"Referring to FIG.\",\n \"2B, when one among the two-dimensional reference image, the three-dimensional reference image, and the virtual endoscopic image, which are displayed on the same screen, is changed, the following operation is performed in order to change the other images in association with one another.\",\n \"If information regarding to the two-dimensional reference image is changed in step s501, the changed information is applied to the two-dimensional reference image in step s502.Next, the changed information is applied to the virtual endoscopic image using the predetermined perspective ray-casting algorithm in step s503 and is applied to the three-dimensional reference image using three-dimensional volume rendering in step s504.If information regarding to the three-dimensional reference image is changed in step s505, the changed information is applied to the three-dimensional reference image in step s506.Next, the changed information is applied to the virtual endoscopic image using the predetermined perspective ray-casting algorithm in step s507 and is applied to the two-dimensional reference image using two-dimensional image generation in step s508.If information regarding to the virtual endoscopic image is changed by the user's operation in step s509, the changed information is applied to the virtual endoscopic image using the predetermined perspective ray-casting algorithm in step s510.Thereafter, the changed information is applied to the two-dimensional reference image using two-dimensional image generation in step s511 and is applied to the three-dimensional reference image using three-dimensional volume rendering in step s512.FIGS.\",\n \"3A through 3E show examples of a method of displaying and operating a virtual camera according to the embodiment of the present invention.\",\n \"FIG.\",\n \"3A shows an example in which a virtual camera is displayed on a two-dimensional reference image.\",\n \"Referring to FIG.\",\n \"3A, with respect to a two-dimensional reference image 10, a sphere (hereinafter, a camera display sphere) 12 having a predetermined radius from the current position of a virtual camera 11 is assumed.\",\n \"A set of intersection points between the camera display sphere 12 and the current two-dimensional reference image 10 is defined as a circle (hereinafter, referred to as a camera display circle) 13.Here, if it is assumed that an infinite ray exists in the direction of the virtual camera 11, the infinite ray meets the camera display sphere 12 at one point.\",\n \"An arrow 14 starting from the center of the virtual camera 11 and passing the intersection point between the camera display sphere 12 and the infinite ray indicates the direction of the virtual camera 11.In a coordinate system having the center of the virtual camera 11 as an origin, the infinite ray with respect to the direction d(dx,dy,dz) of the virtual camera 11 is expressed by Formula (3).\",\n \"x=dxk, y=dyk, z=dzk (where k is a constant) (3) In the meantime, the camera display sphere 12 is expressed by Formula (4), and thus the coordinates of the intersection point between the infinite ray and the camera display sphere 12 are given by Formula (5) based on Formulae (3) and (4), x2+y2+z2=r2 (4) x = d x k _ , y = d y k _ , z = d z k _ ( k _ = r 2 d x 2 + d y 2 + d z 2 ) ( 5 ) Here, the virtual camera 11 may face the surface of a hemisphere in front or the rear of the two-dimensional reference image 10.In order to identify whether the virtual camera 11 faces the surface of the hemisphere in front or the rear of the two-dimensional reference image 10, different colors are used for the camera display circle 13 and the virtual camera 11.For example, the direction of the virtual camera 11 is the same in the plane views of FIGS.\",\n \"3A and 3B.\",\n \"However, the virtual camera 11 in FIG.\",\n \"3A faces the surface of the hemisphere in front of the two-dimensional reference image 10 while the virtual camera 11 in FIG.\",\n \"3B faces the surface of the hemisphere in the rear of the two-dimensional reference image 10.Accordingly, the color of the camera display circle 13 and the virtual camera 11 shown in FIG.\",\n \"3A is different from the color of the camera display circle 13 and the virtual camera 11 shown in FIG.\",\n \"3B.\",\n \"For example, the camera display circle 13 and the virtual camera 11 are colored in red in FIG.\",\n \"3A and colored in blue in FIG.\",\n \"3B so that the front and the rear of the two-dimensional reference image 10 can be discriminated from each other.\",\n \"FIG.\",\n \"3C shows an example, in which a virtual camera is displayed on a three-dimensional reference image.\",\n \"Referring to FIG.\",\n \"3C, in order to display a virtual camera 21 on a three-dimensional reference image, a sphere having a predetermined radius from the current position of a virtual camera 21 is defined as a camera display sphere 22, and a set of the intersection points between the camera display sphere 22 and a plane 20, which passes the current position of the virtual camera 21 and is parallel with a screen displaying the three-dimensional reference image, is defined as a camera display circle 23.Here, if it is assumed that an infinite ray exists in the direction of the virtual camera 21, the infinite ray intersects the camera display sphere 22 at one point.\",\n \"An arrow 24 starting from the center of the virtual camera 21 and passing the intersection point between the camera display sphere 22 and the infinite ray indicates the direction of the virtual camera 21.In order to discriminate the direction of the virtual camera 21 facing the surface of a hemisphere in front of the plane 20 from the direction of the virtual camera 21 facing the surface of a hemisphere in the rear of the plane 20, different colors are used for the camera display circle 23 and the virtual camera 21.FIGS.\",\n \"3D and 3E are diagrams for explaining a method of operating a virtual camera on a two-dimensional reference image, according to the present invention.\",\n \"When the virtual camera 11 faces the surface of the hemisphere in front of the two-dimensional reference image 10 as shown in FIG.\",\n \"3D(a), if a user clicks a predetermined position 41 within the camera display circle 13, an intersection point between the hemisphere and an infinite ray, which passes the predetermined position 41 and goes toward the front of the two-dimensional reference image 10, is calculated and the virtual camera 11 is adjusted to face the intersection point, as shown in FIG.\",\n \"3D(b).\",\n \"In a coordinate system having the center of the virtual camera 11 as an origin, the infinite ray, with respect to the direction d(dx,dy,dz) of the virtual camera 11 and the predetermined position 41 p(px,pypz), is expressed by Formula (6).\",\n \"x=px+dxk, y=py+dyk, z=pz+dzk (where k is a constant) (6) In the meantime, the camera display sphere 12 is expressed by Formula (4), and thus the coordinates of the intersection point between the infinite ray and the camera display sphere 12 are given by Formula (7) based on Formulae (6) and (4).\",\n \"x=px+dxk, y=py+dy{overscore (k)}, z=pz+dz{overscore (k)}({overscore (k)} satisfies (d2x+d2y+d2z){overscore (k)}2+2(pxdx+pydy+pzdz) {overscore (k)}+p2x+p2y+p2z−r2=0) (7) In the meantime, when the virtual camera 11 faces the surface of the hemisphere in front of the two-dimensional reference image 10 as shown in FIG.\",\n \"3E(a), if a user clicks a predetermined position 42 on the camera display circle 13, the direction of the virtual camera 11 is converted to face the surface of the hemisphere in the rear of the two-dimensional reference image 10.In other words, in the case where the virtual camera 11 is colored in blue because it faces the front hemisphere, if one point on the camera display circle is clicked, the direction of the virtual camera 11 is symmetrically changed with respect to the two-dimensional reference image 10 so that the virtual camera 11 faces the rear hemisphere, as shown in FIG.\",\n \"3E(b).\",\n \"FIGS.\",\n \"4A through 4G show examples of a three-dimensional virtual endoscopic image according to the embodiment of the present invention.\",\n \"FIG.\",\n \"4A shows a user interface of a virtual endoscopy system composed of a two-dimensional reference image, a three-dimensional reference image, and a virtual endoscopic image.\",\n \"Referring to FIG.\",\n \"4A, in the virtual endoscopy system, the three-dimensional reference image is displayed on the right, the virtual endoscopic image is displayed on the upper right, and the two-dimensional reference image is displayed on the lower right.\",\n \"A two-dimensional reference image display line 51 is displayed on the three-dimensional reference image in order to indicate the position of an axial image, i.e., the current two-dimensional reference image.\",\n \"Although the axial image is shown as the two-dimensional reference image, a user can view the two-dimensional reference image in a desired format using buttons, i.e., Axial, Coronal, Sagittal, and Path MPR, in an image title bar.\",\n \"FIG.\",\n \"4B shows an example of a sagittal image to which the axial image shown in FIG.\",\n \"4A is converted by pressing the Sagittal button.\",\n \"FIGS.\",\n \"4C and 4D show the results of adjusting the position of the two-dimensional reference image shown in FIG.\",\n \"4A.\",\n \"FIG.\",\n \"4C shows the result of moving a scroll bar 52 located at the lower end of the two-dimensional reference image, and FIG.\",\n \"4D shows the result of moving the two-dimensional reference image display line 51 displayed on the three-dimensional reference image using a mouse.\",\n \"FIG.\",\n \"4E shows an example of the case where a virtual camera positioned in a particular region of a bronchus is displayed on two- and three-dimensional reference image areas.\",\n \"Referring to FIG.\",\n \"4E, virtual cameras 53 and 54 are displayed on the respective two- and three-dimensional reference images.\",\n \"Here, in order to identify whether the virtual camera 53 on the two-dimensional reference image faces above or below the two-dimensional reference image, different colors are used for the virtual camera 53.For example, in the case where the virtual camera 53 facing above the two-dimensional reference image is set to red color while the virtual camera 53 facing below the two-dimensional reference image is set to blue color, if the virtual camera 53 is colored in red on the two-dimensional reference image, it can be inferred that the virtual camera 53 faces above the two-dimensional reference image.\",\n \"Like the case of the virtual camera 53 on the two-dimensional reference image, it is identified whether the virtual camera 54 on the three-dimensional reference image faces the front or rear of the three-dimensional reference image using the color of the virtual camera 54.For example, in the case where the virtual camera 54 facing the front of the three-dimensional reference image is set to red color while the virtual camera 54 facing the rear of the three-dimensional reference image is set to blue color, if the virtual camera 54 is colored in blue on the three-dimensional reference image, it can be inferred that the virtual camera 54 faces the rear of the three-dimensional reference image.\",\n \"FIG.\",\n \"4F shows the result of adjusting the direction of the virtual camera 53 on the two-dimensional reference image shown in FIG.\",\n \"4E.\",\n \"In other words, FIG.\",\n \"4F shows the result of changing the direction of the virtual camera 53 on the two-dimensional reference image by clicking a point within a camera display circle on the two-dimensional reference image shown in FIG.\",\n \"4E.\",\n \"Accordingly, if the virtual camera 53 is colored in red before its direction is changed, it is colored in blue after its direction is changed.\",\n \"FIG.\",\n \"4G shows the result of adjusting the direction of the virtual camera 54 on the three-dimensional reference image shown in FIG.\",\n \"4E.\",\n \"In other words, FIG.\",\n \"4G shows the result of changing the direction of the virtual camera 54 on the three-dimensional reference image by clicking a point within a camera display circle on the three-dimensional reference image shown in FIG.\",\n \"4E.\",\n \"Here, the direction of the virtual camera 54 is symmetrically changed with respect to the three-dimensional reference image.\",\n \"Accordingly, if the virtual camera 54 is colored in blue before its direction is changed, it is colored in red after its direction is changed.\",\n \"The above description just concerns embodiments of the present invention.\",\n \"The present invention is not restricted to the above embodiments, and various modifications can be made thereto within the scope defined by the attached claims.\",\n \"For example, the shape and structure of each member specified in the embodiments can be changed without departing from the spirit of the invention.\",\n \"Industrial Applicability As described above, in an apparatus and method for displaying a three-dimensional virtual endoscopic image according to the present invention, a virtual endoscopic image is displayed along with two- and three-dimensional reference images, and these images are associated with one another.\",\n \"Accordingly, correlations among the reference images and the virtual endoscopic image can be effectively presented.\",\n \"In addition, since a virtual camera is intuitively displayed on each of the two- and three-dimensional reference images, and the virtual camera can be operated using a normal user interface such as a mouse, the virtual camera is easily operated.\",\n \"Therefore, by using the present invention, a virtual endoscopy system can be improved to integrate various images and techniques into a synthetic system.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471228"}}},{"rowIdx":583,"cells":{"text":{"kind":"list like","value":[["Modulation of gene expression associated with inflammation proliferation and neurite outgrowth using nucleic acid based technologies","The present invention relates to nucleic acid molecules, including antisense, enzymatic nucleic acid molecules, and RNA interference molecules, such as hammerhead ribozymes, DNAzymes, allozymes, siRNA, decoys and antisense, which modulate the expression of prostaglandin D2 (PTGDS), prostaglandin D2 receptor (PTGDR), adenosine receptor, NOGO and NOGO receptor, and IKK genes, such as IKK-gamma, IKK-alpha, or IKK-beta, and PKR genes."],["1.A nucleic acid molecule that down regulates expression or inhibits function of a receptor for a neurite growth inhibitor.","2.A nucleic acid molecule of claim 1, wherein the receptor is a NOGO receptor.","3.The nucleic acid of claim 1, wherein said nucleic acid molecule is adapted for use to treat conditions selected from the group consisting of CNS injury, spinal cord injury, and cerebrovascular accident.","4.The nucleic acid molecule of claim 1 or claim 2, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule having at least one binding arm.","5.The nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule has an endonuclease activity to cleave RNA encoded by a NOGO receptor gene.","6.The nucleic acid of claim 4, wherein the at least one binding arm of the enzymatic nucleic acid molecule comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.","1-1023.7.An enzymatic nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs.","5484-7055.8.The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is an antisense nucleic acid molecule.","9.An antisense nucleic acid molecule comprising a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.","1-1023.10.The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is in a hammerhead (HH) motif.","11.The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is in a hairpin, hepatitis Delta virus, group I intron, VS nucleic acid, amberzyme, zinzyme or RNAse P nucleic acid motif.","12.The enzymatic nucleic acid molecule of claim 11, wherein said zinzyme motif comprises a sequence selected from the group consisting of SEQ ID NOs.","6030-6272.13.The enzymatic nucleic acid molecule of claim 11, wherein said amberzyme motif comprises a sequence selected from the group consisting of SEQ ID NOs.","6630-7055.14.The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is in a NCH motif.","15.The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is in a G-cleaver motif.","16.The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is a DNAzyme.","17.The nucleic acid molecule of claim 2, wherein said nucleic acid molecule comprises between 12 and 100 bases complementary to RNA encoded by a NOGO receptor gene.","18.The nucleic acid molecule of claim 2, wherein said nucleic acid molecule comprises between 14 and 24 bases complementary to the RNA encoded by a NOGO receptor gene.","19.The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is chemically synthesized.","20.The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one 2′-sugar modification.","21.The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one nucleic acid base modification.","22.The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one phosphate backbone modification.","23.A mammalian cell comprising the nucleic acid molecule of claim 1.24.The mammalian cell of claim 23, wherein said mammalian cell is a human cell.","25.A method of reducing NOGO receptor activity in a cell, comprising the step of contacting said cell with the nucleic acid molecule of claim 2, under conditions suitable for said inhibition.","26.A method of treatment of a patient having a condition associated with levels of a NOGO receptor, comprising contacting cells of said patient with the nucleic acid molecule of claim 2, under conditions suitable for said treatment.","27.The method of claim 26 further comprising the use of one or more drug therapies under conditions suitable for said treatment.","28.A method of cleaving RNA encoded by a NOGO receptor gene comprising contacting the nucleic acid molecule of claim 2 with said RNA under conditions suitable for the cleavage of said RNA.","29.The method of claim 28, wherein said cleavage is carried out in the presence of a divalent cation.","30.The method of claim 29, wherein said divalent cation is Mg2+.","31.The nucleic acid molecule of claim 1, wherein said nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, 3′-end, or both the 5′-end and the 3′-end.","32.The enzymatic nucleic acid molecule of claim 10, wherein said hammerhead motif comprises a sequence selected from the group consisting of SEQ ID NOs.","5484-5583.33.The enzymatic nucleic acid molecule of claim 14, wherein said NCH motif comprises a sequence selected from the group consisting of SEQ ID NOs.","5584-6029.34.The enzymatic nucleic acid molecule of claim 16, wherein said DNAzyme comprises a sequence selected from the group consisting of SEQ ID NOs.","6273-6629.35.The method of claim 25, wherein said nucleic acid molecule is in a hammerhead motif.","36.The method of claim 25, wherein said nucleic acid molecule is a DNAzyme.","37.An expression vector comprising at least one nucleic acid molecule of claim 1 in a manner that allows expression of the nucleic acid molecule.","38.A mammalian cell comprising an expression vector of claim 37.39.The mammalian cell of claim 38, wherein said mammalian cell is a human cell.","40.The expression vector of claim 37, wherein said expression vector encodes a nucleic acid molecule having a hammerhead motif.","41.The expression vector of claim 37, wherein said expression vector further comprises a sequence for an antisense nucleic acid molecule complementary to RNA encoded by a NOGO receptor gene.","42.The expression vector of claim 37, wherein said expression vector comprises a two or more of said nucleic acid molecules, which can be the same or different.","43.The expression vector of claim 42, wherein said expression vector comprises a sequence encoding an antisense nucleic acid molecule complementary to RNA encoded by a NOGO receptor gene.","44.A method for treatment of conditions selected from the group consisting of CNS injury and cerebrovascular accident comprising the step of administering to a patient the nucleic acid molecule of claim 1 under conditions suitable for said treatment.","45.The method of claim 44, wherein said treatment of CNS injury is treatment of spinal cord injury.","46.A method for treatment of conditions selected from the group consisting of CNS injury and cerebrovascular accident comprising the step of administering to a patient the antisense nucleic acid molecule of claim 9 under conditions suitable for said treatment.","47.The method of claim 44, wherein said nucleic acid molecule is in a hammerhead motif.","48.The method of claim 44, wherein said method further comprises administering to said patient one or more other therapies.","49.The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification.","50.The nucleic acid molecule of claim 49, wherein said nucleic acid molecule further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.","51.The nucleic acid molecule of claim 49, wherein said 3′-end modification is 3′-3′ inverted abasic moiety.","52.The enzymatic nucleic acid molecule of claim 16, wherein said DNAzyme comprises at least ten 2′-O-methyl modifications and a 3′-end modification.","53.The enzymatic nucleic acid molecule of claim 52, wherein said DNAzyme further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.","54.The enzymatic nucleic acid molecule of claim 52, wherein said 3′-end modification is 3′-3′ inverted abasic moiety.","55.An enzymatic nucleic acid molecule that down regulates expression of a nucleic acid molecule encoding an IkappaB kinase (IKK) subunit.","56.An enzymatic nucleic acid molecule that down regulates expression of a nucleic acid molecule encoding protein kinase PKR.","57.An enzymatic nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs.","7056-11665.58.An enzymatic nucleic acid molecule comprising at least one binding arm wherein one or more of said binding arms comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.","1024-4414.59.An antisense nucleic acid molecule comprising a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.","1024-4414.60.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule is adapted to treat cancer.","61.The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule is adapted to treat cancer.","62.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule has an endonuclease activity to cleave RNA encoded by a IKK-gamma gene or PKR gene.","63.The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in an Inozyme configuration.","64.The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.","65.The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in a G-cleaver configuration.","66.The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in an Amberzyme configuration.","67.The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in a DNAzyme configuration.","68.The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in a Hammerhead configuration.","69.The enzymatic nucleic acid molecule of claim 63, wherein said Inozyme comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.","1218-1721 and 3051-3549.70.The enzymatic nucleic acid molecule of claim 63, wherein said Inozyme comprises a sequence selected from the group consisting of SEQ ID NOs.","7250-7753 and 9701-10199.71.The enzymatic nucleic acid molecule of claim 64, wherein said Zinzyme comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.","1722-1998 and 3550-3768.72.The enzymatic nucleic acid molecule of claim 64, wherein said Zinzyme comprises a sequence selected from the group consisting of SEQ ID NOs 7754-8030 and 10200-10418.73.The enzymatic nucleic acid molecule of claim 66, wherein said Amberzyme comprises a sequence selected from the group consisting of SEQ ID NOs 8441-9069 and 11001-11547.74.The enzymatic nucleic acid molecule of claim 67, wherein said DNAzyme comprises a sequence selected from the group consisting of SEQ ID NOs 8031-8440 and 10419-11000.75.The enzymatic nucleic acid molecule of claim 68, wherein said Hammerhead comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.","1024-1217 and 2420-3050.76.The enzymatic nucleic acid molecule of claim 68, wherein said Hammerhead comprises a sequence selected from the group consisting of SEQ ID NOs 7056-7249 and 9070-9700.77.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises between 12 and 100 bases complementary to RNA encoded by an IKK-gamma gene or PKR gene.","78.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises between 14 and 24 bases complementary to RNA encoded by an IKK-gamma gene or PKR gene.","79.The enzymatic nucleic acid molecule of any of claims 55-58 wherein said enzymatic nucleic acid molecule is chemically synthesized.","80.The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule is chemically synthesized.","81.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises at least one 2′-sugar modification.","82.The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule comprises at least one 2′-sugar modification.","83.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises at least one nucleic acid base modification.","84.The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule comprises at least one nucleic acid base modification.","85.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises at least one phosphate backbone modification.","86.The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule comprises at least one phosphate backbone modification.","87.A mammalian cell including the enzymatic nucleic acid molecule of any of claims 55-58.88.The mammalian cell of claim 87, wherein said mammalian cell is a human cell.","89.A method of down-regulating PKR activity in a cell, comprising contacting said cell with the enzymatic nucleic acid molecule of claim 56, under conditions suitable for down-regulating of PKR activity.","90.A method of treatment of a patient having a condition associated with the level of PKR, comprising contacting cells of said patient with the enzymatic nucleic acid molecule of any of claims 55-59, under conditions suitable for said treatment.","91.A method of down-regulating IKK-gamma activity in a cell, comprising contacting said cell with the enzymatic nucleic acid molecule of any of claims 55-59, under conditions suitable for down-regulating of IKK-gamma activity.","92.A method of treatment of a patient having a condition associated with the level of IKK-gamma, comprising contacting cells of said patient with the enzymatic nucleic acid molecule of any of claims 55-59, under conditions suitable for said treatment.","93.The method of claim 89 further comprising the use of one or more drug therapies under conditions suitable for said treatment.","94.The method of claim 90 further comprising the use of one or more drug therapies under conditions suitable for said treatment.","95.The method of claim 91 further comprising the use of one or more drug therapies under conditions suitable for said treatment.","96.The method of claim 92 further comprising the use of one or more drug therapies under conditions suitable for said treatment.","97.A method of cleaving RNA encoded by a PKR gene comprising contacting an enzymatic nucleic acid molecule of claim 56 with said RNA under conditions suitable for the cleavage.","98.A method of cleaving RNA encoded by an IKK-gamma gene comprising contacting an enzymatic nucleic acid molecule of claim 55 with said RNA under conditions suitable for the cleavage.","99.The method of claim 98, wherein said cleavage is carried out in the presence of a divalent cation.","100.The method of claim 99, wherein said cleavage is carried out in the presence of a divalent cation.","101.The method of claim 100, wherein said divalent cation is Mg2+.","102.The method of claim 101, wherein said divalent cation is Mg2+.","103.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, the 3′-end, or both the 5′-end and the 3′-end.","104.The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, the 3′-end, or both the 5′-end and the 3′-end.","105.The enzymatic nucleic acid molecule of claim 103, wherein the cap structure at the 5′-end, 3′-end, or both the 5′-end and the 3′-end comprises a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative.","106.The antisense nucleic acid molecule of claim 104, wherein the cap structure at the 5′-end, 3′-end, or both the 5′-end and the 3′-end comprises a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative.","107.The method of claim 89, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.","108.An expression vector comprising at least one enzymatic nucleic acid molecule of claim 55 or claim 56 in a manner that allows expression of the nucleic acid molecule.","109.A mammalian cell comprising the expression vector of claim 108.110.The mammalian cell of claim 109, wherein said mammalian cell is a human cell.","111.The expression vector of claim 108, wherein said enzymatic nucleic acid molecule is in a hammerhead configuration.","(DOES THIS MAKE SENSE?)","112.The expression vector of claim 108, wherein said expression vector further comprises a sequence for an antisense nucleic acid molecule complementary to RNA encoded by an IKK-gamma subunit gene or PKR gene.","113.The expression vector of claim 108, wherein said expression vector comprises a nucleic acid sequence encoding two or more of said enzymatic nucleic acid molecules, which can be the same or different.","114.The expression vector of claim 108, wherein said expression vector further comprises a sequence encoding an antisense nucleic acid molecule complementary to RNA encoded by an IKK-gamma gene or PKR gene.","115.A method for treatment of cancer comprising administering to a patient the enzymatic nucleic acid molecule of any of claims 55-58 under conditions suitable for said treatment.","116.The method of claim 115, wherein said cancer is breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer.","117.A method for treatment of cancer comprising administering to a patient the antisense nucleic acid molecule of claim 59 under conditions suitable for said treatment.","118.The method of claim 117, wherein said cancer is breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer.","119.The method of claim 115, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.","120.The method of claim 115, wherein said method further comprises administering to said patient one or more other therapies.","121.The method of claim 117, wherein said method further comprises administering to said patient one or more other therapies.","122.The nucleic acid molecule of any of claims 55, 56, or 58, wherein said nucleic acid molecule comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification.","123.The nucleic acid molecule of claim 122, wherein said nucleic acid molecule further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.","124.The nucleic acid molecule of claim 122, wherein said 3′-end modification is a 3′-3′ inverted abasic moiety.","125.The method of claim 93 wherein said other drug therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.","126.The method of claim 125, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.","127.The method of claim 94 wherein said other drug therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.","128.The method of claim 127, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.","129.The method of claim 95 wherein said other drug therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.","130.The method of claim 129, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.","131.The method of claim 96 wherein said other drug therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.","132.The method of claim 131, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.","133.The method of claim 120, wherein said other therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.","134.The method of claim 133, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.","135.The method of claim 121, wherein said other therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.","136.The method of claim 135, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.","137.A method for treatment of an inflammatory disease comprising the step of administering to a patient the enzymatic nucleic acid molecule of any of claims 55-58 under conditions suitable for said treatment.","138.The method of claim 137, wherein said inflammatory disease is rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury, glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, or infection.","139.A method for treatment of an inflammatory disease comprising the step of administering to a patient the antisense nucleic acid molecule of claim 59 under conditions suitable for said treatment.","140.The method of claim 139, wherein said inflammatory disease is rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, or infection.","141.The method of claim 137, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.","142.The method of claim 137, wherein said method further comprises administering to said patient one or more other therapies.","143.The method of claim 139, wherein said method further comprises administering to said patient one or more other therapies.","144.A pharmaceutical composition comprising an enzymatic nucleic acid molecule of any of claims 55-58 in a pharmaceutically acceptable carrier.","145.A pharmaceutical composition comprising an antisense nucleic acid molecule of claim 59 in a pharmaceutically acceptable carrier.","146.The enzymatic nucleic acid molecule of claim 55, wherein said subunit of IKK-is IKK-gamma.","147.The enzymatic nucleic acid molecule of claim 55, wherein said subunit of IKK-is IKK-alpha.","148.The enzymatic nucleic acid molecule of claim 55, wherein said subunit of IKK-is IKK-beta.","149.A method of administering to a cell an enzymatic nucleic acid molecule of any of claims 55-57 comprising contacting said cell with the enzymatic nucleic acid molecule under conditions suitable for said administration.","150.The method of claim 149, wherein said cell is a mammalian cell.","151.The method of claim 149, wherein said cell is a human cell.","152.The method of claim 149, wherein said administration is in the presence of a delivery reagent.","153.The method of claim 152, wherein said delivery reagent is a lipid.","154.The method of claim 153, wherein said lipid is a cationic lipid.","155.The method of claim 153, wherein said lipid is a phospholipid.","156.The method of claim 152, wherein said delivery reagent is a liposome.","157.A nucleic acid molecule that down regulates expression of a prostaglandin D2 receptor (PTGDR) gene.","158.The nucleic acid molecule of claim 157, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule.","159.The nucleic acid molecule of claim 157, wherein said nucleic acid molecule is an antisense nucleic acid molecule.","160.The enzymatic nucleic acid molecule of claim 158, wherein said enzymatic nucleic acid molecule comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 11666-13262.161.The enzymatic nucleic acid molecule of claim 158, wherein said enzymatic nucleic acid molecule comprises at least one binding arm wherein the at least one binding arm comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4415-5483.162.The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4415-5483.163.The nucleic acid molecule of claim 157, wherein said nucleic acid molecule is adapted to treat asthma.","164.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule has an endonuclease activity to cleave RNA encoded by a PTGDR gene.","165.The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in a hammerhead configuration.","166.The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in an Inozyme configuration.","167.The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.","168.The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in a DNAzyme configuration.","169.The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in a G-cleaver configuration.","170.The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in an Amberzyme configuration.","171.The enzymatic nucleic acid molecule of claim 165, wherein said hammerhead configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4415-4641.172.The enzymatic nucleic acid molecule of claim 165, wherein said hammerhead configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 11666-11892.173.The enzymatic nucleic acid molecule of claim 166, wherein said Inozyme configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4642-5017.174.The enzymatic nucleic acid molecule of claim 166, wherein said Inozyme configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 11893-12268.175.The enzymatic nucleic acid molecule of claim 167, wherein said Zinzyme configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 5018-5248.176.The enzymatic nucleic acid molecule of claim 167, wherein said Zinzyme configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 12269-12499.177.The enzymatic nucleic acid molecule of claim 168, wherein said DNAzyme configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4415-5294.178.The enzymatic nucleic acid molecule of claim 168, wherein said DNAzyme configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 12500-12842.179.The enzymatic nucleic acid molecule of claim 170, wherein said Amberzyme configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 5018-5248, and 5295-5483.180.The enzymatic nucleic acid molecule of claim 170, wherein said Amberzyme configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 12843-13262.181.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises between 8 and 100 bases complementary to a RNA molecule encoded by a PTGDR gene.","182.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises between 14 and 24 bases complementary to a RNA molecule encoded by a PTGDR gene.","183.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule is chemically synthesized.","184.The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule is chemically synthesized.","185.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises at least one 2′-sugar modification.","186.The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule comprises at least one 2′-sugar modification.","187.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises at least one nucleic acid base modification.","188.The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule comprises at least one nucleic acid base modification.","189.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises at least one phosphate backbone modification.","190.The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule comprises at least one phosphate backbone modification.","191.A mammalian cell comprising the enzymatic nucleic acid molecule of any of claims 158, 160 or 161.192.The mammalian cell of claim 191, wherein said mammalian cell is a human cell.","193.A method of reducing PTGDR activity in a cell, comprising contacting said cell with the enzymatic nucleic acid molecule of any of claims 158, 160 or 161, under conditions suitable for said reduction.","194.A method of reducing PTGDR activity in a cell, comprising contacting said cell with the antisense nucleic acid molecule of claim 159 under conditions suitable for said reduction.","195.A method of treatment of a patient having a condition associated with the level of PTGDR, comprising contacting cells of said patient with the enzymatic nucleic acid molecule of any of claims 158, 160 or 161, under conditions suitable for said treatment.","196.A method of treatment of a patient having a condition associated with the level of PTGDR, comprising contacting cells of said patient with the antisense nucleic acid molecule of claim 159, under conditions suitable for said treatment.","197.The method of claim 193 further comprising the use of one or more drug therapies under conditions suitable for said treatment.","198.The method of claim 194 further comprising the use of one or more drug therapies under conditions suitable for said treatment.","199.The method of claim 195 further comprising the use of one or more drug therapies under conditions suitable for said treatment.","200.The method of claim 196 further comprising the use of one or more drug therapies under conditions suitable for said treatment.","201.A method of cleaving a RNA molecule encoded by a PTGDR gene comprising contacting the enzymatic nucleic acid molecule of any of claims 158, 160 or 161 with said RNA molecule encoded by a PTGDR gene under conditions suitable for the cleavage.","202.The method of claim 201, wherein said cleavage is carried out in the presence of a divalent cation.","203.The method of claim 202, wherein said divalent cation is Mg2+.","204.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, 3′-end, or both the 5′-end and the 3′-end.","205.The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, 3′-end, or both the 5′-end and the 3′-end.","206.The enzymatic nucleic acid molecule of claim 204, wherein the cap structure at the 5′-end, 3′-end, or both the 5′-end and the 3′-end comprises a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative.","207.The antisense nucleic acid molecule of claim 205, wherein the cap structure at the 5′-end, 3′-end, or both the 5′-end and the 3′-end comprises a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative.","208.The method of claim 193, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.","209.An expression vector comprising a nucleic acid molecule encoding at least one enzymatic nucleic acid molecule of claim 158 or claim 160 in a manner that allows expression of the nucleic acid molecule.","210.A mammalian cell comprising the expression vector of claim 209.211.The mammalian cell of claim 210, wherein said mammalian cell is a human cell.","212.The expression vector of claim 209, wherein said enzymatic nucleic acid molecule is in a hammerhead configuration.","213.The expression vector of claim 209, wherein said expression vector further comprises a sequence for an antisense nucleic acid molecule complementary to a RNA molecule encoded by a PTGDR gene.","214.The expression vector of claim 209, wherein said expression vector comprises a nucleic acid sequence encoding two or more of said enzymatic nucleic acid molecules, which can be the same or different.","215.The expression vector of claim 214, wherein said expression vector further comprises a sequence encoding an antisense nucleic acid molecule complementary to a RNA molecule encoded by a PTGDR gene.","216.A method for treatment of an allergic condition comprising the step of administering to a patient the enzymatic nucleic acid molecule of any of claims 156-159 under conditions suitable for said treatment.","217.The method of claim 216, wherein said allergic condition is asthma, allergic rhinitis, or atopic dermatitis.","218.A method for treatment of an allergic condition comprising administering to a patient the antisense nucleic acid molecule of claim 159 under conditions suitable for said treatment.","219.The method of claim 218, wherein said allergic condition is asthma, allergic rhinitis, or atopic dermatitis.","220.The method of claim 216, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.","221.The method of claim 216, wherein said method further comprises administering to said patient one or more other treatment therapies.","222.The method of claim 218, wherein said method further comprises administering to said patient one or more other treatment therapies.","223.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification.","224.The enzymatic nucleic acid molecule of claim 223, wherein said enzymatic nucleic acid molecule further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.","225.The nucleic acid molecule of claim 223, wherein said 3′-end modification is a 3′-3′ inverted abasic moiety.","226.The method of claim 197 wherein said other drug therapies are bronchodilators, anti-inflammatories, adenosine inhibitors, or adenosine A1 receptor inhibitors.","227.A pharmaceutical composition comprising an enzymatic nucleic acid molecule of any of claims 158, 160 or 161.228.A pharmaceutical composition comprising an antisense nucleic acid molecule of claim 159.229.A method of administering to a mammal the nucleic acid molecule of claim 157, comprising contacting said mammal with the nucleic acid molecule under conditions suitable for said administration.","230.The method of claim 229, wherein said mammal is a human.","231.The method of claim 229 wherein said administration is in the presence of a delivery reagent.","232.The method of claim 231, wherein said delivery reagent is a lipid.","233.The method of claim 232, wherein said lipid is a cationic lipid.","234.The method of claim 232, wherein said lipid is a phospholipid.","235.The method of claim 231, wherein said delivery reagent is a liposome."],["<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention provides compounds, compositions, and methods for the study, diagnosis, and treatment of conditions relating to the expression of NOGO and NOGO receptor genes.","In particular, the invention provides nucleic acid molecules that are used to modulate the expression of NOGO and NOGO receptor gene products.","The present invention further relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to IKK gamma (IKKG) and PKR levels, such as cancer, inflammatory, and autoimmune diseases and/or disorders.","In addition, the present invention also relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to allergic response.","Specifically, the invention provides compositions and methods for the treatment of diseases or conditions related to levels of factors involved in allergic conditions such as asthma, for example prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS) and adenosine A1 receptor (ADORA1).","The discussions that follow are not meant to be complete and are provided only to assist understanding the invention that follows.","The summary is not an admission that any of the work described below is prior art to the claimed invention.","The following is a brief description of the current understanding of NOGO and NOGO receptors.","The ceased growth of neurons following development has severe implications for lesions of the central nervous system (CNS) caused by neurodegenerative disorders and traumatic accidents.","Although CNS neurons have the capacity to rearrange their axonal and dendritic foci in the developed brain, the regeneration of severed CNS axons spanning distance does not exist.","Axonal growth following CNS injury is limited by the local tissue environment rather than intrinsic factors, as indicated by transplantation experiments (Richardson et al., 1980 , Nature, 284, 264-265).","Non-neuronal glial cells of the CNS, including oligodendrocytes and astrocytes, have been shown to inhibit the axonal growth of dorsal root ganglion neurons in culture (Schwab and Thoenen, 1985 , J.","Neurosci., 5, 2415-2423).","Cultured dorsal root ganglion cells can extend their axons across glial cells from the peripheral nervous system, (ie; Schwann cells), but are inhibited by oligodendrocytes and myelin of the CNS (Schwab and Caroni, 1988 , J.","Neurosci., 8, 2381-2393).","The non-conducive properties of CNS tissue in adult vertebrates is thought to result from the existence of inhibitory factors rather than the lack of growth factors.","The identification of proteins with neurite outgrowth inhibitory or repulsive properties include NI-35, NI-250 (Caroni and Schwab, 1988 , Neuron, 1, 85-96), myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945).","Monoclonal antibodies (mAb IN-1) raised against NI-35/250 have been shown to partially neutralize the growth inhibitory effect of CNS myelin and oligodendrocytes.","IN-1 treatment in vivo has resulted in long distance fiber regeneration in lesioned adult mammalian CNS tissue (Weibel et al., 1994 , Brain Res., 642, 259-266).","Additionally, IN-1 treatment in vivo has resulted in the recovery of specific reflex and locomotor functions after spinal cord injury in adult rats (Bregman et al., 1995 , Nature, 378, 498-501).","Recently, the cloning of NOGO-A (Genbank Accession No AJ242961), the rat complementary DNA encoding NI-220/250 has been reported (Chen et al., 2000 , Nature, 403, 434-439).","The NOGO gene encodes at least three major protein products (NOGO-A, NOGO-B, and NOGO-C) resulting from both alternative promoter usage and alternative splicing.","Recombinant NOGO-A inhibits neurite outgrowth from dorsal root ganglia and the spreading of 3T3 firboblasts.","Monoclonal antibody IN-1 recognizes NOGO-A and neutralizes NOGO-A inhibition of neuronal growth in vitro.","Evidence supports the proposal that NOGO-A is the previously described rat NI-250 since NOGO-A contains all six peptide sequences obtained from purified bNI-220, the bovine equivalent of rat NI-250 (Chen et al supra).","Prinjha et al., 2000 , Nature, 403, 383-384, report the cloning of the human NOGO gene which encodes three different NOGO isoforms that are potent inhibitors of neurite outgrowth.","Using oligonucleotide primers to amplify and clone overlapping regions of the open reading frame of NOGO cDNA, Phrinjha et al., supra identified three forms of cDNA clone corresponding to the three protein isoforms.","The longest ORF of 1,192 amino acids corresponds to NOGO-A (Accession No.","AJ251383).","An intermediate-length splice variant that lacks residues 186-1,004 corresponds to NOGO-B (Accession No.","AJ251384).","The shortest splice variant, NOGO-C (Accession No.","AJ251385), appears to be the previously described rat vp20 (Accession No.","AF051335) and foocen-s (Accession No.","AF132048), and also lacks residues 186-1,004.According to Prinjha et al., supra, the NOGO amino-terminal region shows no significant homology to any known protein, while the carboxy-terminal tail shares homology with neuroendocrine-specific proteins and other members of the reticulon gene family.","In addition, the carboxy-terminal tail contains a consensus sequence that may serve as an endoplasmic-reticulum retention region.","Based on the NOGO protein sequence, Prinjha et al., supra, postulate NOGO to be a membrane associated protein comprising a putative large extracellular domain of 1,024 residues with seven predicted N-linked glycosylation sites, two or three transmembrane domains, and a short carboxy-terminal region of 43 residues.","Grandpre et al., 2000 , Nature , also report the identification of NOGO as a potent inhibitor of axon regeneration.","The 4.1 kilobase NOGO human cDNA clone identified by Grandpre et al., supra, KIAA0886, is homologous to a cDNA derived from a previous effort to sequence random high molecular-weight brain derived cDNAs (Nagase et al., 1998 , DNA Res., 31, 355-364).","This cDNA clone encodes a protein that matches all six of the peptide sequences derived from bovine NOGO.","Grandpre et al., supra demonstrate that NOGO expression is predominantly associated with the CNS and not the peripheral nervous system (PNS).","Cellular localization of NOGO protein appears to be predominantly reticluar in origin, however, NOGO is found on the surface of some oligodentrocytes.","An active domain of NOGO has been identified, defined as residues 31-55 of a hydrophilic 66-residue lumenal/extracellular domain.","A synthetic fragment corresponding to this sequence exhibits growth-cone collapsing and outgrowth inhibiting activities (Grandpre et al., supra).","A receptor for the NOGO-A extracellular domain (NOGO-66) is described in Fournier et al., 2001, Nature, 409, 341-346.Fournier et al., have shown that isolated NOGO-66 inhibits axonal extension but does not alter non-neuronal cell morphology.","The receptor identified has a high affinity for soluble NOGO-66, and is expressed as a glycophosphatidylinostitol-linked protein on the surface of CNS neurons.","Furthermore, the expression of the NOGO-66 receptor in neurons that are NOGO insensitive results in NOGO dependent inhibition of axonal growth in these cells.","Cleavage of the NOGO-66 receptor and other glycophosphatidylinostitol-linked proteins from axonal surfaces renders neurons insensitive to NOGO-66 inhibition.","As such, disruption of the interaction between NOGO-66 and the NOGO-66 receptor provides the possibility of treating a wide variety of neurological diseases, injuries, and conditions.","Hauswirth and Flannery, International PCT Publication No.","WO 98/48027, describe materials and methods for the specific expression of proteins in retinal photoreceptor cells consisting of an adeno-associated viral vector contacting a rod or cone-opsin promoter.","In addition, ribozymes which degrade mutant mRNA are described for use in the treatment of retinitis pigmentosa.","Fechteler et al., Interanational PCT Publication No.","WO 00/03004 describe ribozymes targeting presenilin-2 RNA for the use in treating neurodegenerative diseases such as Alzheimer's disease.","Eldadah et al., 2000 , J.","Neurosci., 20, 179-186, describe the protection of cerebellar granule cells from apoptosis induced by serum-potassium deprivation from ribozyme mediated inhibition of caspase-3.Seidman et al., 1999 , Antisense Nucleic Acid Drug Dev., 9, 333-340, describe in general terms, the use of antisense and ribozyme constructs for treatment of neurodegenerative diseases.","Denman et al., 1994 , Nucleic Acids Research, 22, 2375-82, describe the ribozyme mediated degradation of beta-amyloid peptide precursor mRNA in COS-7 cells.","Schwab and Chen, International PCT publication No.","WO 00/31235, describe NOGO proteins and inhibitors of NOGO.","Blatt et al., International PCT publication No.","WO 01/59103, describe nucleic acid molecules for modulating expression of NOGO genes.","The following is a brief description of the physiological role of nuclear factor kappa B (NFKB), IKK kinases, and protein kinase PKR.","Nuclear factor kappa B (NFKB) is a multiunit transcription factor which regulates the expression of genes involved in a number of physiologic and pathologic processes.","NFKB is a key component of the TNF signaling pathway.","These processes include, but are not limited to: apoptosis, immune, inflammatory and acute phase responses.","The REL-A gene product (a.k.a.","RelA or p65), and p50 subunits of NFKB, have been implicated in the induction of inflammatory responses and cellular transformation.","NFKB exists in the cytoplasm as an inactive heterodimer of the p50 and p65 subunits.","NFKB is complexed with an inhibitory protein complex, IkappaB (IKK complex), until activated by the appropriate stimuli.","NFKB activation can occur following stimulation of a variety of cell types by inflammatory mediators, for example TNF and IL-1, and reactive oxygen intermediates.","In response to induction, NFKB can stimulate production of pro-inflammatory cytokines such as TNF-alpha, IL-1-beta, IL-6 and iNOS, thereby perpetuating a positive feedback loop.","NFKB appears to play a role in a number of disease processes including: ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, arthritis, and cancer.","The nuclear DNA-binding protein, NFKB, was first identified as a factor that binds and activates the immunoglobulin kappa light chain enhancer in B cells.","NFKB now is known to activate transcription of a variety of other cellular genes (e.g., cytokines, adhesion proteins, oncogenes and viral proteins) in response to a variety of stimuli (e.g., phorbol esters, mitogens, cytokines and oxidative stress).","In addition, molecular and biochemical characterization of NFKB has shown that the activity is due to a homodimer or heterodimer of a family of DNA binding subunits.","Each subunit bears a stretch of 300 amino acids that is homologous to the oncogene, v-rel.","The activity first described as NFKB is a heterodimer of p49 or p50 with p65.The p49 and p50 subunits of NFKB (encoded by the NF-kappa B2 or NF kappa B1 genes, respectively) are generated from the precursors NFKB1 (p105) or NFKB2 (p100).","The p65 subunit of NFKB (now termed REL-A) is encoded by the rel-A locus.","The roles of each specific transcription-activating complex now are being elucidated in cells (Perkins, et al., 1992 , Proc.","Natl.","Acad.","Sci USA, 89, 1529-1533).","For instance, the heterodimer of NFKB1 and Rel A (p50/p65) activates transcription of the promoter for the adhesion molecule, VCAM-1, while NFKB2/RelA heterodimers (p49/p65) actually inhibit transcription (Shu, et al., 1993 , Mol.","Cell.","Biol., 13, 6283-6289).","Conversely, heterodimers of NFKB2/RelA (p49/p65) act with Tat-I to activate transcription of the HIV genome, while NFKB1/RelA (p50/p65) heterodimers have little effect (Liu et al., 1992 , J.","Virol., 66, 3883-3887).","Similarly, blocking rel A gene expression with antisense oligonucleotides specifically blocks embryonic stem cell adhesion; blocking NFKB1 gene expression with antisense oligonucleotides had no effect on cellular adhesion (Narayanan et al., 1993 , Mol.","Cell.","Biol., 13, 3802-3810).","Thus, the promiscuous role initially assigned to NFKB in transcriptional activation (Lenardo, and Baltimore, 1989 , Cell, 58, 227-229) represents the sum of the activities of the rel family of DNA-binding proteins.","This conclusion is supported by recent transgenic “knock-out” mice of individual members of the rel family.","Such “knock-outs” show few developmental defects, suggesting that essential transcriptional activation functions can be performed by more than one member of the rel family.","A number of specific inhibitors of NFKB function in cells exist, including treatment with phosphorothioate antisense oliogonucleotide, treatment with double-stranded NFKB binding sites, and over expression of the natural inhibitor MAD-3 (an Ikappa-B family member).","These agents have been used to show that NFKB is required for induction of a number of molecules involved in cancer and/or inflammation, as described below.","NFkB is required for phorbol ester-mediated induction of IL-6 (Kitajima, et al., 1992 , Science, 258, 1792-5) and IL-8 (Kunsch and Rosen, 1993 , Mol.","Cell.","Biol., 13, 6137-46).","NFkB is required for induction of the adhesion molecules ICAM-1 (Eck, et al., 1993 , Mol.","Cell.","Biol., 13, 6530-6536), VCAM-1 (Shu et al., supra), and E-selectin (Read, et al., 1994 , J. Exp.","Med., 179, 503-512) on endothelial cells.","NFkB is involved in the induction of the integrin subunit, CD18, and other adhesive properties of leukocytes (Eck et al., 1993 supra).","HER2/Neu overexpression induces NFKB via a PI3-kinase/Akt pathway involving calpain-mediated degradation of IKB-alpha.","Breast cancer has been shown to typify the aberrant expression of NFKB/REL factors (Pianetti et al., 2001 , Oncogene, 20, 1287-1299; Sovak et al., 1999 , J. Clin.","Invest., 100, 2952-2960).","Inhibition of NFKB activity has been shown to induce apoptosis in murine hepatocytes (Bellas et al., 1997 , Am.","J.","Pathol., 151, 891-896).","NFKB has been shown to regulate cyclooxygenase-2 expression and cell proliferation in human gastric cancer cells (Joo Weon et al., 2001 , Laboratory Investigation, 81, 349-360).","The above studies suggest that NFKB is integrally involved in the induction of cytokines and adhesion molecules by inflammatory mediators and is involved in the transformation of cancerous cells.","Two reported studies point to another connection between NFKB and inflammation: glucocorticoids can exert their anti-inflammatory effects by inhibiting NFKB.","The glucocorticoid receptor and p65 both act at NFKB binding sites in the ICAM-1 promoter (van de Stolpe, et al., 1994 , J. Biol.","Chem., 269, 6185-6192).","Glucocorticoid receptor inhibits NFKB-mediated induction of IL-6 (Ray and Prefontaine, 1994 Proc.","Natl.","Acad.","Sci USA, 91, 752-756).","Conversely, overexpression of p65 inhibits glucocorticoid induction of the mouse mammary tumor virus promoter.","Finally, protein cross-linking and co-immunoprecipitation experiments demonstrated direct physical interaction between p65 and the glucocorticoid receptor.","The IKK complex that sequesters NFKB in the cytoplasm comprises IkappaB (IκB) proteins (IκB-alpha, IκB-beta, IκKB-epsilon, p105, and p100).","The phosphorylation of IκB proteins results in the release of NFKB from the IκB complex which is transported to the nucleus via the unmasking of nuclear translocation signals.","Phosphorylation marks IkB proteins for ubiquitination and degradation via the proteosome pathway.","Most NFKB inducing stimuli initiate activation of an IκB kinase (IKK) complex that contains two catalytic subunits, IKK-alpha (IKK1) and IKK-beta (IKK2), that phosphorylate IκB-alpha and IκB-beta, with IKK-beta playing a predominant role in pro-inflammatory signaling.","In addition to the two kinases, the IKK complex contains regulatory subunits, including IKK-gamma (NEMO/IKKAP1).","IKK-gamma is a protein that is critical for the assembly of the IKK complex.","IKK-gamma directly binds to IKK-beta and is required for activation of NFKB, for example by TNF-alpha, IL-1-beta, lipopolysaccharide, phorbol 12-myristate 13-acetate, the human T-cell lymphotrophic virus (HTLV-1), or double stranded RNA.","Genomic rearrangements in IKK-gamma have been shown to impair NFKB activation and result in incontinentia pigmenti.","Additional proteins that associate with the IKK complex include, MEK kinase (MEKK1), NFKB inducing kinase (NIK), receptor interacting protein (RIP), protein kinase CK2, and IKK-associated protein (IKAP), which appears to be associated with the IκB Kinase (IKK) complex, but does not appear to be an integral component of the tripartate IKK complex as does IKK-gamma (Krappmann et al., 2001 , J. Biol.","Chem., 275, 29779-87).","The RNA-dependent protein kinase PKR is a signal transducer for NFKB and IFN regulatory factor-1.PKR is required for activation of NFKB by IFN-gamma via a STAT-1 independent pathway (Amitabha et al., 2001 , J.","Immunol., 166, 6170-6180).","The induction of NFKB by PKR takes place though phosphorylation of IκB-alpha, and appears not to require the catalytic activity of PKR, thereby proceeding independently of the dsRNA-binding properties of PKR (Ishii et al., 2001 , Oncogene, 20, 1900-1912).","PKR also plays an important role in the regulation of protein synthesis by modulating the activity of eukaryotic initiation factor 2 (eIF-2-alpha) through interferon induction.","Kamiya, JP 2000253884, describes specific antisense oligonucleotides for inhibiting IκB-kinase subunit expression.","Krappmann et al., 2001 , J. Biol.","Chem ., describe specific antisense oligonucleotides to IKK-gamma.","The following is a description of molecular targets involved in diseases or conditions related to allergic response.","Asthma is a chronic inflammatory disorder of the lungs characterized by airflow obstruction, bronchial hyper-responsiveness, and airway inflammation.","T-lymphocytes that produce TH2 cytokines and eosinophilic leukocytes infiltrate the airways.","In the airway and in bronchial alveolar lavage (BAL) fluid of individuals with asthma, high concentrations of TH2 cytokines, interleukin-4 (14), IL-5, and IL-13, are present along with increased levels of adenosine.","In contrast to normal individuals, asthmatics respond to adenosine challenge with marked airway obstruction.","Upon allergen challenge, mast cells are activated by cross-linked IgE-allergen complexes.","Large amounts of prostaglandin D2 (PGD2), the major cyclooxygenase product of arachidonic acid are released.","PGD2 is generated from PGH2 via the activity of prostaglandin D2 synthetase (PTGDS).","PGD2 receptors and adenosine A1 receptors are present in the lungs and airway along with various other tissues in response to allergic stimuli (Howarth, 1997 , Allergy, 52, 12).","The significance of PGD2 as a mediator of allergic asthma has been established with the development of mice deficient in the PGD2 receptor (DP).","DP is a heterotrimeric GTP-binding protein-coupled, rhodopsin-type receptor specific for PGD2 (Hirata et al., 1994 , PNAS USA., 91, 11192).","These mice fail to develop airway hyperreactivity and have greatly reduced eosinophil infiltration and cytokine accumulation in response to allergens.","Upon allergen challenge mice deficient in the prostaglandin D2 (PGD2) receptor (DP) did not develop airway hyperactivity.","Cytokine, lymphocyte and eosinophil accumulation in the lungs were greatly reduced (Matsuoka et al., 2000 , Science, 287, 2013).","The DP −/− mice exhibited no behavioral, anatomic, or histological abnormalities.","Primary immune response is not affected by DP disruption.","Asthma affects more than 100 million people worldwide and more than 17 million Americans (5% of the population).","Since 1980 the incidence has more than doubled and deaths have tripled (5,000 deaths in 1995).","Annual asthma-related healthcare costs in the US alone were estimated to exceed $14.5 billion in 2000.Current therapies such as inhalant anti-inflammatories and bronchodilators can be used to treat symptoms, however, these therapies do not prevent or cure asthma.","Sandberg et al., 2001 , Prog.","Respir.","Res., 31, 370-373, describes ribozyme therapy for asthma and COPD.","Sullivan et al., International U.S. Pat.","No.","5,616,488, describes ribozymes targeting interleukin-5 for treatment and diagnosis of asthma and other inflammatory disorders.","Stinchcomb et al., International PCT Publication No.","WO 95/23225, describes ribozymes and methods for inhibiting the expression of disease related genes including genes associated with asthma.","Nyce, International PCT Publication Nos.","WO 00/62736, WO 00/09525, WO 99/13886, WO 98/23294, WO 96/40266 and U.S. Pat.","No.","6,025,339 describe specific antisense oligonucleotides targeting certain mRNAs encoding particular adenosine receptors."],["<SOH> SUMMARY OF THE INVENTION <EOH>The invention features novel nucleic acid-based molecules, for example, enzymatic nucleic acid molecules, allozymes, antisense nucleic acids, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNA, dsRNA, siRNA, aptamers, and antisense nucleic acids containing RNA cleaving chemical groups, and methods to modulate gene expression; for example, gene(s) encoding prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), and adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3; gene(s) encoding NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors; and genes encoding an IkappaB kinase (IKK) subunit or protein kinase PKR.","In one embodiment, the instant invention features nucleic-acid based techniques to inhibit the expression of NOGO-A (Accession No.","AJ251383), NOGO-B (Accession No.","AJ251384), and/or NOGO-C (Accession No.","AJ251385), NOGO-66 receptor (Accession No AF283463, Fournier et al., 2001, Nature, 409, 341-346), NI-35, NI-220, and/or NI-250, myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945).","The description below of the various aspects and embodiments is provided with reference to the exemplary NOGO-A and NOGO-66 receptor genes.","However, the various aspects and embodiments are also directed to other genes which express NOGOA-like inhibitor proteins and other receptors involved in neurite outgrowth inhibition.","Those additional genes can be analyzed for target sites using the methods described for NOGO and the NOGO-66 receptor, referred to alternatively as NOGO receptor.","Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.","The invention features one or more enzymatic nucleic acid-based molecules and methods that independently or in combination modulate the expression of gene(s) encoding a member of the IκB kinase IKK complex or PKR.","In particular embodiments, the invention features nucleic acid-based molecules and methods that modulate the expression of a member of the IκB kinase IKK complex, for example IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKKγ) and/or a protein kinase PKR protein, such as IKK-alpha (IKK1) gene (Genbank Accession No.","NM — 001278); IKK-beta (IKK2) gene, for example (Genbank Accession No.","AF080158), IKK-gamma (IKKγ) gene, for example (Genbank Accession No.","NM — 003639), and protein kinase PKR gene, for example (Genbank Accession No.","NM — 002759).","The description below of the various aspects and embodiments is provided with reference to the exemplary IKK-gamma and PKR genes.","IKK-gamma is also known as NEMO/IKKAP1.However, the various aspects and embodiments are also directed to other genes which encode other subunits of the IKK complex, such as IKK-alpha (IKK1) or IKK-beta (IKK2).","Those additional genes can be analyzed for target sites using the methods described for IKK-gamma or PKR.","Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.","In one embodiment, an enzymatic nucleic acid molecule of the invention is in a hammerhead, Inozyme, Zinzyme, DNAzyme, Amberzyme, or G-cleaver configuration.","In another embodiment, a nucleic acid molecule of the invention comprises between 8 and 100 bases complementary to the RNA of the target gene.","In another embodiment, a nucleic acid molecule of the invention comprises between 14 and 24 bases complementary to a RNA molecule of the target gene.","In one embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention is chemically synthesized.","In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one 2′-sugar modification.","In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one nucleic acid base modification.","In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one phosphate backbone modification.","In one embodiment, the invention features a mammalian cell, for example a human cell, including the nucleic acid molecule of the invention.","In another embodiment, the invention features a method of reducing target gene expression or activity in a cell, comprising contacting the cell with a nucleic acid molecule of the invention, such as an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups, under conditions suitable for the reduction.","In yet another embodiment, the invention features a method of treatment of a patient having a condition associated with the level of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR, comprising contacting cells of the patient with an enzymatic nucleic acid molecule of the invention, under conditions suitable for the treatment.","In another embodiment, a method of treatment of a patient having a condition associated with the level of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR is featured, wherein the method further comprises the use of one or more drug therapies under conditions suitable for the treatment.","In another embodiment, the invention features a method of cleaving a RNA molecule of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene, comprising contacting an enzymatic nucleic acid molecule of the invention with a RNA molecule of the corresponding gene under conditions suitable for the cleavage, for example, wherein the cleavage is carried out in the presence of a divalent cation, such as Mg 2+ .","In one embodiment, a nucleic acid molecule of the invention comprises a cap structure, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end of the enzymatic nucleic acid molecule.","In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one nucleic acid molecule of the invention, in a manner which allows expression of the nucleic acid molecule.","In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.","In yet another embodiment, the expression vector of the invention further comprises a sequence for an antisense nucleic acid molecule complementary to a RNA molecule of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene.","In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more nucleic acid molecules, such as enzymatic nucleic acid molecules, antisense, aptamers, decoys, siRNA, or 2-5A chimeras which can be the same or different.","In one embodiment, the method of treatment features an enzymatic nucleic acid molecule of the invention comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification, such as a 3′-3′ inverted abasic moiety.","In another embodiment, an enzymatic nucleic acid molecule or antisense nucleic acid molecule of the invention further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.","In another embodiment, the invention features a method of administering to a mammal, for example a human, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention, comprising contacting the mammal with the nucleic acid molecule under conditions suitable for the administration, for example, in the presence of a delivery reagent such as a lipid, cationic lipid, phospholipid, or liposome.","In yet another embodiment, the invention features a method of administering to a mammal an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention in conjunction with a therapeutic agent, comprising contacting the mammal, for example a human, with the nucleic acid molecule and the therapeutic agent under conditions suitable for the administration.","In one embodiment, the invention features the use of an enzymatic nucleic acid molecule, which can be in a hammerhead, NCH, G-cleaver, Amberzyme, Zinzyme, and/or DNAzyme motif, to down-regulate the expression of a a target gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene.","By “inhibit”, “down-regulate”, or “reduce”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR subunits, is reduced below that observed in the absence of the nucleic acid molecules of the invention.","In one embodiment, inhibition, down-regulation or reduction with an enzymatic nucleic acid molecule is below that level observed in the presence of an enzymatically inactive or attenuated molecule that is able to bind to the same site on the target RNA molecule, but is unable to cleave that RNA molecule.","In another embodiment, inhibition, down-regulation, or reduction with antisense oligonucleotides is below that level observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches.","In another embodiment, inhibition, down-regulation, or reduction of the target gene with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.","By “up-regulate” is meant that the expression of a gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins, protein subunits, or activity of one or more proteins or protein subunits, such as a target gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR subunits, is greater than that observed in the absence of the nucleic acid molecules of the invention.","For example, the expression of a gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene, can be increased in order to treat, prevent, ameliorate, or modulate a pathological condition caused or exacerbated by an absence or low level of gene expression.","By “modulate” is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more protein subunits, or activity of one or more protein subunits is up-regulated or down-regulated, such that the expression, level, or activity is greater than or less than that observed in the absence of a nucleic acid molecule of the invention.","By “enzymatic nucleic acid molecule” it is meant a nucleic acid molecule that has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that is active to specifically cleave target a RNA molecule.","That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave a RNA molecule and thereby inactivate a target RNA molecule.","These complementary regions allow sufficient hybridization of an enzymatic nucleic acid molecule to a target RNA molecule and thus permit cleavage.","One hundred percent complementarity is preferred, but complementarity as low as 50-75% can also be useful in this invention (see for example Werner and Uhlenbeck, 1995 , Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999 , Antisense and Nucleic Acid Drug Dev., 9, 25-31).","The nucleic acids can be modified at the base, sugar, and/or phosphate groups.","The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme.","All of these terminologies describe nucleic acid molecules with enzymatic activity.","The specific enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site that is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule (Cech et al., U.S. Pat.","No.","4,987,071; Cech et al., 1988, 260 JAMA 3030).","By “nucleic acid molecule” as used herein is meant a molecule having nucleotides.","The nucleic acid can be single, double, or multiple stranded and can comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.","Exemplary nucleic acid molecules of the invention include enzymatic nucleic acid molecules, allozymes, antisense nucleic acids, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNA, dsRNA, siRNA, aptamers, and/or antisense nucleic acids containing RNA cleaving chemical groups.","By “enzymatic portion” or “catalytic domain” is meant that portion/region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate (for example see FIGS.","1-4 ).","By “substrate binding arm” or “substrate binding domain” is meant that portion/region of a enzymatic nucleic acid that is able to interact, for example via complementarity (i.e., able to base-pair with), with a portion of its substrate.","Such complementarity can be 100%, but can be less if desired.","For example, as few as 10 bases out of 14 can be base-paired (see for example Werner and Uhlenbeck, 1995 , Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999 , Antisense and Nucleic Acid Drug Dev., 9, 25-31).","Examples of such arms are shown generally in FIGS.","1-4 .","That is, these arms contain sequences within an enzymatic nucleic acid that are intended to bring enzymatic nucleic acid and target RNA together through complementary base-pairing interactions.","The enzymatic nucleic acid of the invention can have binding arms that are contiguous or non-contiguous and can be of varying lengths.","The length of the binding arm(s) can be greater than or equal to four nucleotides and of sufficient length to stably interact with a target RNA; in one embodiment they can be 12-100 nucleotides; in another embodiment they can be 14-24 nucleotides long (see for example Werner and Uhlenbeck, supra; Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herranze et al., 1993 , EMBO J., 12, 2567-73) or between 8 and 14 nucleotides long.","If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., four and four, five and five nucleotides, or six and six nucleotides, or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., three and five, six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).","By “Inozyme” or “NCH” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as NCH Rz in FIG.","1 .","Inozymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCH/, where N is a nucleotide, C is cytidine and H is adenosine, uridine or cytidine, and/represents the cleavage site.","H is used interchangeably with X. Inozymes can also possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCN/, where N is a nucleotide, C is cytidine, and / represents the cleavage site.","“I” r in FIG.","1 represents an Inosine nucleotide, including a ribo-Inosine or xylo-Inosine nucleoside.","By “G-cleaver” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as G-cleaver Rz in FIG.","1 .","G-cleavers possess endonuclease activity to cleave RNA substrates having a cleavage triplet NYN/, where N is a nucleotide, Y is uridine or cytidine and / represents the cleavage site.","G-cleavers can be chemically modified as is generally shown in FIG.","1 .","By “amberzyme” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG.","2 .","Amberzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NG/N, where N is a nucleotide, G is guanosine, and / represents the cleavage site.","Amberzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG.","2 .","In addition, differing nucleoside and/or non-nucleoside linkers can be used to substitute the 5′-gaaa-3′ loops shown in the figure.","Amberzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2′-OH) group within its own nucleic acid sequence for activity.","By “zinzyme” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG.","3 .","Zinzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet including but not limited to YG/Y, where Y is uridine or cytidine, and G is guanosine and / represents the cleavage site.","Zinzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG.","3 , including substituting 2′-O-methyl guanosine nucleotides for guanosine nucleotides.","In addition, differing nucleotide and/or non-nucleotide linkers can be used to substitute the 5′-gaaa-2′ loop shown in the figure.","Zinzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2′-OH) group within its own nucleic acid sequence for activity.","By ‘DNAzyme’ is meant, an enzymatic nucleic acid molecule that does not require the presence of a 2′-OH group within its own nucleic acid sequence for activity.","In particular embodiments the enzymatic nucleic acid molecule can have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups.","DNAzymes can be synthesized chemically or expressed endogenously in vivo, by means of a single stranded DNA vector or equivalent thereof.","An example of a DNAzyme is shown in FIG.","4 and is generally reviewed in Usman et al., U.S. Pat.","No.","6,159,714; Chartrand et al., 1995 , NAR 23, 4092; Breaker et al., 1995 , Chem.","Bio.","2, 655; Santoro et al., 1997 , PNAS 94, 4262; Breaker, 1999 , Nature Biotechnology, 17, 422-423; and Santoro et.","al., 2000 , J.","Am.","Chem.","Soc., 122, 2433-39.The “10-23” DNAzyme motif is one particular type of DNAzyme that was evolved using in vitro selection (see Santoro et al., supra).","Additional DNAzyme motifs can be selected for using techniques similar to those described in these references, and hence, are within the scope of the present invention.","By “sufficient length” is meant an oligonucleotide of greater than or equal to 3 nucleotides that is of a length great enough to provide the intended function under the expected condition.","For example, for binding arms of enzymatic nucleic acid “sufficient length” means that the binding arm sequence is long enough to provide stable binding to a target site under the expected binding conditions.","The binding arms are not so long as to prevent useful turnover of the nucleic acid molecule.","By “stably interact” is meant interaction of the oligonucleotides with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions) that is sufficient to the intended purpose (e.g., cleavage of target RNA by an enzyme).","By “equivalent” or “related” RNA to NOGO is meant to include those naturally occurring RNA molecules having homology (partial or complete) to NOGO-A, NOGO-B, NOGO-C and/or NOGO receptor proteins or encoding for proteins with similar function as NOGO or NOGO receptor proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.","The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.","By “equivalent” or “related” RNA to IKK-gamma is meant to include those naturally occurring RNA molecules having homology (partial or complete) to IKK-gamma proteins or encoding for proteins with similar function as IKK-gamma proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.","The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.","By “equivalent” or “related” RNA to PKR is meant to include those naturally occurring RNA molecules having homology (partial or complete) to PKR proteins or encoding for proteins with similar function as PKR proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.","The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.","By “equivalent” or “related” RNA to PTGDS is meant to include RNA molecules having homology (partial or complete) to RNA molecules encoding PTGDS proteins or encoding proteins with similar function as PTGDS proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites.","The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.","By “equivalent” or “related” RNA to PTGDR is meant to include RNA molecules having homology (partial or complete) to RNA molecules encoding PTGDR proteins or encoding proteins with similar function as PTGDR proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites.","The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.","By “equivalent” or “related” RNA to ADORA1 is meant to include RNA molecules having homology (partial or complete) to RNA molecule encoding ADORA1 proteins or encoding proteins with similar function as ADORA1 proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites.","The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.","By “homology” is meant the nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.","By “antisense nucleic acid”, it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., U.S. Pat.","No.","5,849,902).","Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.","However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop.","Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.","For a review of current antisense strategies, see Schmajuk et al., 1999 , J. Biol.","Chem., 274, 21783-21789, Delihas et al., 1997 , Nature, 15, 751-753, Stein et al., 1997 , Antisense N. A.","Drug Dev., 7, 151, Crooke, 2000 , Methods Enzymol., 313, 3-45; Crooke, 1998 , Biotech.","Genet.","Eng.","Rev., 15, 121-157, Crooke, 1997 , Ad.","Pharmacol., 40, 1-49.In addition, antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.","The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA.","Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.","By “RNase H activating region” is meant a region (generally greater than or equal to 4-25 nucleotides in length, and in one embodiment from 5-11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al., U.S. Pat.","No.","5,849,902; Arrow et al., U.S. Pat.","No.","5,989,912).","The RNase H enzyme binds to the nucleic acid molecule-target RNA complex and cleaves the target RNA sequence.","The RNase H activating region comprises, for example, phosphodiester, phosphorothioate (at least four of the nucleotides are phosphorothiote substitutions; and in another embodiment, 4-11 of the nucleotides are phosphorothiote substitutions); phosphorodithioate, 5′-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof.","In addition to one or more backbone chemistries described above, the RNase H activating region can also comprise a variety of sugar chemistries.","For example, the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry.","Those skilled in the art will recognize that the foregoing are non-limiting examples and that any combination of phosphate, sugar and base chemistry of a nucleic acid that supports the activity of RNase H enzyme is within the scope of the definition of the RNase H activating region and the instant invention.","By “2-5A antisense chimera” is meant an antisense oligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylate residue.","These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc.","Natl.","Acad.","Sci.","USA 90, 1300; Silverman et al., 2000 , Methods Enzymol., 313, 522-533; Player and Torrence, 1998 , Pharmacol.","Ther., 78, 55-113).","By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that is distinct from sequence recognized by the target molecule in its natural setting.","Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid.","The target molecule can be any molecule of interest.","For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein.","This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art, see for example Gold et al., 1995 , Annu.","Rev.","Biochem., 64, 763; Brody and Gold, 2000 , J.","Biotechnol., 74, 5; Sun, 2000 , Curr.","Opin.","Mol.","Ther., 2, 100; Kusser, 2000 , J.","Biotechnol., 74, 27; Hermann and Patel, 2000 , Science, 287, 820; and Jayasena, 1999 , Clinical Chemistry, 45, 1628.By “triplex forming oligonucleotides” is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix.","Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc.","Natl.","Acad.","Sci.","USA 89, 504; Fox, 2000 , Curr.","Med.","Chem., 7, 17-37; Praseuth et al., 2000 , Biochim.","Biophys.","Acta, 1489, 181-206).","By “gene” it is meant a nucleic acid that encodes an RNA, for example, nucleic acid sequences including but not limited to structural genes encoding a polypeptide.","“Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another RNA molecule by either traditional Watson-Crick or other non-traditional types.","In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., enzymatic nucleic acid cleavage, antisense or triple helix inhibition.","Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987 , CSH Symp.","Quant.","Biol.","LII pp.","123-133; Frier et al., 1986 , Proc.","Nat.","Acad.","Sci.","USA 83:9373-9377; Turner et al., 1987 , J.","Am.","Chem.","Soc.","109:3783-3785).","A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).","“Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.","By “RNA” is meant a molecule comprising at least one ribonucleotide residue.","By “ribonucleotide” or “2′-OH” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribo-furanose moiety.","By “decoy RNA” is meant an RNA molecule or aptamer that is designed to preferentially bind to a predetermined ligand.","Such binding can result in the inhibition or activation of a target molecule.","The decoy RNA or aptamer can compete with a naturally occurring binding target for the binding of a specific ligand.","For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a “decoy” and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990, Cell, 63, 601-608).","This is but a specific example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art, see for example Gold et al., 1995 , Annu.","Rev.","Biochem., 64, 763; Brody and Gold, 2000 , J.","Biotechnol., 74, 5; Sun, 2000 , Curr.","Opin.","Mol.","Ther., 2, 100; Kusser, 2000 , J.","Biotechnol., 74, 27; Hermann and Patel, 2000 , Science, 287, 820; and Jayasena, 1999 , Clinical Chemistry, 45, 1628.Similarly, a decoy RNA can be designed to bind to a D2 receptor and block the binding of PTGDS or a decoy RNA can be designed to bind to PTGDS and prevent interaction with the D2 receptor.","The term “short interfering RNA” or “siRNA” as used herein refers to a double stranded nucleic acid molecule capable of RNA interference “RNAi”, see for example Bass, 2001 , Nature, 411, 428-429; Elbashir et al., 2001 , Nature, 411, 494-498; and Kreutzer et al., International PCT Publication No.","WO 00/44895; Zernicka-Goetz et al., International PCT Publication No.","WO 01/36646; Fire, International PCT Publication No.","WO 99/32619; Plaetinck et al., International PCT Publication No.","WO 00/01846; Mello and Fire, International PCT Publication No.","WO 01/29058; Deschamps-Depaillette, International PCT Publication No.","WO 99/07409; and Li et al., International PCT Publication No.","WO 00/44914.As used herein, siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides.","The term “allozyme” as used herein refers to an allosteric enzymatic nucleic acid molecule, see, e.g., George et al., U.S. Pat.","Nos.","5,834,186 and 5,741,679, Shih et al., U.S. Pat.","No.","5,589,332, Nathan et al., U.S. Pat.","No.","5,871,914, Nathan and Ellington, International PCT publication No.","WO 00/24931, Breaker et al., International PCT Publication Nos.","WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No.","WO 99/29842.The term “2-5A chimera” as used herein refers to an oligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylate residue.","These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc.","Natl.","Acad.","Sci.","USA 90, 1300; Silverman et al., 2000 , Methods Enzymol., 313, 522-533; Player and Torrence, 1998 , Pharmacol.","Ther., 78, 55-113).","The term “triplex forming oligonucleotides” as used herein refers to an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix.","Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc.","Natl.","Acad.","Sci.","USA 89, 504; Fox, 2000 , Curr.","Med.","Chem., 7, 17-37; Praseuth et.","al., 2000 , Biochim.","Biophys.","Acta, 1489, 181-206).","Several varieties of naturally-occurring enzymatic RNAs are known presently.","Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions.","Table I summarizes some of the characteristics of these ribozymes.","In general, enzymatic nucleic acids act by first binding to a target RNA.","Such binding occurs through the target binding portion of a enzymatic nucleic acid that is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.","Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.","Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein.","After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.","Thus, a single ribozyme molecule is able to cleave many molecules of target RNA.","In addition, the ribozyme is a highly specific inhibitor of gene expression, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage.","Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme.","In one embodiment of the inventions described herein, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but can also be formed in the motif of a hepatitis delta virus, group I intron, group II intron or RNase P RNA (in association with an RNA guide sequence), Neurospora VS RNA, DNAzymes, NCH cleaving motifs, or G-cleavers.","Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al., 1992 , AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al., 1989 , Gene 82, 53, Haseloff and Gerlach, 1989 , Gene, 82, 43, and Hampel et al., 1990 Nucleic Acids Res.","18, 299; Chowrira & McSwiggen, U.S. Pat.","No.","5,631,359; of the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNase P motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman, 1990 , Science 249, 783; Li and Altman, 1996 , Nucleic Acids Res.","24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc.","Natl.","Acad.","Sci.","USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995 , EMBO.","J.","14, 363); Group II introns are described by Griffin et al., 1995 , Chem.","Biol.","2, 761; Michels and Pyle, 1995 , Biochemistry 34, 2965; Pyle et al., International PCT Publication No.","WO 96/22689; of the Group I intron by Cech et al., U.S. Pat.","No.","4,987,071 and of DNAzymes by Usman et al., International PCT Publication No.","WO 95/11304; Chartrand et al., 1995 , NAR 23, 4092; Breaker et al., 1995 , Chem.","Bio.","2, 655; Santoro et al., 1997 , PNAS 94, 4262, and Beigelman et al., International PCT publication No.","WO 99/55857.NCH cleaving motifs are described in Ludwig & Sproat, International PCT Publication No.","WO 98/58058; and G-cleavers are described in Kore et al., 1998 , Nucleic Acids Research 26, 4116-4120 and Eckstein et al., International PCT Publication No.","WO 99/16871.Additional motifs such as the Aptazyme (Breaker et al., WO 98/43993), Amberzyme (Class I motif; FIG.","2 ; Beigelman et al., U.S. Ser.","No.","09/301,511) and Zinzyme ( FIG.","3 ) (Beigelman et al., U.S. Ser.","No.","09/301,511), all included by reference herein including drawings, can also be used in the present invention.","These specific motifs or configurations are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule (Cech et al., U.S. Pat.","No.","4,987,071).","In one embodiment of the present invention, a nucleic acid molecule of the instant invention can be between 12 and 100 nucleotides in length.","Exemplary enzymatic nucleic acid molecules of the invention are shown in Tables III-XXIII.","For example, enzymatic nucleic acid molecules of the invention can be between 15 and 50 nucleotides in length, and in another embodiment between 25 and 40 nucleotides in length, e.g., 34, 36, or 38 nucleotides in length (for example see Jarvis et al., 1996 , J. Biol.","Chem., 271, 29107-29112).","Exemplary DNAzymes of the invention are can between 15 and 40 nucleotides in length, and in one embodiment, between 25 and 35 nucleotides in length, e.g., 29, 30, 31, or 32 nucleotides in length (see, e.g., Santoro et al., 1998 , Biochemistry, 37, 13330-13342; Chartrand et al., 1995, Nucleic Acids Research, 23, 4092-4096).","Exemplary antisense molecules of the invention can be between 15 and 75 nucleotides in length, and in one embodiment between 20 and 35 nucleotides in length, e.g., 25, 26, 27, or 28 nucleotides in length (see for example Woolf et al., 1992 , PNAS., 89, 7305-7309; Milner et al., 1997 , Nature Biotechnology, 15, 537-541).","Exemplary triplex forming oligonucleotide molecules of the invention are between 10 and 40 nucleotides in length, and in one embodiment are between 12 and 25 nucleotides in length, e.g., 18, 19, 20, or 21 nucleotides in length (see for example Maher et al., 1990 , Biochemistry, 29, 8820-8826; Strobel and Dervan, 1990 , Science, 249, 73-75).","Those skilled in the art will recognize that all that is required is for the nucleic acid molecule to be of length and conformation sufficient and suitable for the nucleic acid molecule to catalyze a reaction contemplated herein.","The length of the nucleic acid molecules of the instant invention are not limiting within the general limits stated.","In one embodiment, a nucleic acid molecule that modulates, for example, down-regulates, the expression of a target gene comprises between 8 and 100 bases complementary to a RNA molecule of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR.","In another embodiment, a nucleic acid molecule that modulates the expression of a target gene comprises between 14 and 24 bases complementary to a RNA molecule of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR.","The invention provides a method for producing a class of nucleic acid-based gene modulating agents that exhibit a high degree of specificity for the RNA of a desired target.","For example, the enzymatic nucleic acid molecule is can be targeted to a highly conserved sequence region of target RNAs encoding prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR (e.g., prostaglandin D2 receptor (PTGDR)), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR genes) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention.","Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required.","Alternatively, the nucleic acid molecules (e.g., ribozymes, antisense, aptamers, and/or siRNA) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.","As used in herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism.","The cell can, for example, be in vitro, e.g., in cell culture, or present in a multicellular organism, including, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats.","The cell may be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).","By “NOGO proteins” is meant, a protein, protein receptor or a mutant protein derivative thereof, comprising neuronal inhibitor activity, preferably CNS neuronal growth inhibitor activity.","By “IKK-gamma proteins” is meant, a peptide or protein comprising a IKK-gamma or NEMO/IKKAP1 component of the IKK complex, for example a regulatory IKK subunit involved in the assembly of the high molecular weight IKK complex and/or induction of NFKB.","By “PKR proteins” is meant, a peptide or protein comprising a protein kinase PKR activity, for example the activation of NFKB.","By “PTGDR proteins” is meant, a protein receptor or a mutant protein or peptide derivative thereof, having prostaglandin D2 receptor activity, for example, having the ability to bind prostaglandin D2 and/or having GTP-binding protein coupled activity.","By “PTGDS proteins” is meant, a prostaglandin synthetase protein or a mutant protein or peptide derivative thereof, having prostaglandin D2 synthetase activity, for example, having the ability to convert PGH2 to PGD2.By “highly conserved sequence region” is meant, a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.","The nucleic acid-based inhibitors of the invention can be added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues, for example by pulmonary delivery of an aerosol formulation with an inhaler or nebulizer.","The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through inhalation, injection or infusion pump, with or without their incorporation in biopolymers.","In preferred embodiments, the enzymatic nucleic acid inhibitors comprise sequences that are complementary to the substrate sequences in Tables III to XXIII.","Examples of such enzymatic nucleic acid molecules also are shown in Tables III to XXIII.","Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in these tables.","In another embodiment, the invention features antisense nucleic acid molecules, siRNA and 2-5A chimeras including sequences complementary to the substrate sequences shown in Tables III to XXIII.","Such nucleic acid molecules can include sequences as shown for the binding arms of the enzymatic nucleic acid molecules in Tables III to XXIII.","Similarly, triplex molecules can be provided targeted to the corresponding DNA target regions, and containing the DNA equivalent of a target sequence or a sequence complementary to the specified target (substrate) sequence.","Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.","However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop.","Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.","By “consists essentially of” is meant that the active nucleic acid molecule of the invention, for example, an enzymatic nucleic acid molecule, contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind RNA such that cleavage at the target site occurs.","Other sequences can be present that do not interfere with such cleavage.","Thus, a core region can, for example, include one or more loop, stem-loop structure, or linker which does not prevent enzymatic activity.","Thus, the underlined regions in the sequences in Tables III, IV, VIII, IX, XIII, XIV, XIX, and XX can be such a loop, stem-loop, nucleotide linker, and/or non-nucleotide linker and can be represented generally as sequence “X”.","For example, a core sequence for a hammerhead enzymatic nucleic acid can comprise a conserved sequence, such as 5′-CUGAUGAG-3′ and 5′-CGAA-3′ connected by “X”, where X is 5′- GCCGUUAGGC -3′ (SEQ ID NO: 13274), or any other Stem II region known in the art, or a nucleotide and/or non-nucleotide linker.","Similarly, for other nucleic acid molecules of the instant invention, such as Inozyme, G-cleaver, amberzyme, zinzyme, DNAzyme, antisense, 2-5A antisense, triplex forming nucleic acid, siRNA and decoy nucleic acids, other sequences or non-nucleotide linkers can be present that do not interfere with the function of the nucleic acid molecule.","Sequence X can be a linker of ≧2 nucleotides in length, including 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the nucleotides can be internally base-paired to form a stem of ≧2 base pairs.","Alternatively or in addition, sequence X can be a non-nucleotide linker.","In yet another embodiment, the nucleotide linker X can be a nucleic acid aptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others (for a review see Gold et al., 1995 , Annu.","Rev.","Biochem., 64, 763; and Szostak & Ellington, 1993, in The RNA World , ed.","Gesteland and Atkins, pp.","511, CSH Laboratory Press).","A “nucleic acid aptamer” as used herein is meant to indicate a nucleic acid sequence capable of interacting with a ligand.","The ligand can be any natural or a synthetic molecule, including but not limited to a resin, metabolites, nucleosides, nucleotides, drugs, toxins, transition state analogs, peptides, lipids, proteins, amino acids, nucleic acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria and others.","In yet another embodiment, the non-nucleotide linker X is as defined herein.","The term “non-nucleotide” as used herein include either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds.","Specific examples include those described by Seela and Kaiser, Nucleic Acids Res.","1990, 18:6353 and Nucleic Acids Res.","1987, 15:3113; Cload and Schepartz, J.","Am.","Chem.","Soc.","1991, 113:6324; Richardson and Schepartz, J.","Am.","Chem.","Soc.","1991, 113:5109; Ma et al., Nucleic Acids Res.","1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res.","1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett.","1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No.","WO 89/02439; Usman et al., International Publication No.","WO 95/06731; Dudycz et al., International Publication No.","WO 95/11910 and Ferentz and Verdine, J.","Am.","Chem.","Soc.","1991, 113:4000, all hereby incorporated by reference herein.","A “non-nucleotide” further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.","The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.","Thus, in a preferred embodiment, the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties, and having enzymatic activity to cleave an RNA or DNA molecule.","In another aspect of the invention, nucleic acid molecules that interact with target RNA molecules and down-regulate target genes (e.g., prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene) activity are expressed from transcription units inserted into DNA or RNA vectors.","The recombinant vectors can be DNA plasmids or viral vectors.","Enzymatic nucleic acid molecule or antisense expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.","The recombinant vectors capable of expressing the enzymatic nucleic acid molecules or antisense can be delivered as described above, and persist in target cells.","Alternatively, viral vectors can be used that provide for transient expression of enzymatic nucleic acid molecules or antisense.","Such vectors can be repeatedly administered as necessary.","Once expressed, the enzymatic nucleic acid molecules or antisense bind to the target RNA and down-regulate its function or expression.","Delivery of enzymatic nucleic acid molecule or antisense expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell.","Antisense DNA can be expressed via the use of a single stranded DNA intracellular expression vector.","By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.","By “patient” is meant an organism, which is a donor or recipient of explanted cells, or the cells themselves.","“Patient” also refers to an organism to which the nucleic acid molecules of the invention can be administered.","A patient can be a mammal or mammalian cells.","In one embodiment, a patient is a human or human cells.","By “enhanced enzymatic activity” is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both the catalytic activity and the stability of the nucleic acid molecules of the invention.","In this invention, the product of these properties can be increased in vivo compared to an all RNA enzymatic nucleic acid or all DNA enzyme.","In some cases, the activity or stability of the nucleic acid molecule can be decreased (i.e., less than ten-fold), but the overall activity of the nucleic acid molecule is enhanced, in vivo.","The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above.","For example, to treat a disease or condition associated with the levels of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR, the patient can be treated, or other appropriate cells can be treated, as is evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.","In a further embodiment, the described molecules, such as antisense or enzymatic nucleic acid molecules, can be used in combination with other known treatments to treat conditions or diseases discussed above.","For example, the described molecules can be used in combination with one or more known therapeutic agents to treat allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR expression.","By “comprising” is meant including, but not limited to, whatever follows the word “comprising”.","Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.","By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”.","Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.","Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims."],["This patent application claims priority from U.S. Ser.","No.","09/827,395, filed Apr.","5, 2001, entitled “METHOD AND REAGENT FOR THE INHIBITION OF NOGO AND NOGO RECEPTOR GENES”, which is a continuation-in-part of Blatt, U.S. Ser.","No.","09/780,533 filed Feb. 9, 2001, entitled “METHOD AND REAGENT FOR THE INHIBITION OF NOGO GENE” which claims priority from Blatt, U.S. Ser.","No.","60/181,797, filed Feb. 11, 2000, entitled “METHOD AND REAGENT FOR THE INHIBITION OF NOGO GENE”.","This patent application also claims priority from U.S. Ser.","No.","60/294,412, filed May 29, 2001, entitled “ENZYMATIC NUCLEIC ACID TREATMENT OF DISEASES OR CONDITIONS RELATED TO LEVELS OF IKK-GAMMA AND PKR”, and U.S. Ser.","No.","60/315,315, filed Aug. 28, 2001, entitled “METHOD AND REAGENT FOR THE TREATMENT OF ASTHMA AND ALLERGIC CONDITIONS”.","These applications are hereby incorporated by reference herein in their entirety including the drawings.","BACKGROUND OF THE INVENTION The present invention provides compounds, compositions, and methods for the study, diagnosis, and treatment of conditions relating to the expression of NOGO and NOGO receptor genes.","In particular, the invention provides nucleic acid molecules that are used to modulate the expression of NOGO and NOGO receptor gene products.","The present invention further relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to IKK gamma (IKKG) and PKR levels, such as cancer, inflammatory, and autoimmune diseases and/or disorders.","In addition, the present invention also relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to allergic response.","Specifically, the invention provides compositions and methods for the treatment of diseases or conditions related to levels of factors involved in allergic conditions such as asthma, for example prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS) and adenosine A1 receptor (ADORA1).","The discussions that follow are not meant to be complete and are provided only to assist understanding the invention that follows.","The summary is not an admission that any of the work described below is prior art to the claimed invention.","The following is a brief description of the current understanding of NOGO and NOGO receptors.","The ceased growth of neurons following development has severe implications for lesions of the central nervous system (CNS) caused by neurodegenerative disorders and traumatic accidents.","Although CNS neurons have the capacity to rearrange their axonal and dendritic foci in the developed brain, the regeneration of severed CNS axons spanning distance does not exist.","Axonal growth following CNS injury is limited by the local tissue environment rather than intrinsic factors, as indicated by transplantation experiments (Richardson et al., 1980, Nature, 284, 264-265).","Non-neuronal glial cells of the CNS, including oligodendrocytes and astrocytes, have been shown to inhibit the axonal growth of dorsal root ganglion neurons in culture (Schwab and Thoenen, 1985, J.","Neurosci., 5, 2415-2423).","Cultured dorsal root ganglion cells can extend their axons across glial cells from the peripheral nervous system, (ie; Schwann cells), but are inhibited by oligodendrocytes and myelin of the CNS (Schwab and Caroni, 1988, J.","Neurosci., 8, 2381-2393).","The non-conducive properties of CNS tissue in adult vertebrates is thought to result from the existence of inhibitory factors rather than the lack of growth factors.","The identification of proteins with neurite outgrowth inhibitory or repulsive properties include NI-35, NI-250 (Caroni and Schwab, 1988, Neuron, 1, 85-96), myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945).","Monoclonal antibodies (mAb IN-1) raised against NI-35/250 have been shown to partially neutralize the growth inhibitory effect of CNS myelin and oligodendrocytes.","IN-1 treatment in vivo has resulted in long distance fiber regeneration in lesioned adult mammalian CNS tissue (Weibel et al., 1994, Brain Res., 642, 259-266).","Additionally, IN-1 treatment in vivo has resulted in the recovery of specific reflex and locomotor functions after spinal cord injury in adult rats (Bregman et al., 1995, Nature, 378, 498-501).","Recently, the cloning of NOGO-A (Genbank Accession No AJ242961), the rat complementary DNA encoding NI-220/250 has been reported (Chen et al., 2000, Nature, 403, 434-439).","The NOGO gene encodes at least three major protein products (NOGO-A, NOGO-B, and NOGO-C) resulting from both alternative promoter usage and alternative splicing.","Recombinant NOGO-A inhibits neurite outgrowth from dorsal root ganglia and the spreading of 3T3 firboblasts.","Monoclonal antibody IN-1 recognizes NOGO-A and neutralizes NOGO-A inhibition of neuronal growth in vitro.","Evidence supports the proposal that NOGO-A is the previously described rat NI-250 since NOGO-A contains all six peptide sequences obtained from purified bNI-220, the bovine equivalent of rat NI-250 (Chen et al supra).","Prinjha et al., 2000, Nature, 403, 383-384, report the cloning of the human NOGO gene which encodes three different NOGO isoforms that are potent inhibitors of neurite outgrowth.","Using oligonucleotide primers to amplify and clone overlapping regions of the open reading frame of NOGO cDNA, Phrinjha et al., supra identified three forms of cDNA clone corresponding to the three protein isoforms.","The longest ORF of 1,192 amino acids corresponds to NOGO-A (Accession No.","AJ251383).","An intermediate-length splice variant that lacks residues 186-1,004 corresponds to NOGO-B (Accession No.","AJ251384).","The shortest splice variant, NOGO-C (Accession No.","AJ251385), appears to be the previously described rat vp20 (Accession No.","AF051335) and foocen-s (Accession No.","AF132048), and also lacks residues 186-1,004.According to Prinjha et al., supra, the NOGO amino-terminal region shows no significant homology to any known protein, while the carboxy-terminal tail shares homology with neuroendocrine-specific proteins and other members of the reticulon gene family.","In addition, the carboxy-terminal tail contains a consensus sequence that may serve as an endoplasmic-reticulum retention region.","Based on the NOGO protein sequence, Prinjha et al., supra, postulate NOGO to be a membrane associated protein comprising a putative large extracellular domain of 1,024 residues with seven predicted N-linked glycosylation sites, two or three transmembrane domains, and a short carboxy-terminal region of 43 residues.","Grandpre et al., 2000, Nature, also report the identification of NOGO as a potent inhibitor of axon regeneration.","The 4.1 kilobase NOGO human cDNA clone identified by Grandpre et al., supra, KIAA0886, is homologous to a cDNA derived from a previous effort to sequence random high molecular-weight brain derived cDNAs (Nagase et al., 1998, DNA Res., 31, 355-364).","This cDNA clone encodes a protein that matches all six of the peptide sequences derived from bovine NOGO.","Grandpre et al., supra demonstrate that NOGO expression is predominantly associated with the CNS and not the peripheral nervous system (PNS).","Cellular localization of NOGO protein appears to be predominantly reticluar in origin, however, NOGO is found on the surface of some oligodentrocytes.","An active domain of NOGO has been identified, defined as residues 31-55 of a hydrophilic 66-residue lumenal/extracellular domain.","A synthetic fragment corresponding to this sequence exhibits growth-cone collapsing and outgrowth inhibiting activities (Grandpre et al., supra).","A receptor for the NOGO-A extracellular domain (NOGO-66) is described in Fournier et al., 2001, Nature, 409, 341-346.Fournier et al., have shown that isolated NOGO-66 inhibits axonal extension but does not alter non-neuronal cell morphology.","The receptor identified has a high affinity for soluble NOGO-66, and is expressed as a glycophosphatidylinostitol-linked protein on the surface of CNS neurons.","Furthermore, the expression of the NOGO-66 receptor in neurons that are NOGO insensitive results in NOGO dependent inhibition of axonal growth in these cells.","Cleavage of the NOGO-66 receptor and other glycophosphatidylinostitol-linked proteins from axonal surfaces renders neurons insensitive to NOGO-66 inhibition.","As such, disruption of the interaction between NOGO-66 and the NOGO-66 receptor provides the possibility of treating a wide variety of neurological diseases, injuries, and conditions.","Hauswirth and Flannery, International PCT Publication No.","WO 98/48027, describe materials and methods for the specific expression of proteins in retinal photoreceptor cells consisting of an adeno-associated viral vector contacting a rod or cone-opsin promoter.","In addition, ribozymes which degrade mutant mRNA are described for use in the treatment of retinitis pigmentosa.","Fechteler et al., Interanational PCT Publication No.","WO 00/03004 describe ribozymes targeting presenilin-2 RNA for the use in treating neurodegenerative diseases such as Alzheimer's disease.","Eldadah et al., 2000, J.","Neurosci., 20, 179-186, describe the protection of cerebellar granule cells from apoptosis induced by serum-potassium deprivation from ribozyme mediated inhibition of caspase-3.Seidman et al., 1999, Antisense Nucleic Acid Drug Dev., 9, 333-340, describe in general terms, the use of antisense and ribozyme constructs for treatment of neurodegenerative diseases.","Denman et al., 1994, Nucleic Acids Research, 22, 2375-82, describe the ribozyme mediated degradation of beta-amyloid peptide precursor mRNA in COS-7 cells.","Schwab and Chen, International PCT publication No.","WO 00/31235, describe NOGO proteins and inhibitors of NOGO.","Blatt et al., International PCT publication No.","WO 01/59103, describe nucleic acid molecules for modulating expression of NOGO genes.","The following is a brief description of the physiological role of nuclear factor kappa B (NFKB), IKK kinases, and protein kinase PKR.","Nuclear factor kappa B (NFKB) is a multiunit transcription factor which regulates the expression of genes involved in a number of physiologic and pathologic processes.","NFKB is a key component of the TNF signaling pathway.","These processes include, but are not limited to: apoptosis, immune, inflammatory and acute phase responses.","The REL-A gene product (a.k.a.","RelA or p65), and p50 subunits of NFKB, have been implicated in the induction of inflammatory responses and cellular transformation.","NFKB exists in the cytoplasm as an inactive heterodimer of the p50 and p65 subunits.","NFKB is complexed with an inhibitory protein complex, IkappaB (IKK complex), until activated by the appropriate stimuli.","NFKB activation can occur following stimulation of a variety of cell types by inflammatory mediators, for example TNF and IL-1, and reactive oxygen intermediates.","In response to induction, NFKB can stimulate production of pro-inflammatory cytokines such as TNF-alpha, IL-1-beta, IL-6 and iNOS, thereby perpetuating a positive feedback loop.","NFKB appears to play a role in a number of disease processes including: ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, arthritis, and cancer.","The nuclear DNA-binding protein, NFKB, was first identified as a factor that binds and activates the immunoglobulin kappa light chain enhancer in B cells.","NFKB now is known to activate transcription of a variety of other cellular genes (e.g., cytokines, adhesion proteins, oncogenes and viral proteins) in response to a variety of stimuli (e.g., phorbol esters, mitogens, cytokines and oxidative stress).","In addition, molecular and biochemical characterization of NFKB has shown that the activity is due to a homodimer or heterodimer of a family of DNA binding subunits.","Each subunit bears a stretch of 300 amino acids that is homologous to the oncogene, v-rel.","The activity first described as NFKB is a heterodimer of p49 or p50 with p65.The p49 and p50 subunits of NFKB (encoded by the NF-kappa B2 or NF kappa B1 genes, respectively) are generated from the precursors NFKB1 (p105) or NFKB2 (p100).","The p65 subunit of NFKB (now termed REL-A) is encoded by the rel-A locus.","The roles of each specific transcription-activating complex now are being elucidated in cells (Perkins, et al., 1992, Proc.","Natl.","Acad.","Sci USA, 89, 1529-1533).","For instance, the heterodimer of NFKB1 and Rel A (p50/p65) activates transcription of the promoter for the adhesion molecule, VCAM-1, while NFKB2/RelA heterodimers (p49/p65) actually inhibit transcription (Shu, et al., 1993, Mol.","Cell.","Biol., 13, 6283-6289).","Conversely, heterodimers of NFKB2/RelA (p49/p65) act with Tat-I to activate transcription of the HIV genome, while NFKB1/RelA (p50/p65) heterodimers have little effect (Liu et al., 1992, J.","Virol., 66, 3883-3887).","Similarly, blocking rel A gene expression with antisense oligonucleotides specifically blocks embryonic stem cell adhesion; blocking NFKB1 gene expression with antisense oligonucleotides had no effect on cellular adhesion (Narayanan et al., 1993, Mol.","Cell.","Biol., 13, 3802-3810).","Thus, the promiscuous role initially assigned to NFKB in transcriptional activation (Lenardo, and Baltimore, 1989, Cell, 58, 227-229) represents the sum of the activities of the rel family of DNA-binding proteins.","This conclusion is supported by recent transgenic “knock-out” mice of individual members of the rel family.","Such “knock-outs” show few developmental defects, suggesting that essential transcriptional activation functions can be performed by more than one member of the rel family.","A number of specific inhibitors of NFKB function in cells exist, including treatment with phosphorothioate antisense oliogonucleotide, treatment with double-stranded NFKB binding sites, and over expression of the natural inhibitor MAD-3 (an Ikappa-B family member).","These agents have been used to show that NFKB is required for induction of a number of molecules involved in cancer and/or inflammation, as described below.","NFkB is required for phorbol ester-mediated induction of IL-6 (Kitajima, et al., 1992, Science, 258, 1792-5) and IL-8 (Kunsch and Rosen, 1993, Mol.","Cell.","Biol., 13, 6137-46).","NFkB is required for induction of the adhesion molecules ICAM-1 (Eck, et al., 1993, Mol.","Cell.","Biol., 13, 6530-6536), VCAM-1 (Shu et al., supra), and E-selectin (Read, et al., 1994, J. Exp.","Med., 179, 503-512) on endothelial cells.","NFkB is involved in the induction of the integrin subunit, CD18, and other adhesive properties of leukocytes (Eck et al., 1993 supra).","HER2/Neu overexpression induces NFKB via a PI3-kinase/Akt pathway involving calpain-mediated degradation of IKB-alpha.","Breast cancer has been shown to typify the aberrant expression of NFKB/REL factors (Pianetti et al., 2001, Oncogene, 20, 1287-1299; Sovak et al., 1999, J. Clin.","Invest., 100, 2952-2960).","Inhibition of NFKB activity has been shown to induce apoptosis in murine hepatocytes (Bellas et al., 1997, Am.","J.","Pathol., 151, 891-896).","NFKB has been shown to regulate cyclooxygenase-2 expression and cell proliferation in human gastric cancer cells (Joo Weon et al., 2001, Laboratory Investigation, 81, 349-360).","The above studies suggest that NFKB is integrally involved in the induction of cytokines and adhesion molecules by inflammatory mediators and is involved in the transformation of cancerous cells.","Two reported studies point to another connection between NFKB and inflammation: glucocorticoids can exert their anti-inflammatory effects by inhibiting NFKB.","The glucocorticoid receptor and p65 both act at NFKB binding sites in the ICAM-1 promoter (van de Stolpe, et al., 1994, J. Biol.","Chem., 269, 6185-6192).","Glucocorticoid receptor inhibits NFKB-mediated induction of IL-6 (Ray and Prefontaine, 1994 Proc.","Natl.","Acad.","Sci USA, 91, 752-756).","Conversely, overexpression of p65 inhibits glucocorticoid induction of the mouse mammary tumor virus promoter.","Finally, protein cross-linking and co-immunoprecipitation experiments demonstrated direct physical interaction between p65 and the glucocorticoid receptor.","The IKK complex that sequesters NFKB in the cytoplasm comprises IkappaB (IκB) proteins (IκB-alpha, IκB-beta, IκKB-epsilon, p105, and p100).","The phosphorylation of IκB proteins results in the release of NFKB from the IκB complex which is transported to the nucleus via the unmasking of nuclear translocation signals.","Phosphorylation marks IkB proteins for ubiquitination and degradation via the proteosome pathway.","Most NFKB inducing stimuli initiate activation of an IκB kinase (IKK) complex that contains two catalytic subunits, IKK-alpha (IKK1) and IKK-beta (IKK2), that phosphorylate IκB-alpha and IκB-beta, with IKK-beta playing a predominant role in pro-inflammatory signaling.","In addition to the two kinases, the IKK complex contains regulatory subunits, including IKK-gamma (NEMO/IKKAP1).","IKK-gamma is a protein that is critical for the assembly of the IKK complex.","IKK-gamma directly binds to IKK-beta and is required for activation of NFKB, for example by TNF-alpha, IL-1-beta, lipopolysaccharide, phorbol 12-myristate 13-acetate, the human T-cell lymphotrophic virus (HTLV-1), or double stranded RNA.","Genomic rearrangements in IKK-gamma have been shown to impair NFKB activation and result in incontinentia pigmenti.","Additional proteins that associate with the IKK complex include, MEK kinase (MEKK1), NFKB inducing kinase (NIK), receptor interacting protein (RIP), protein kinase CK2, and IKK-associated protein (IKAP), which appears to be associated with the IκB Kinase (IKK) complex, but does not appear to be an integral component of the tripartate IKK complex as does IKK-gamma (Krappmann et al., 2001, J. Biol.","Chem., 275, 29779-87).","The RNA-dependent protein kinase PKR is a signal transducer for NFKB and IFN regulatory factor-1.PKR is required for activation of NFKB by IFN-gamma via a STAT-1 independent pathway (Amitabha et al., 2001, J.","Immunol., 166, 6170-6180).","The induction of NFKB by PKR takes place though phosphorylation of IκB-alpha, and appears not to require the catalytic activity of PKR, thereby proceeding independently of the dsRNA-binding properties of PKR (Ishii et al., 2001, Oncogene, 20, 1900-1912).","PKR also plays an important role in the regulation of protein synthesis by modulating the activity of eukaryotic initiation factor 2 (eIF-2-alpha) through interferon induction.","Kamiya, JP 2000253884, describes specific antisense oligonucleotides for inhibiting IκB-kinase subunit expression.","Krappmann et al., 2001, J. Biol.","Chem., describe specific antisense oligonucleotides to IKK-gamma.","The following is a description of molecular targets involved in diseases or conditions related to allergic response.","Asthma is a chronic inflammatory disorder of the lungs characterized by airflow obstruction, bronchial hyper-responsiveness, and airway inflammation.","T-lymphocytes that produce TH2 cytokines and eosinophilic leukocytes infiltrate the airways.","In the airway and in bronchial alveolar lavage (BAL) fluid of individuals with asthma, high concentrations of TH2 cytokines, interleukin-4 (14), IL-5, and IL-13, are present along with increased levels of adenosine.","In contrast to normal individuals, asthmatics respond to adenosine challenge with marked airway obstruction.","Upon allergen challenge, mast cells are activated by cross-linked IgE-allergen complexes.","Large amounts of prostaglandin D2 (PGD2), the major cyclooxygenase product of arachidonic acid are released.","PGD2 is generated from PGH2 via the activity of prostaglandin D2 synthetase (PTGDS).","PGD2 receptors and adenosine A1 receptors are present in the lungs and airway along with various other tissues in response to allergic stimuli (Howarth, 1997, Allergy, 52, 12).","The significance of PGD2 as a mediator of allergic asthma has been established with the development of mice deficient in the PGD2 receptor (DP).","DP is a heterotrimeric GTP-binding protein-coupled, rhodopsin-type receptor specific for PGD2 (Hirata et al., 1994, PNAS USA., 91, 11192).","These mice fail to develop airway hyperreactivity and have greatly reduced eosinophil infiltration and cytokine accumulation in response to allergens.","Upon allergen challenge mice deficient in the prostaglandin D2 (PGD2) receptor (DP) did not develop airway hyperactivity.","Cytokine, lymphocyte and eosinophil accumulation in the lungs were greatly reduced (Matsuoka et al., 2000, Science, 287, 2013).","The DP −/− mice exhibited no behavioral, anatomic, or histological abnormalities.","Primary immune response is not affected by DP disruption.","Asthma affects more than 100 million people worldwide and more than 17 million Americans (5% of the population).","Since 1980 the incidence has more than doubled and deaths have tripled (5,000 deaths in 1995).","Annual asthma-related healthcare costs in the US alone were estimated to exceed $14.5 billion in 2000.Current therapies such as inhalant anti-inflammatories and bronchodilators can be used to treat symptoms, however, these therapies do not prevent or cure asthma.","Sandberg et al., 2001, Prog.","Respir.","Res., 31, 370-373, describes ribozyme therapy for asthma and COPD.","Sullivan et al., International U.S. Pat.","No.","5,616,488, describes ribozymes targeting interleukin-5 for treatment and diagnosis of asthma and other inflammatory disorders.","Stinchcomb et al., International PCT Publication No.","WO 95/23225, describes ribozymes and methods for inhibiting the expression of disease related genes including genes associated with asthma.","Nyce, International PCT Publication Nos.","WO 00/62736, WO 00/09525, WO 99/13886, WO 98/23294, WO 96/40266 and U.S. Pat.","No.","6,025,339 describe specific antisense oligonucleotides targeting certain mRNAs encoding particular adenosine receptors.","SUMMARY OF THE INVENTION The invention features novel nucleic acid-based molecules, for example, enzymatic nucleic acid molecules, allozymes, antisense nucleic acids, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNA, dsRNA, siRNA, aptamers, and antisense nucleic acids containing RNA cleaving chemical groups, and methods to modulate gene expression; for example, gene(s) encoding prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), and adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3; gene(s) encoding NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors; and genes encoding an IkappaB kinase (IKK) subunit or protein kinase PKR.","In one embodiment, the instant invention features nucleic-acid based techniques to inhibit the expression of NOGO-A (Accession No.","AJ251383), NOGO-B (Accession No.","AJ251384), and/or NOGO-C (Accession No.","AJ251385), NOGO-66 receptor (Accession No AF283463, Fournier et al., 2001, Nature, 409, 341-346), NI-35, NI-220, and/or NI-250, myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945).","The description below of the various aspects and embodiments is provided with reference to the exemplary NOGO-A and NOGO-66 receptor genes.","However, the various aspects and embodiments are also directed to other genes which express NOGOA-like inhibitor proteins and other receptors involved in neurite outgrowth inhibition.","Those additional genes can be analyzed for target sites using the methods described for NOGO and the NOGO-66 receptor, referred to alternatively as NOGO receptor.","Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.","The invention features one or more enzymatic nucleic acid-based molecules and methods that independently or in combination modulate the expression of gene(s) encoding a member of the IκB kinase IKK complex or PKR.","In particular embodiments, the invention features nucleic acid-based molecules and methods that modulate the expression of a member of the IκB kinase IKK complex, for example IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKKγ) and/or a protein kinase PKR protein, such as IKK-alpha (IKK1) gene (Genbank Accession No.","NM—001278); IKK-beta (IKK2) gene, for example (Genbank Accession No.","AF080158), IKK-gamma (IKKγ) gene, for example (Genbank Accession No.","NM—003639), and protein kinase PKR gene, for example (Genbank Accession No.","NM—002759).","The description below of the various aspects and embodiments is provided with reference to the exemplary IKK-gamma and PKR genes.","IKK-gamma is also known as NEMO/IKKAP1.However, the various aspects and embodiments are also directed to other genes which encode other subunits of the IKK complex, such as IKK-alpha (IKK1) or IKK-beta (IKK2).","Those additional genes can be analyzed for target sites using the methods described for IKK-gamma or PKR.","Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.","In one embodiment, an enzymatic nucleic acid molecule of the invention is in a hammerhead, Inozyme, Zinzyme, DNAzyme, Amberzyme, or G-cleaver configuration.","In another embodiment, a nucleic acid molecule of the invention comprises between 8 and 100 bases complementary to the RNA of the target gene.","In another embodiment, a nucleic acid molecule of the invention comprises between 14 and 24 bases complementary to a RNA molecule of the target gene.","In one embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention is chemically synthesized.","In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one 2′-sugar modification.","In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one nucleic acid base modification.","In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one phosphate backbone modification.","In one embodiment, the invention features a mammalian cell, for example a human cell, including the nucleic acid molecule of the invention.","In another embodiment, the invention features a method of reducing target gene expression or activity in a cell, comprising contacting the cell with a nucleic acid molecule of the invention, such as an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups, under conditions suitable for the reduction.","In yet another embodiment, the invention features a method of treatment of a patient having a condition associated with the level of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR, comprising contacting cells of the patient with an enzymatic nucleic acid molecule of the invention, under conditions suitable for the treatment.","In another embodiment, a method of treatment of a patient having a condition associated with the level of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR is featured, wherein the method further comprises the use of one or more drug therapies under conditions suitable for the treatment.","In another embodiment, the invention features a method of cleaving a RNA molecule of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene, comprising contacting an enzymatic nucleic acid molecule of the invention with a RNA molecule of the corresponding gene under conditions suitable for the cleavage, for example, wherein the cleavage is carried out in the presence of a divalent cation, such as Mg2+.","In one embodiment, a nucleic acid molecule of the invention comprises a cap structure, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end of the enzymatic nucleic acid molecule.","In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one nucleic acid molecule of the invention, in a manner which allows expression of the nucleic acid molecule.","In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.","In yet another embodiment, the expression vector of the invention further comprises a sequence for an antisense nucleic acid molecule complementary to a RNA molecule of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene.","In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more nucleic acid molecules, such as enzymatic nucleic acid molecules, antisense, aptamers, decoys, siRNA, or 2-5A chimeras which can be the same or different.","In one embodiment, the method of treatment features an enzymatic nucleic acid molecule of the invention comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification, such as a 3′-3′ inverted abasic moiety.","In another embodiment, an enzymatic nucleic acid molecule or antisense nucleic acid molecule of the invention further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.","In another embodiment, the invention features a method of administering to a mammal, for example a human, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention, comprising contacting the mammal with the nucleic acid molecule under conditions suitable for the administration, for example, in the presence of a delivery reagent such as a lipid, cationic lipid, phospholipid, or liposome.","In yet another embodiment, the invention features a method of administering to a mammal an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention in conjunction with a therapeutic agent, comprising contacting the mammal, for example a human, with the nucleic acid molecule and the therapeutic agent under conditions suitable for the administration.","In one embodiment, the invention features the use of an enzymatic nucleic acid molecule, which can be in a hammerhead, NCH, G-cleaver, Amberzyme, Zinzyme, and/or DNAzyme motif, to down-regulate the expression of a a target gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene.","By “inhibit”, “down-regulate”, or “reduce”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR subunits, is reduced below that observed in the absence of the nucleic acid molecules of the invention.","In one embodiment, inhibition, down-regulation or reduction with an enzymatic nucleic acid molecule is below that level observed in the presence of an enzymatically inactive or attenuated molecule that is able to bind to the same site on the target RNA molecule, but is unable to cleave that RNA molecule.","In another embodiment, inhibition, down-regulation, or reduction with antisense oligonucleotides is below that level observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches.","In another embodiment, inhibition, down-regulation, or reduction of the target gene with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.","By “up-regulate” is meant that the expression of a gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins, protein subunits, or activity of one or more proteins or protein subunits, such as a target gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR subunits, is greater than that observed in the absence of the nucleic acid molecules of the invention.","For example, the expression of a gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene, can be increased in order to treat, prevent, ameliorate, or modulate a pathological condition caused or exacerbated by an absence or low level of gene expression.","By “modulate” is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more protein subunits, or activity of one or more protein subunits is up-regulated or down-regulated, such that the expression, level, or activity is greater than or less than that observed in the absence of a nucleic acid molecule of the invention.","By “enzymatic nucleic acid molecule” it is meant a nucleic acid molecule that has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that is active to specifically cleave target a RNA molecule.","That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave a RNA molecule and thereby inactivate a target RNA molecule.","These complementary regions allow sufficient hybridization of an enzymatic nucleic acid molecule to a target RNA molecule and thus permit cleavage.","One hundred percent complementarity is preferred, but complementarity as low as 50-75% can also be useful in this invention (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31).","The nucleic acids can be modified at the base, sugar, and/or phosphate groups.","The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme.","All of these terminologies describe nucleic acid molecules with enzymatic activity.","The specific enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site that is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule (Cech et al., U.S. Pat.","No.","4,987,071; Cech et al., 1988, 260 JAMA 3030).","By “nucleic acid molecule” as used herein is meant a molecule having nucleotides.","The nucleic acid can be single, double, or multiple stranded and can comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.","Exemplary nucleic acid molecules of the invention include enzymatic nucleic acid molecules, allozymes, antisense nucleic acids, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNA, dsRNA, siRNA, aptamers, and/or antisense nucleic acids containing RNA cleaving chemical groups.","By “enzymatic portion” or “catalytic domain” is meant that portion/region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate (for example see FIGS.","1-4).","By “substrate binding arm” or “substrate binding domain” is meant that portion/region of a enzymatic nucleic acid that is able to interact, for example via complementarity (i.e., able to base-pair with), with a portion of its substrate.","Such complementarity can be 100%, but can be less if desired.","For example, as few as 10 bases out of 14 can be base-paired (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31).","Examples of such arms are shown generally in FIGS.","1-4.That is, these arms contain sequences within an enzymatic nucleic acid that are intended to bring enzymatic nucleic acid and target RNA together through complementary base-pairing interactions.","The enzymatic nucleic acid of the invention can have binding arms that are contiguous or non-contiguous and can be of varying lengths.","The length of the binding arm(s) can be greater than or equal to four nucleotides and of sufficient length to stably interact with a target RNA; in one embodiment they can be 12-100 nucleotides; in another embodiment they can be 14-24 nucleotides long (see for example Werner and Uhlenbeck, supra; Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herranze et al., 1993, EMBO J., 12, 2567-73) or between 8 and 14 nucleotides long.","If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., four and four, five and five nucleotides, or six and six nucleotides, or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., three and five, six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).","By “Inozyme” or “NCH” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as NCH Rz in FIG.","1.Inozymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCH/, where N is a nucleotide, C is cytidine and H is adenosine, uridine or cytidine, and/represents the cleavage site.","H is used interchangeably with X. Inozymes can also possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCN/, where N is a nucleotide, C is cytidine, and / represents the cleavage site.","“I” r in FIG.","1 represents an Inosine nucleotide, including a ribo-Inosine or xylo-Inosine nucleoside.","By “G-cleaver” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as G-cleaver Rz in FIG.","1.G-cleavers possess endonuclease activity to cleave RNA substrates having a cleavage triplet NYN/, where N is a nucleotide, Y is uridine or cytidine and / represents the cleavage site.","G-cleavers can be chemically modified as is generally shown in FIG.","1.By “amberzyme” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG.","2.Amberzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NG/N, where N is a nucleotide, G is guanosine, and / represents the cleavage site.","Amberzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG.","2.In addition, differing nucleoside and/or non-nucleoside linkers can be used to substitute the 5′-gaaa-3′ loops shown in the figure.","Amberzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2′-OH) group within its own nucleic acid sequence for activity.","By “zinzyme” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG.","3.Zinzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet including but not limited to YG/Y, where Y is uridine or cytidine, and G is guanosine and / represents the cleavage site.","Zinzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG.","3, including substituting 2′-O-methyl guanosine nucleotides for guanosine nucleotides.","In addition, differing nucleotide and/or non-nucleotide linkers can be used to substitute the 5′-gaaa-2′ loop shown in the figure.","Zinzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2′-OH) group within its own nucleic acid sequence for activity.","By ‘DNAzyme’ is meant, an enzymatic nucleic acid molecule that does not require the presence of a 2′-OH group within its own nucleic acid sequence for activity.","In particular embodiments the enzymatic nucleic acid molecule can have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups.","DNAzymes can be synthesized chemically or expressed endogenously in vivo, by means of a single stranded DNA vector or equivalent thereof.","An example of a DNAzyme is shown in FIG.","4 and is generally reviewed in Usman et al., U.S. Pat.","No.","6,159,714; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem.","Bio.","2, 655; Santoro et al., 1997, PNAS 94, 4262; Breaker, 1999, Nature Biotechnology, 17, 422-423; and Santoro et.","al., 2000, J.","Am.","Chem.","Soc., 122, 2433-39.The “10-23” DNAzyme motif is one particular type of DNAzyme that was evolved using in vitro selection (see Santoro et al., supra).","Additional DNAzyme motifs can be selected for using techniques similar to those described in these references, and hence, are within the scope of the present invention.","By “sufficient length” is meant an oligonucleotide of greater than or equal to 3 nucleotides that is of a length great enough to provide the intended function under the expected condition.","For example, for binding arms of enzymatic nucleic acid “sufficient length” means that the binding arm sequence is long enough to provide stable binding to a target site under the expected binding conditions.","The binding arms are not so long as to prevent useful turnover of the nucleic acid molecule.","By “stably interact” is meant interaction of the oligonucleotides with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions) that is sufficient to the intended purpose (e.g., cleavage of target RNA by an enzyme).","By “equivalent” or “related” RNA to NOGO is meant to include those naturally occurring RNA molecules having homology (partial or complete) to NOGO-A, NOGO-B, NOGO-C and/or NOGO receptor proteins or encoding for proteins with similar function as NOGO or NOGO receptor proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.","The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.","By “equivalent” or “related” RNA to IKK-gamma is meant to include those naturally occurring RNA molecules having homology (partial or complete) to IKK-gamma proteins or encoding for proteins with similar function as IKK-gamma proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.","The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.","By “equivalent” or “related” RNA to PKR is meant to include those naturally occurring RNA molecules having homology (partial or complete) to PKR proteins or encoding for proteins with similar function as PKR proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.","The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.","By “equivalent” or “related” RNA to PTGDS is meant to include RNA molecules having homology (partial or complete) to RNA molecules encoding PTGDS proteins or encoding proteins with similar function as PTGDS proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites.","The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.","By “equivalent” or “related” RNA to PTGDR is meant to include RNA molecules having homology (partial or complete) to RNA molecules encoding PTGDR proteins or encoding proteins with similar function as PTGDR proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites.","The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.","By “equivalent” or “related” RNA to ADORA1 is meant to include RNA molecules having homology (partial or complete) to RNA molecule encoding ADORA1 proteins or encoding proteins with similar function as ADORA1 proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites.","The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.","By “homology” is meant the nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.","By “antisense nucleic acid”, it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., U.S. Pat.","No.","5,849,902).","Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.","However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop.","Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.","For a review of current antisense strategies, see Schmajuk et al., 1999, J. Biol.","Chem., 274, 21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et al., 1997, Antisense N. A.","Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol., 313, 3-45; Crooke, 1998, Biotech.","Genet.","Eng.","Rev., 15, 121-157, Crooke, 1997, Ad.","Pharmacol., 40, 1-49.In addition, antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.","The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA.","Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.","By “RNase H activating region” is meant a region (generally greater than or equal to 4-25 nucleotides in length, and in one embodiment from 5-11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al., U.S. Pat.","No.","5,849,902; Arrow et al., U.S. Pat.","No.","5,989,912).","The RNase H enzyme binds to the nucleic acid molecule-target RNA complex and cleaves the target RNA sequence.","The RNase H activating region comprises, for example, phosphodiester, phosphorothioate (at least four of the nucleotides are phosphorothiote substitutions; and in another embodiment, 4-11 of the nucleotides are phosphorothiote substitutions); phosphorodithioate, 5′-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof.","In addition to one or more backbone chemistries described above, the RNase H activating region can also comprise a variety of sugar chemistries.","For example, the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry.","Those skilled in the art will recognize that the foregoing are non-limiting examples and that any combination of phosphate, sugar and base chemistry of a nucleic acid that supports the activity of RNase H enzyme is within the scope of the definition of the RNase H activating region and the instant invention.","By “2-5A antisense chimera” is meant an antisense oligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylate residue.","These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc.","Natl.","Acad.","Sci.","USA 90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and Torrence, 1998, Pharmacol.","Ther., 78, 55-113).","By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that is distinct from sequence recognized by the target molecule in its natural setting.","Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid.","The target molecule can be any molecule of interest.","For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein.","This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art, see for example Gold et al., 1995, Annu.","Rev.","Biochem., 64, 763; Brody and Gold, 2000, J.","Biotechnol., 74, 5; Sun, 2000, Curr.","Opin.","Mol.","Ther., 2, 100; Kusser, 2000, J.","Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.By “triplex forming oligonucleotides” is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix.","Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc.","Natl.","Acad.","Sci.","USA 89, 504; Fox, 2000, Curr.","Med.","Chem., 7, 17-37; Praseuth et al., 2000, Biochim.","Biophys.","Acta, 1489, 181-206).","By “gene” it is meant a nucleic acid that encodes an RNA, for example, nucleic acid sequences including but not limited to structural genes encoding a polypeptide.","“Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another RNA molecule by either traditional Watson-Crick or other non-traditional types.","In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., enzymatic nucleic acid cleavage, antisense or triple helix inhibition.","Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp.","Quant.","Biol.","LII pp.","123-133; Frier et al., 1986, Proc.","Nat.","Acad.","Sci.","USA 83:9373-9377; Turner et al., 1987, J.","Am.","Chem.","Soc.","109:3783-3785).","A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).","“Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.","By “RNA” is meant a molecule comprising at least one ribonucleotide residue.","By “ribonucleotide” or “2′-OH” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribo-furanose moiety.","By “decoy RNA” is meant an RNA molecule or aptamer that is designed to preferentially bind to a predetermined ligand.","Such binding can result in the inhibition or activation of a target molecule.","The decoy RNA or aptamer can compete with a naturally occurring binding target for the binding of a specific ligand.","For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a “decoy” and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990, Cell, 63, 601-608).","This is but a specific example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art, see for example Gold et al., 1995, Annu.","Rev.","Biochem., 64, 763; Brody and Gold, 2000, J.","Biotechnol., 74, 5; Sun, 2000, Curr.","Opin.","Mol.","Ther., 2, 100; Kusser, 2000, J.","Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.Similarly, a decoy RNA can be designed to bind to a D2 receptor and block the binding of PTGDS or a decoy RNA can be designed to bind to PTGDS and prevent interaction with the D2 receptor.","The term “short interfering RNA” or “siRNA” as used herein refers to a double stranded nucleic acid molecule capable of RNA interference “RNAi”, see for example Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., International PCT Publication No.","WO 00/44895; Zernicka-Goetz et al., International PCT Publication No.","WO 01/36646; Fire, International PCT Publication No.","WO 99/32619; Plaetinck et al., International PCT Publication No.","WO 00/01846; Mello and Fire, International PCT Publication No.","WO 01/29058; Deschamps-Depaillette, International PCT Publication No.","WO 99/07409; and Li et al., International PCT Publication No.","WO 00/44914.As used herein, siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides.","The term “allozyme” as used herein refers to an allosteric enzymatic nucleic acid molecule, see, e.g., George et al., U.S. Pat.","Nos.","5,834,186 and 5,741,679, Shih et al., U.S. Pat.","No.","5,589,332, Nathan et al., U.S. Pat.","No.","5,871,914, Nathan and Ellington, International PCT publication No.","WO 00/24931, Breaker et al., International PCT Publication Nos.","WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No.","WO 99/29842.The term “2-5A chimera” as used herein refers to an oligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylate residue.","These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc.","Natl.","Acad.","Sci.","USA 90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and Torrence, 1998, Pharmacol.","Ther., 78, 55-113).","The term “triplex forming oligonucleotides” as used herein refers to an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix.","Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc.","Natl.","Acad.","Sci.","USA 89, 504; Fox, 2000, Curr.","Med.","Chem., 7, 17-37; Praseuth et.","al., 2000, Biochim.","Biophys.","Acta, 1489, 181-206).","Several varieties of naturally-occurring enzymatic RNAs are known presently.","Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions.","Table I summarizes some of the characteristics of these ribozymes.","In general, enzymatic nucleic acids act by first binding to a target RNA.","Such binding occurs through the target binding portion of a enzymatic nucleic acid that is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.","Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.","Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein.","After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.","Thus, a single ribozyme molecule is able to cleave many molecules of target RNA.","In addition, the ribozyme is a highly specific inhibitor of gene expression, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage.","Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme.","In one embodiment of the inventions described herein, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but can also be formed in the motif of a hepatitis delta virus, group I intron, group II intron or RNase P RNA (in association with an RNA guide sequence), Neurospora VS RNA, DNAzymes, NCH cleaving motifs, or G-cleavers.","Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al., 1992, AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al., 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al., 1990 Nucleic Acids Res.","18, 299; Chowrira & McSwiggen, U.S. Pat.","No.","5,631,359; of the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNase P motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res.","24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc.","Natl.","Acad.","Sci.","USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO.","J.","14, 363); Group II introns are described by Griffin et al., 1995, Chem.","Biol.","2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al., International PCT Publication No.","WO 96/22689; of the Group I intron by Cech et al., U.S. Pat.","No.","4,987,071 and of DNAzymes by Usman et al., International PCT Publication No.","WO 95/11304; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem.","Bio.","2, 655; Santoro et al., 1997, PNAS 94, 4262, and Beigelman et al., International PCT publication No.","WO 99/55857.NCH cleaving motifs are described in Ludwig & Sproat, International PCT Publication No.","WO 98/58058; and G-cleavers are described in Kore et al., 1998, Nucleic Acids Research 26, 4116-4120 and Eckstein et al., International PCT Publication No.","WO 99/16871.Additional motifs such as the Aptazyme (Breaker et al., WO 98/43993), Amberzyme (Class I motif; FIG.","2; Beigelman et al., U.S. Ser.","No.","09/301,511) and Zinzyme (FIG.","3) (Beigelman et al., U.S. Ser.","No.","09/301,511), all included by reference herein including drawings, can also be used in the present invention.","These specific motifs or configurations are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule (Cech et al., U.S. Pat.","No.","4,987,071).","In one embodiment of the present invention, a nucleic acid molecule of the instant invention can be between 12 and 100 nucleotides in length.","Exemplary enzymatic nucleic acid molecules of the invention are shown in Tables III-XXIII.","For example, enzymatic nucleic acid molecules of the invention can be between 15 and 50 nucleotides in length, and in another embodiment between 25 and 40 nucleotides in length, e.g., 34, 36, or 38 nucleotides in length (for example see Jarvis et al., 1996, J. Biol.","Chem., 271, 29107-29112).","Exemplary DNAzymes of the invention are can between 15 and 40 nucleotides in length, and in one embodiment, between 25 and 35 nucleotides in length, e.g., 29, 30, 31, or 32 nucleotides in length (see, e.g., Santoro et al., 1998, Biochemistry, 37, 13330-13342; Chartrand et al., 1995, Nucleic Acids Research, 23, 4092-4096).","Exemplary antisense molecules of the invention can be between 15 and 75 nucleotides in length, and in one embodiment between 20 and 35 nucleotides in length, e.g., 25, 26, 27, or 28 nucleotides in length (see for example Woolf et al., 1992, PNAS., 89, 7305-7309; Milner et al., 1997, Nature Biotechnology, 15, 537-541).","Exemplary triplex forming oligonucleotide molecules of the invention are between 10 and 40 nucleotides in length, and in one embodiment are between 12 and 25 nucleotides in length, e.g., 18, 19, 20, or 21 nucleotides in length (see for example Maher et al., 1990, Biochemistry, 29, 8820-8826; Strobel and Dervan, 1990, Science, 249, 73-75).","Those skilled in the art will recognize that all that is required is for the nucleic acid molecule to be of length and conformation sufficient and suitable for the nucleic acid molecule to catalyze a reaction contemplated herein.","The length of the nucleic acid molecules of the instant invention are not limiting within the general limits stated.","In one embodiment, a nucleic acid molecule that modulates, for example, down-regulates, the expression of a target gene comprises between 8 and 100 bases complementary to a RNA molecule of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR.","In another embodiment, a nucleic acid molecule that modulates the expression of a target gene comprises between 14 and 24 bases complementary to a RNA molecule of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR.","The invention provides a method for producing a class of nucleic acid-based gene modulating agents that exhibit a high degree of specificity for the RNA of a desired target.","For example, the enzymatic nucleic acid molecule is can be targeted to a highly conserved sequence region of target RNAs encoding prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR (e.g., prostaglandin D2 receptor (PTGDR)), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR genes) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention.","Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required.","Alternatively, the nucleic acid molecules (e.g., ribozymes, antisense, aptamers, and/or siRNA) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.","As used in herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism.","The cell can, for example, be in vitro, e.g., in cell culture, or present in a multicellular organism, including, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats.","The cell may be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).","By “NOGO proteins” is meant, a protein, protein receptor or a mutant protein derivative thereof, comprising neuronal inhibitor activity, preferably CNS neuronal growth inhibitor activity.","By “IKK-gamma proteins” is meant, a peptide or protein comprising a IKK-gamma or NEMO/IKKAP1 component of the IKK complex, for example a regulatory IKK subunit involved in the assembly of the high molecular weight IKK complex and/or induction of NFKB.","By “PKR proteins” is meant, a peptide or protein comprising a protein kinase PKR activity, for example the activation of NFKB.","By “PTGDR proteins” is meant, a protein receptor or a mutant protein or peptide derivative thereof, having prostaglandin D2 receptor activity, for example, having the ability to bind prostaglandin D2 and/or having GTP-binding protein coupled activity.","By “PTGDS proteins” is meant, a prostaglandin synthetase protein or a mutant protein or peptide derivative thereof, having prostaglandin D2 synthetase activity, for example, having the ability to convert PGH2 to PGD2.By “highly conserved sequence region” is meant, a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.","The nucleic acid-based inhibitors of the invention can be added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues, for example by pulmonary delivery of an aerosol formulation with an inhaler or nebulizer.","The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through inhalation, injection or infusion pump, with or without their incorporation in biopolymers.","In preferred embodiments, the enzymatic nucleic acid inhibitors comprise sequences that are complementary to the substrate sequences in Tables III to XXIII.","Examples of such enzymatic nucleic acid molecules also are shown in Tables III to XXIII.","Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in these tables.","In another embodiment, the invention features antisense nucleic acid molecules, siRNA and 2-5A chimeras including sequences complementary to the substrate sequences shown in Tables III to XXIII.","Such nucleic acid molecules can include sequences as shown for the binding arms of the enzymatic nucleic acid molecules in Tables III to XXIII.","Similarly, triplex molecules can be provided targeted to the corresponding DNA target regions, and containing the DNA equivalent of a target sequence or a sequence complementary to the specified target (substrate) sequence.","Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.","However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop.","Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.","By “consists essentially of” is meant that the active nucleic acid molecule of the invention, for example, an enzymatic nucleic acid molecule, contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind RNA such that cleavage at the target site occurs.","Other sequences can be present that do not interfere with such cleavage.","Thus, a core region can, for example, include one or more loop, stem-loop structure, or linker which does not prevent enzymatic activity.","Thus, the underlined regions in the sequences in Tables III, IV, VIII, IX, XIII, XIV, XIX, and XX can be such a loop, stem-loop, nucleotide linker, and/or non-nucleotide linker and can be represented generally as sequence “X”.","For example, a core sequence for a hammerhead enzymatic nucleic acid can comprise a conserved sequence, such as 5′-CUGAUGAG-3′ and 5′-CGAA-3′ connected by “X”, where X is 5′-GCCGUUAGGC-3′ (SEQ ID NO: 13274), or any other Stem II region known in the art, or a nucleotide and/or non-nucleotide linker.","Similarly, for other nucleic acid molecules of the instant invention, such as Inozyme, G-cleaver, amberzyme, zinzyme, DNAzyme, antisense, 2-5A antisense, triplex forming nucleic acid, siRNA and decoy nucleic acids, other sequences or non-nucleotide linkers can be present that do not interfere with the function of the nucleic acid molecule.","Sequence X can be a linker of ≧2 nucleotides in length, including 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the nucleotides can be internally base-paired to form a stem of ≧2 base pairs.","Alternatively or in addition, sequence X can be a non-nucleotide linker.","In yet another embodiment, the nucleotide linker X can be a nucleic acid aptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others (for a review see Gold et al., 1995, Annu.","Rev.","Biochem., 64, 763; and Szostak & Ellington, 1993, in The RNA World, ed.","Gesteland and Atkins, pp.","511, CSH Laboratory Press).","A “nucleic acid aptamer” as used herein is meant to indicate a nucleic acid sequence capable of interacting with a ligand.","The ligand can be any natural or a synthetic molecule, including but not limited to a resin, metabolites, nucleosides, nucleotides, drugs, toxins, transition state analogs, peptides, lipids, proteins, amino acids, nucleic acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria and others.","In yet another embodiment, the non-nucleotide linker X is as defined herein.","The term “non-nucleotide” as used herein include either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds.","Specific examples include those described by Seela and Kaiser, Nucleic Acids Res.","1990, 18:6353 and Nucleic Acids Res.","1987, 15:3113; Cload and Schepartz, J.","Am.","Chem.","Soc.","1991, 113:6324; Richardson and Schepartz, J.","Am.","Chem.","Soc.","1991, 113:5109; Ma et al., Nucleic Acids Res.","1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res.","1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett.","1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No.","WO 89/02439; Usman et al., International Publication No.","WO 95/06731; Dudycz et al., International Publication No.","WO 95/11910 and Ferentz and Verdine, J.","Am.","Chem.","Soc.","1991, 113:4000, all hereby incorporated by reference herein.","A “non-nucleotide” further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.","The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.","Thus, in a preferred embodiment, the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties, and having enzymatic activity to cleave an RNA or DNA molecule.","In another aspect of the invention, nucleic acid molecules that interact with target RNA molecules and down-regulate target genes (e.g., prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene) activity are expressed from transcription units inserted into DNA or RNA vectors.","The recombinant vectors can be DNA plasmids or viral vectors.","Enzymatic nucleic acid molecule or antisense expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.","The recombinant vectors capable of expressing the enzymatic nucleic acid molecules or antisense can be delivered as described above, and persist in target cells.","Alternatively, viral vectors can be used that provide for transient expression of enzymatic nucleic acid molecules or antisense.","Such vectors can be repeatedly administered as necessary.","Once expressed, the enzymatic nucleic acid molecules or antisense bind to the target RNA and down-regulate its function or expression.","Delivery of enzymatic nucleic acid molecule or antisense expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell.","Antisense DNA can be expressed via the use of a single stranded DNA intracellular expression vector.","By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.","By “patient” is meant an organism, which is a donor or recipient of explanted cells, or the cells themselves.","“Patient” also refers to an organism to which the nucleic acid molecules of the invention can be administered.","A patient can be a mammal or mammalian cells.","In one embodiment, a patient is a human or human cells.","By “enhanced enzymatic activity” is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both the catalytic activity and the stability of the nucleic acid molecules of the invention.","In this invention, the product of these properties can be increased in vivo compared to an all RNA enzymatic nucleic acid or all DNA enzyme.","In some cases, the activity or stability of the nucleic acid molecule can be decreased (i.e., less than ten-fold), but the overall activity of the nucleic acid molecule is enhanced, in vivo.","The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above.","For example, to treat a disease or condition associated with the levels of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR, the patient can be treated, or other appropriate cells can be treated, as is evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.","In a further embodiment, the described molecules, such as antisense or enzymatic nucleic acid molecules, can be used in combination with other known treatments to treat conditions or diseases discussed above.","For example, the described molecules can be used in combination with one or more known therapeutic agents to treat allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR expression.","By “comprising” is meant including, but not limited to, whatever follows the word “comprising”.","Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.","By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”.","Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.","Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 shows examples of chemically stabilized ribozyme motifs.","HH Rz, represents hammerhead ribozyme motif (Usman et al., 1996, Curr.","Op.","Struct.","Bio., 1, 527); NCH Rz represents the NCH ribozyme motif (Ludwig & Sproat, International PCT Publication No.","WO 98/58058); G-Cleaver, represents G-cleaver ribozyme motif (Kore et al., 1998, Nucleic Acids Research 26, 4116-4120, Eckstein et al., International PCT publication No.","WO 99/16871).","N or n, represent independently a nucleotide that can be same or different and have complementarity to each other; rI, represents ribo-Inosine nucleotide; arrow indicates the site of cleavage within the target.","Position 4 of the HH Rz and the NCH Rz is shown as having 2′-C-allyl modification, but those skilled in the art will recognize that this position can be modified with other modifications well known in the art, so long as such modifications do not significantly inhibit the activity of the ribozyme.","FIG.","2 shows an example of the Amberzyme ribozyme motif that is chemically stabilized (see for example Beigelman et al., International PCT publication No.","WO 99/55857).","FIG.","3 shows an example of the Zinzyme A ribozyme motif that is chemically stabilized (see for example Beigelman et al., Beigelman et al., International PCT publication No.","WO 99/55857).","FIG.","4 shows an example of a specific DNAzyme motif, commonly referred to as the “10-23 motif”, as described by Santoro et al., 1997, PNAS, 94, 4262.DESCRIPTION OF THE PREFERRED EMBODIMENTS Nucleic Acid Molecules and Mechanism of Action Antisense: Antisense molecules can be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides and primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, November 1994, BioPharm, 20-33).","The antisense oligonucleotide binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme.","Antisense molecules can also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit.","Rev.","in Oncogenesis 7, 151-190).","In addition, binding of single stranded DNA to RNA can result in nuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke, supra).","To date, the only backbone modified DNA chemistry which act as substrates for RNase H are phosphorothioates, phosphorodithioates, and borontrifluoridates.","Recently it has been reported that 2′-arabino and 2′-fluoro arabino-containing oligos can also activate RNase H activity.","A number of antisense molecules have been described that utilize novel configurations of chemically modified nucleotides, secondary structure, and/or RNase H substrate domains (Woolf et al., International PCT Publication No.","WO 98/13526; Thompson et al., International PCT Publication No.","WO 99/54459; Hartmann et al., U.S. Ser.","No.","60/101,174, filed on Sep. 21, 1998) all of these are incorporated by reference herein in their entirety.","In addition, antisense deoxyoligoribonucleotides can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.","Antisense DNA can be expressed via the use of a single stranded DNA intracellular expression vector or equivalents and variations thereof.","Triplex Forming Oligonucleotides (TFO): Single stranded DNA can be designed to bind to genomic DNA in a sequence specific manner.","TFOs are comprised of pyrimidine-rich oligonucleotides which bind DNA helices through Hoogsteen Base-pairing (Wu-Pong, supra).","The resulting triple helix composed of the DNA sense, DNA antisense, and TFO disrupts RNA synthesis by RNA polymerase.","The TFO mechanism can result in gene expression or cell death since binding can be irreversible (Mukhopadhyay & Roth, supra).","2-5A Antisense Chimera: The 2-5A system is an interferon mediated mechanism for RNA degradation found in higher vertebrates (Mitra et al., 1996, Proc Nat Acad Sci USA 93, 6780-6785).","Two types of enzymes, 2-5A synthetase and RNase L, are required for RNA cleavage.","The 2-5A synthetases require double stranded RNA to form 2′-5′ oligoadenylates (2-5A).","2-5A then acts as an allosteric effector for utilizing RNase L, which has the ability to cleave single stranded RNA.","The ability to form 2-5A structures with double stranded RNA makes this system particularly useful for inhibition of viral replication.","(2′-5′) oligoadenylate structures can be covalently linked to antisense molecules to form chimeric oligonucleotides capable of RNA cleavage (Torrence, supra).","These molecules putatively bind and activate a 2-5A dependent RNase, the oligonucleotide/enzyme complex then binds to a target RNA molecule which can then be cleaved by the RNase enzyme.","Aptamer: Nucleic acid aptamers can be selected to specifically bind to a particular ligand of interest (see for example Gold et al., U.S. Pat.","No.","5,567,588 and U.S. Pat.","No.","5,475,096, Gold et al., 1995, Annu.","Rev.","Biochem., 64, 763; Brody and Gold, 2000, J.","Biotechnol., 74, 5; Sun, 2000, Curr.","Opin.","Mol.","Ther., 2, 100; Kusser, 2000, J.","Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628).","For example, the use of in vitro selection can be applied to evolve nucleic acid aptamers with binding specificity for the NOGO receptor, prostaglandin D2 receptor (PTGDR), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors.","Nucleic acid aptamers can include chemical modifications and linkers as described herein.","Aptamer molecules of the invention that bind to a cellular receptor, such as prostaglandin D2 receptor (PTGDR), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, or protein kinase PKR receptor, and modulate the activity of the receptor or ligand having specificity for the receptor.","RNAi: RNA interference refers to the process of sequence specific post transcriptional gene silencing in animals mediated by short interfering RNAs (siRNA) (Fire et al., 1998, Nature, 391, 806).","The corresponding process in plants is commonly referred to as post transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi.","The process of post transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358).","Such protection from foreign gene expression may have evolved in response to the production of double stranded RNAs (dsRNA) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single stranded RNA or viral genomic RNA.","The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized.","This mechanism appears to be different from the interferon response that results from dsRNA mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L. The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer.","Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363).","Short interfering RNAs derived from dicer activity are typically about 21-23 nucleotides in length and comprise about 19 base pair duplexes.","Dicer has also been implicated in the excision of 21 and 22 nucleotide small temporal RNAs (stRNA) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834).","The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single stranded RNA having sequence homologous to the siRNA.","Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).","Short interfering RNA mediated RNAi has been studied in a variety of systems.","Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. Elegans.","Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describes RNAi mediated by dsRNA in mouse embryos.","Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA.","Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells.","Recent work in Drosophila embryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity.","These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two nucleotide 3′-overhangs.","Furthermore, substitution of one or both siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3′-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated.","Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity.","In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J., 20, 6877).","Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309), however siRNA molecules lacking a 5′-phosphate are active when introduced exogenously, suggesting that 5′-phosphorylation of siRNA constructs may occur in vivo.","Enzymatic Nucleic Acid: Several varieties of naturally-occurring enzymatic RNAs are presently known.","In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc.","R. Soc.","London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing cleavage and ligation of phosphodiester linkages (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr.","Op.","Biotech., 7, 442; Santoro et al., 1997, Proc.","Natl.","Acad.","Sci., 94, 4262; Tang et al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra; Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish et al., 1997, Biochemistry 36, 6495; all of these are incorporated by reference herein).","Each can catalyze a series of reactions including the hydrolysis of phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions.","The enzymatic nature of an enzymatic nucleic acid molecule has significant advantages, one advantage being that the concentration of enzymatic nucleic acid molecule necessary to affect a therapeutic treatment is lower.","This advantage reflects the ability of the enzymatic nucleic acid molecule to act enzymatically.","Thus, a single enzymatic nucleic acid molecule is able to cleave many molecules of target RNA.","In addition, the enzymatic nucleic acid molecule is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage.","Single mismatches, or base-substitutions, near the site of cleavage can be chosen to completely eliminate catalytic activity of a enzymatic nucleic acid molecule.","Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner.","With the proper design, such enzymatic nucleic acid molecules can be targeted to RNA transcripts, and achieve efficient cleavage in vitro (Zaug et al., 324, Nature 429 1986; Uhlenbeck, 1987 Nature 328, 596; Kim et al., 84 Proc.","Natl.","Acad.","Sci.","USA 8788, 1987; Dreyfus, 1988, Einstein Quart.","J. Bio.","Med., 6, 92; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989; Santoro et al., 1997 supra).","Because of their sequence specificity, trans-cleaving enzymatic nucleic acid molecules can be used as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann.","Rep. Med.","Chem.","30, 285-294; Christoffersen and Marr, 1995 J. Med.","Chem.","38, 2023-2037).","Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA.","Such a cleavage event renders the RNA non-functional and abrogates protein expression from that RNA.","In this manner, synthesis of a protein associated with a disease state can be selectively inhibited (Warashina et al., 1999, Chemistry and Biology, 6, 237-250).","Enzymatic nucleic acid molecules of the invention that are allosterically regulated (“allozymes”) can be used to down-regulate prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR expression.","These allosteric enzymatic nucleic acids or allozymes (see for example George et al., U.S. Pat.","Nos.","5,834,186 and 5,741,679, Shih et al., U.S. Pat.","No.","5,589,332, Nathan et al., U.S. Pat.","No.","5,871,914, Nathan and Ellington, International PCT publication No.","WO 00/24931, Breaker et al., International PCT Publication Nos.","WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No.","WO 99/29842) are designed to respond to a signaling agent, for example, mutant and/or wild type protein, other proteins and/or RNAs involved in target gene signal transduction, compounds, metals, polymers, molecules and/or drugs that are targeted to target gene expressing cells etc., which in turn modulates the activity of the enzymatic nucleic acid molecule.","In response to interaction with a predetermined signaling agent, the allosteric enzymatic nucleic acid molecule's activity is activated or inhibited such that the expression of a particular target is selectively down-regulated.","The target can comprise wild-type protein, mutant protein, and/or a predetermined component of the protein's signal transduction pathway.","In a specific example, allosteric enzymatic nucleic acid molecules that are activated by interaction with a RNA encoding a PTGDR protein are used as therapeutic agents in vivo.","The presence of RNA encoding the PTGDS protein activates the allosteric enzymatic nucleic acid molecule that subsequently cleaves the RNA encoding a PTGDR protein resulting in the inhibition of PTGDR protein expression.","In this manner, cells that express both PTGDS and PTGDR protein are selectively targeted.","In another non-limiting example, an allozyme can be activated by a prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR protein, peptide, or mutant polypeptide that causes the allozyme to inhibit the expression of a target gene, by, for example, cleaving RNA encoded by the target gene.","In this non-limiting example, the allozyme acts as a decoy to inhibit the function of the target protein and also inhibit the expression of the protein once activated by the prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR protein.","Target Sites Targets for useful enzymatic nucleic acid molecules and antisense nucleic acids can be determined as disclosed in Draper et al., WO 93/23569; Sullivan et al., WO 93/23057; Thompson et al., WO 94/02595; Draper et al., WO 95/04818; McSwiggen et al., U.S. Pat.","No.","5,525,468, and hereby incorporated by reference herein in totality.","Other examples include the following PCT applications, which concern inactivation of expression of disease-related genes: WO 95/23225, WO 95/13380, WO 94/02595, incorporated by reference herein.","Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art.","Enzymatic nucleic acid molecules and antisense to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described.","The sequences of human prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR RNAs were screened for optimal enzymatic nucleic acid and antisense target sites using a computer-folding algorithm.","Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme, or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified.","These sites are shown in Tables III to XXIII (all sequences are 5′ to 3′ in the tables; underlined regions can be any sequence “X” or linker X, the actual sequence is not relevant here).","The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of enzymatic nucleic acid molecule.","While human sequences can be screened and enzymatic nucleic acid molecule and/or antisense thereafter designed, as discussed in Stinchcomb et al., WO 95/23225, mouse targeted enzymatic nucleic acid molecules can be useful to test efficacy of action of the enzymatic nucleic acid molecule and/or antisense prior to testing in humans.","Antisense, siRNA, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified.","The nucleic acid molecules are individually analyzed by computer folding (Jaeger et al., 1989 Proc.","Natl.","Acad.","Sci.","USA, 86, 7706) to assess whether the sequences fold into the appropriate secondary structure.","Those nucleic acid molecules with unfavorable intramolecular interactions such as between the binding arms and the catalytic core are eliminated from consideration.","Varying binding arm lengths can be chosen to optimize activity.","Antisense, siRNA, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified and were designed to anneal to various sites in the RNA target.","The binding arms are complementary to the target site sequences described above.","The nucleic acid molecules were chemically synthesized.","The method of synthesis used follows the procedure for normal DNA/RNA synthesis as described below and in Usman et al., 1987 J.","Am.","Chem.","Soc., 109, 7845; Scaringe et al., 1990 Nucleic Acids Res., 18, 5433; and Wincott et al., 1995 Nucleic Acids Res.","23, 2677-2684; Caruthers et al., 1992, Methods in Enzymology 211, 3-19.Synthesis of Nucleic Acid Molecules Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive.","In this invention, small nucleic acid motifs (“small refers to nucleic acid motifs less than about 100 nucleotides in length, and in one embodiment less than about 80 nucleotides in length, and in another embodiment less than about 50 nucleotides in length; e.g., antisense oligonucleotides, hammerhead or the NCH ribozymes) can be used for exogenous delivery.","The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of RNA structure.","Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.","Oligonucleotides (e.g., antisense GeneBlocs) are synthesized using protocols known in the art as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No.","WO 99/54459, Wincott et al., 1995, Nucleic Acids Res.","23, 2677-2684, Wincott et al., 1997, Methods Mol.","Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat.","No.","6,001,311.All of these references are incorporated herein by reference.","The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.","In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45 sec coupling step for 2′-deoxy nucleotides.","Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle.","Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle.","A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl.","A 22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl.","Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%.","Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM I2, 49 mM pyridine, 9% water in THF (PERSEPTIVE™).","Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle.","S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.","Deprotection of the antisense oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq.","methylamine (1 mL) at 65° C. for 10 min.","After cooling to −20° C., the supernatant is removed from the polymer support.","The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant.","The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.","The method of synthesis used for normal RNA including certain enzymatic nucleic acid molecules follows the procedure as described in Usman et al., 1987, J.","Am.","Chem.","Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res.","23, 2677-2684 Wincott et al., 1997, Methods Mol.","Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.","In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2′-O-methylated nucleotides.","Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle.","Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle.","A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl.","A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.","Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%.","Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I2, 49 mM pyridine, 9% water in THF (PERSEPTIVE™).","Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle.","S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used.","Deprotection of the RNA is performed using either a two-pot or one-pot protocol.","For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq.","methylamine (1 mL) at 65° C. for 10 min.","After cooling to −20° C., the supernatant is removed from the polymer support.","The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant.","The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.","The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA•3HF to provide a 1.4 M HF concentration) and heated to 65° C. After 1.5 h, the oligomer is quenched with 1.5 M NH4HCO3.Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65° C. for 15 min.","The vial is brought to r.t. TEA•3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 min.","The sample is cooled at −20° C. and then quenched with 1.5 M NH4HCO3.For purification of the trityl-on oligomers, the quenched NH4HCO3 solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA.","After washing, the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 min.","The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again.","The oligonucleotide is then eluted with 30% acetonitrile.","Inactive hammerhead ribozymes or binding attenuated control (BAC) oligonucleotides are synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel, K. J., et al., 1992, Nucleic Acids Res., 20, 3252).","Similarly, one or more nucleotide substitutions can be introduced in other enzymatic nucleic acid molecules to inactivate the molecule and such molecules can serve as a negative control.","The average stepwise coupling yields are typically ≧98% (Wincott et al., 1995 Nucleic Acids Res.","23, 2677-2684).","Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96 well format, all that is important is the ratio of chemicals used in the reaction.","Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No.","WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem.","8, 204).","The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp.","Ser.","31, 163).","Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., Supra, the totality of which is hereby incorporated herein by reference) and are re-suspended in water.","The sequences of the nucleic acid molecules, including enzymatic nucleic acid molecules and antisense, that are chemically synthesized, are shown in Tables III-XXIII.","The sequences of the enzymatic nucleic acid constructs that are chemically synthesized are complementary to the Substrate sequences shown in Tables III-XXIII.","Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the enzymatic nucleic acid (all but the binding arms) is altered to affect activity.","The enzymatic nucleic acid construct sequences listed in Tables III-XXIII can be formed of ribonucleotides or other nucleotides or non-nucleotides.","Such enzymatic nucleic acid molecules with enzymatic activity are equivalent to the enzymatic nucleic acid molecules described specifically in the Tables.","Optimizing Activity of the Nucleic Acid Molecule of the Invention Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases can increase their potency (see e.g., Eckstein et al., International Publication No.","WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem.","Sci.","17, 334; Usman et al., International Publication No.","WO 93/15187; and Rossi et al., International Publication No.","WO 91/03162; Sproat, U.S. Pat.","No.","5,334,711; and Burgin et al., supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules herein).","Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.","(All these publications are hereby incorporated by reference herein).","There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy.","For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS.","17, 34; Usman et al., 1994, Nucleic Acids Symp.","Ser.","31, 163; Burgin et al., 1996, Biochemistry, 35, 14090).","Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No.","WO 92/07065; Perrault et al.","Nature, 1990, 344, 565-568; Pieken et al.","Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem.","Sci., 1992, 17, 334-339; Usman et al.","International Publication PCT No.","WO 93/15187; Sproat, U.S. Pat.","No.","5,334,711 and Beigelman et al., 1995, J. Biol.","Chem., 270, 25702; Beigelman et al., International PCT publication No.","WO 97/26270; Beigelman et al., U.S. Pat.","No.","5,716,824; Usman et al., U.S. Pat.","No.","5,627,053; Woolf et al., International PCT Publication No.","WO 98/13526; Thompson et al., U.S. Ser.","No.","60/082,404 which was filed on Apr.","20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu.","Rev.","Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg.","Med.","Chem., 5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein).","Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into ribozymes without inhibiting catalysis, and are incorporated by reference herein.","In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid molecules of the instant invention.","While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorothioate, and/or 5′-methylphosphonate linkages improves stability, too many of these modifications can cause some toxicity.","Therefore when designing nucleic acid molecules the amount of these internucleotide linkages should be minimized.","The reduction in the concentration of these linkages should lower toxicity resulting in increased efficacy and higher specificity of these molecules.","Nucleic acid molecules having chemical modifications that maintain or enhance activity are provided.","Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid.","Thus, in a cell and/or in vivo the activity may not be significantly lowered.","Therapeutic nucleic acid molecules delivered exogenously are optimally stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein.","This period of time varies between hours to days depending upon the disease state.","Clearly, nucleic acid molecules must be resistant to nucleases in order to function as effective intracellular therapeutic agents.","Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995 Nucleic Acids Res.","23, 2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19 (incorporated by reference herein) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.","In one embodiment, nucleic acid molecules of the invention include one or more G-clamp nucleotides.","A G-clamp nucleotide is a modified cytosine analog wherein modifications result in the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J.","Am.","Chem.","Soc., 120, 8531-8532.A single G-clamp analog substation within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides.","The inclusion of such nucleotides in nucleic acid molecules of the invention can enable both enhanced affinity and specificity to nucleic acid targets.","Therapeutic nucleic acid molecules (e.g., enzymatic nucleic acid molecules and antisense nucleic acid molecules) delivered exogenously are optimally stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein.","This period of time varies between hours to days depending upon the disease state.","These nucleic acid molecules should be resistant to nucleases in order to function as effective intracellular therapeutic agents.","Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.","In another embodiment, the invention features conjugates and/or complexes of nucleic acid molecules targeting prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR.","Compositions and conjugates are used to facilitate delivery of molecules into a biological system, such as cells.","The conjugates provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention.","The present invention encompasses the design and synthesis of novel agents for the delivery of molecules, including but not limited to small molecules, lipids, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes.","In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers.","These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat.","No.","5,854,038).","Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.","The term “biodegradable nucleic acid linker molecule” as used herein, refers to a nucleic acid molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule.","The stability of the biodegradable nucleic acid linker molecule can be modulated by using various combinations of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, for example 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides.","The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus based linkage, for example a phosphoramidate or phosphodiester linkage.","The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.","The term “biodegradable” as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.","The term “biologically active molecule” as used herein, refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system.","Non-limiting examples of biologically active molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siRNA, dsRNA, allozymes, aptamers, decoys and analogs thereof.","Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.","The term “phospholipid” as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group.","For example, a phospholipid can comprise a phosphorus containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.","In another embodiment, nucleic acid catalysts having chemical modifications that maintain or enhance enzymatic activity are provided.","Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids.","Thus, in a cell and/or in vivo the activity of the nucleic acid may not be significantly lowered.","As exemplified herein such enzymatic nucleic acids are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al., 1996, Biochemistry, 35, 14090).","Such enzymatic nucleic acids herein are said to “maintain” the enzymatic activity of an all RNA ribozyme or all DNA DNAzyme.","In another aspect the nucleic acid molecules comprise a 5′ and/or a 3′-cap structure.","By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see for example Wincott et al., WO 97/26270, incorporated by reference herein).","These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and can help in delivery and/or localization within a cell.","The cap can be present at the 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can be present on both terminus.","In non-limiting examples, the 5′-cap includes inverted abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Wincott et al., International PCT publication No.","WO 97/26270, incorporated by reference herein).","In another embodiment the 3′-cap includes, for example 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).","By the term “non-nucleotide” is meant any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.","The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.","An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups.","The alkyl group can have, for example, 1 to 12 carbons.","In one embodiment of the invention, the alkyl group is a lower alkyl of from 1 to 7 carbons.","In another embodiment the alkyl group is 1 to 4 carbons.","The alkyl group can be substituted or unsubstituted.","When substituted the substituted group(s) can be hydroxyl, cyano, alkoxy, ═O, ═S, NO2 or N(CH3)2, amino, or SH.","The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups.","The alkenyl group can have, for example, 1 to 12 carbons.","In one embodiment of the invention the alkenyl group can be a lower alkenyl of from 1 to 7 carbons.","In another embodiment the alkenyl group can be 1 to 4 carbons.","The alkenyl group can be substituted or unsubstituted.","When substituted the substituted group(s) can be, for example, hydroxyl, cyano, alkoxy, ═O, ═S, NO2, halogen, N(CH3)2, amino, or SH.","The term “alkyl” also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups.","The alkynyl group can have, for example, 1 to 12 carbons.","In one embodiment of the invention, the alkynyl group is a lower alkynyl of from 1 to 7 carbons.","In another embodiment of the invention, the alkynyl group is 1 to 4 carbons.","The alkynyl group can be substituted or unsubstituted.","When substituted the substituted group(s) can be, for example, hydroxyl, cyano, alkoxy, ═O, ═S, NO2 or N(CH3)2, amino or SH.","Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups.","An “aryl” group refers to an aromatic group which has at least one ring having a conjugated p electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which can be optionally substituted.","The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups.","An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above).","Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms.","The carbon atoms are optionally substituted.","Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms.","Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.","An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen.","An “ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylaryl or hydrogen.","By “nucleotide” is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar.","Nucleotides are recognized in the art to include natural bases (standard), and modified bases well known in the art.","Such bases are generally located at the 1′ position of a nucleotide sugar moiety.","Nucleotides generally comprise a base, sugar and a phosphate group.","The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No.","WO 92/07065; Usman et al., International PCT Publication No.","WO 93/15187; Uhlman & Peyman, supra all are hereby incorporated by reference herein).","There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res.","22, 2183.Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, for example, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.","6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N-6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).","By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.","By “nucleoside” is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar.","Nucleosides are recognized in the art to include natural bases (standard), and modified bases well known in the art.","Such bases are generally located at the 1′ position of a nucleoside sugar moiety.","Nucleosides generally comprise a base and sugar group.","The nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, modified nucleosides, non-natural nucleosides, non-standard nucleosides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No.","WO 92/07065; Usman et al., International PCT Publication No.","WO 93/15187; Uhlman & Peyman, supra all are hereby incorporated by reference herein).","There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res.","22, 2183.Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.","6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N-6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).","By “modified bases” in this aspect is meant nucleoside bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.","In one embodiment, the invention features modified enzymatic nucleic acid molecules with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.","For a review of oligonucleotide backbone modifications see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39.These references are hereby incorporated by reference herein.","By “abasic” is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1′ position, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative (for more details see Wincott et al., International PCT publication No.","WO 97/26270).","By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1′ carbon of β-D-ribo-furanose.","By “modified nucleoside” is meant any nucleotide base that contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.","In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′-NH2 or 2′-O—NH2, which can be modified or unmodified.","Such modified groups are described, for example, in Eckstein et al., U.S. Pat.","No.","5,672,695 and Matulic-Adamic et al., WO 98/28317, respectively, which are both incorporated by reference in their entireties.","Various modifications to nucleic acid (e.g., antisense and ribozyme) structure can be made to enhance the utility of these molecules.","For example, such modifications can enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, including e.g., enhancing penetration of cellular membranes and conferring the ability to recognize and bind to targeted cells.","Use of the nucleic acid-based molecules of the invention can lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules (including different enzymatic nucleic acid molecule motifs) and/or other chemical or biological molecules).","The treatment of patients with nucleic acid molecules can also include combinations of different types of nucleic acid molecules.","Therapies can be devised which include a mixture of enzymatic nucleic acid molecules (including different enzymatic nucleic acid molecule motifs), antisense and/or 2-5A chimera molecules to one or more targets to alleviate symptoms of a disease.","Administration of Nucleic Acid Molecules Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed.","Akhtar, 1995 which are both incorporated herein by reference.","Sullivan et al., PCT WO 94/02595, further describes the general methods for delivery of enzymatic RNA molecules.","These protocols can be utilized for the delivery of virtually any nucleic acid molecule.","Nucleic acid molecules can be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.","The nucleic acid molecules or the invention are administered via pulmonary delivery, such as by inhalation of an aerosol or spray dried formulation administered by an inhalation device or nebulizer.","Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump.","Other routes of delivery include, but are not limited to oral (tablet or pill form) and/or intrathecal delivery (Gold, 1997, Neuroscience, 76, 1153-1158).","Other approaches include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers.","For a comprehensive review on drug delivery strategies including CNS delivery, see Ho et al., 1999, Curr.","Opin.","Mol.","Ther., 1, 336-343 and Jain, Drug Delivery Systems: Technologies and Commercial Opportunities, Decision Resources, 1998 and Groothuis et al., 1997, J.","NeuroVirol., 3, 387-400.More detailed descriptions of nucleic acid delivery and administration are provided in Sullivan et al., supra, Draper et al., PCT WO93/23569, Beigelman et al., PCT WO99/05094, and Klimuk et al., PCT WO99/04819 all of which have been incorporated by reference herein.","Experiments have demonstrated the efficient in vivo uptake of nucleic acids by neurons.","As an example of local administration of nucleic acids to nerve cells, Sommer et al., 1998, Antisense Nuc.","Acid Drug Dev., 8, 75, describe a study in which a 15mer phosphorothioate antisense nucleic acid molecule to c-fos is administered to rats via microinjection into the brain.","Antisense molecules labeled with tetramethylrhodamine-isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by exclusively by neurons thirty minutes post-injection.","A diffuse cytoplasmic staining and nuclear staining was observed in these cells.","As an example of systemic administration of nucleic acid to nerve cells, Epa et al., 2000, Antisense Nuc.","Acid Drug Dev., 10, 469, describe an in vivo mouse study in which beta-cyclodextrin-adamantane-oligonucleotide conjugates were used to target the p75 neurotrophin receptor in neuronally differentiated PC12 cells.","Following a two week course of IP administration, pronounced uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells.","In addition, a marked and consistent down-regulation of p75 was observed in DRG neurons.","Additional approaches to the targeting of nucleic acid to neurons are described in Broaddus et al., 1998, J.","Neurosurg., 88(4), 734; Karle et al., 1997, Eur.","J.","Pharmocol., 340(2/3), 153; Bannai et al., 1998, Brain Research, 784(1,2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199; Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, Brain Res.","Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39.Nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells that express NOGO and NOGO receptors for modulation of NOGO and/or NOGO receptor expression.","The delivery of nucleic acid molecules of the invention, targeting NOGO and NOGO receptors is provided by a variety of different strategies.","Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier.","Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers.","Furthermore, gene therapy approaches, for example as described in Kaplitt et al., U.S. Pat.","No.","6,180,613, can be used to express nucleic acid molecules in the CNS.","The molecules of the instant invention can be used as pharmaceutical agents.","Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, or all of the symptoms) of a disease state in a patient.","The negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition.","When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed.","The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the other compositions known in the art.","The present invention also includes pharmaceutically acceptable formulations of the compounds described.","These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.","A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., local administration or systemic administration, into a cell or patient, including, for example, a human.","Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection.","Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to).","For example, pharmacological compositions injected into the blood stream should be soluble.","Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect.","By “local administration” is meant in vivo local absorption or accumulation of drugs in the specific tissue, organ, or compartment of the body.","Administration routes that can lead to local absorption include, without limitations: inhalation, direct injection, or dermatological applications.","By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body.","Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.","Each of these administration routes expose the desired compound, e.g., nucleic acids, to an accessible diseased tissue.","The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size.","The use of a liposome or other drug carrier comprising the compounds of the instant invention, for example PEG or phospholipids conjugates, can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES).","A nucleic acid formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful.","This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells.","Both local and systemic administration approaches can be used to administer nucleic acid molecules of the invention for the treatment of asthma or related conditions.","In one embodiment, the nucleic acid molecule or formulation comprising the nucleic acid molecule is administered to a patient with an inhaler or nebulizer, providing rapid local uptake of the nucleic acid molecules into relevant pulmonary tissues.","In another embodiment, the nucleic acid molecule or formulation comprising the nucleic acid molecule is administered to a patient systemically, for example by intravenous or subcutaneous injection, providing sustained uptake of the nucleic acid molecules into relevant bodily tissues.","By pharmaceutically acceptable formulation is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity.","Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: PEG conjugated nucleic acids, phospholipid conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into various tissues, for exaple the CNS (Jolliet-Riant and Tillement, 1999, Fundam.","Clin.","Pharmacol., 13, 16-26); biodegradable polymers, such as poly(DL-lactide-coglycolide) microspheres for sustained release delivery after implantation (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass.","; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).","Other non-limiting examples of delivery strategies, including CNS delivery of the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm.","Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv.","Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.All these references are hereby incorporated herein by reference.","The invention also features the use of the composition comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes).","Nucleic acid molecules of the invention can also comprise covalently attached PEG molecules of various molecular weights.","These formulations offer a method for increasing the accumulation of drugs in target tissues.","This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al.","Chem.","Rev.","1995, 95, 2601-2627; Ishiwata et al., Chem.","Pharm.","Bull 1995, 43, 1005-1011).","Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim.","Biophys.","Acta, 1238, 86-90).","The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol.","Chem.","1995, 42, 24864-24870; Choi et al., International PCT Publication No.","WO 96/10391; Ansell et al., International PCT Publication No.","WO 96/10390; Holland et al., International PCT Publication No.","WO 96/10392; all of which are incorporated by reference herein).","Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.","All of these references are incorporated by reference herein.","The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent.","Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R.","Gennaro edit.","1985) hereby incorporated by reference herein.","For example, preservatives, stabilizers, dyes and flavoring agents can be provided.","These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.","In addition, antioxidants and suspending agents can be used.","A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, or all of the symptoms) of a disease state.","The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize.","Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.","The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.","The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.","In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier.","One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients.","The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.","Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations.","Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets.","These excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.","The tablets can be uncoated or they can be coated by known techniques.","In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.","For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.","Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.","Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.","Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.","The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.","Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.","The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.","Sweetening agents and flavoring agents can be added to provide palatable oral preparations.","These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.","Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.","Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above.","Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.","Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions.","The oily phase can be a vegetable oil or a mineral oil or mixtures of these.","Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.","The emulsions can also contain sweetening and flavoring agents.","Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose.","Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents.","The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension.","This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.","The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.","Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution.","In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.","For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides.","In addition, fatty acids such as oleic acid find use in the preparation of injectables.","The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug.","These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.","Such materials include cocoa butter and polyethylene glycols.","Nucleic acid molecules of the invention can be administered parenterally in a sterile medium.","The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.","Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.","Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day).","The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration.","Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.","It is understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.","For administration to non-human animals, the composition can also be added to the animal feed or drinking water.","It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet.","It can also be convenient to present the composition as a premix for addition to the feed or drinking water.","The nucleic acid molecules of the present invention can also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect.","The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.","Alternatively, certain of the nucleic acid molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.","Natl.","Acad.","Sci., USA 83, 399; Scanlon et al., 1991, Proc.","Natl.","Acad.","Sci.","USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res.","Dev., 2, 3-15; Dropulic et al., 1992, J.","Virol., 66, 1432-41; Weerasinghe et al., 1991, J.","Virol., 65, 5531-4; Ojwang et al., 1992, Proc.","Natl.","Acad.","Sci.","USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45; all of these references are hereby incorporated in their totalities by reference herein).","Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector.","The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp.","Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol.","Chem., 269, 25856; all of these references are hereby incorporated in their totalities by reference herein).","Gene therapy approaches specific to the CNS are described by Blesch et al., 2000, Drug News Perspect., 13, 269-280; Peterson et al., 2000, Cent.","Nerv.","Syst.","Dis., 485-508; Peel and Klein, 2000, J. Neurosci.","Methods, 98, 95-104; Hagihara et al., 2000, Gene Ther., 7, 759-763; and Herrlinger et al., 2000, Methods Mol.","Med., 35, 287-312.AAV-mediated delivery of nucleic acid to cells of the nervous system is further described by Kaplitt et al., U.S. Pat.","No.","6,180,613.In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.","The recombinant vectors can be DNA plasmids or viral vectors.","Ribozyme expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.","The recombinant vectors capable of expressing the nucleic acid molecules can be delivered as described above, and persist in target cells.","Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules.","Such vectors can be repeatedly administered as necessary.","Once expressed, the nucleic acid molecule binds to the target mRNA.","Delivery of nucleic acid molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).","In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the instant invention is disclosed.","The nucleic acid sequence encoding the nucleic acid molecule of the instant invention is operably linked in a manner that allows expression of that nucleic acid molecule.","In another aspect the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); c) a nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner that allows expression and/or delivery of said nucleic acid molecule.","The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the nucleic acid catalyst of the invention; and/or an intron (intervening sequences).","Transcription of the nucleic acid molecule sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III).","Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.)","present nearby.","Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc.","Natl.","Acad.","Sci.","USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol.","Cell.","Biol., 10, 4529-37).","All of these references are incorporated by reference herein.","Several investigators have demonstrated that nucleic acid molecules, such as ribozymes expressed from such promoters can function in mammalian cells (e.g.","Kashani-Sabet et al., 1992, Antisense Res.","Dev., 2, 3-15; Ojwang et al., 1992, Proc.","Natl.","Acad.","Sci.","USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc.","Natl.","Acad.","Sci.","USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc.","Natl.","Acad.","Sci.","U.S.A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566).","More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat.","No.","5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al., International PCT Publication No.","WO 96/18736; all of these publications are incorporated by reference herein).","The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).","In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention, in a manner that allows expression of that nucleic acid molecule.","The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner that allows expression and/or delivery of said nucleic acid molecule.","In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3′-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said open reading frame and said termination region, in a manner that allows expression and/or delivery of said nucleic acid molecule.","In yet another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.","In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3′-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.","EXAMPLES The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.","Example 1 Nucleic Acid Molecules Targeting PTGDS, ADORA1 and PTGDR RNA The invention features novel nucleic acid-based molecules, for example, enzymatic nucleic acid molecules, allozymes, antisense nucleic acids, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNA, dsRNA, siRNA, aptamers, and antisense nucleic acids containing RNA cleaving chemical groups, and methods to modulate gene expression, for example, genes encoding prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), and adenosine receptors (AR) such as adenosine receptor A1, A2a, A2b, and A3.In particular, the instant invention features nucleic-acid based molecules and methods to modulate the expression of PTGDR, PTGDS, and adenosine A1 receptor (ADORA1).","In one embodiment, the invention features one or more nucleic acid-based molecules and methods that independently or in combination modulate the expression of gene(s) encoding prostaglandin D2 receptors (PTGDR), prostaglandin D2 synthetase (PTGDS) and adenosine receptors such as ADORA1.Specifically, the present invention features nucleic acid molecules that modulate the expression of prostaglandin D2 receptor (PTGDR) gene, for example Genbank Accession Nos.","U31332 and U31099, prostaglandin D2 synthetase (PTGDS) gene, for example Genbank Accession No.","NM—000954, and Adenosine A1 receptor (ADORA1), for example Genbank Accession No.","NM—000674.In another embodiment, the invention features an enzymatic nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 11666-13262.In yet another embodiment, the invention features an enzymatic nucleic acid molecule comprising at least one binding arm wherein one or more of said binding arms comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs: 4415-5483.In one embodiment, the invention features an antisense nucleic acid molecule comprising a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs: 4415-5483.In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention is adapted to treat asthma.","In one embodiment, an enzymatic nucleic acid molecule of the invention has an endonuclease activity to cleave RNA encoded by a PTGDS and/or PTGDR gene.","In another embodiment, an enzymatic nucleic acid molecule of the invention is in a hammerhead, Inozyme, Zinzyme, DNAzyme, Amberzyme, or G-cleaver configuration.","In another embodiment, an enzymatic nucleic acid molecule of the invention having a hammerhead configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 4415-4641.In yet another embodiment, an enzymatic nucleic acid molecule of invention having a hammerhead configuration comprises a sequence having SEQ ID NOs: 11666-11892.In another embodiment, an enzymatic nucleic acid molecule of the invention having an Inozyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 4642-5017.In yet another embodiment, an enzymatic nucleic acid molecule of invention having an Inozyme configuration comprises a sequence having SEQ ID NOs: 11893-12268.In another embodiment, an enzymatic nucleic acid molecule of the invention having a Zinzyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 5018-5248.In yet another embodiment, an enzymatic nucleic acid molecule of invention having a Zinzyme configuration comprises a sequence having SEQ ID NOs: 12269-12499.In another embodiment, an enzymatic nucleic acid molecule of the invention having a DNAzyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 4415-5294.In yet another embodiment, an enzymatic nucleic acid molecule of invention having a DNAzyme configuration comprises a sequence having SEQ ID NOs: 12500-12842.In another embodiment, an enzymatic nucleic acid molecule of the invention having an Amberzyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 5018-5248, and 5295-5483.In yet another embodiment, an enzymatic nucleic acid molecule of invention having an Amberzyme configuration comprises a sequence having SEQ ID NOs: 12843-13262.In one embodiment, an enzymatic nucleic acid molecule of the invention comprises between 8 and 100 bases complementary to the RNA of PTGDS, ADORA1 and/or PTGDR gene.","In another embodiment, an enzymatic nucleic acid molecule of the invention comprises between 14 and 24 bases complementary to a RNA molecule of a PTGDS or PTGDR gene.","In one embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention is chemically synthesized.","In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one 2′-sugar modification.","In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one nucleic acid base modification.","In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one phosphate backbone modification.","In one embodiment, the invention features a mammalian cell, for example a human cell, including the enzymatic nucleic acid molecule of the invention.","In another embodiment, the invention features a method of reducing PTGDS, ADORA1 and/or PTGDR expression or activity in a cell, comprising contacting the cell with an enzymatic nucleic acid molecule of the invention, under conditions suitable for the reduction.","In another embodiment, the invention features a method of reducing PTGDS, ADORA1 and/or PTGDR expression or activity in a cell, comprising the step of contacting the cell with an antisense nucleic acid molecule of the invention under conditions suitable for the reduction.","In yet another embodiment, the invention features a method of treatment of a patient having a condition associated with the level of PTGDS, ADORA1 and/or PTGDR, comprising contacting cells of the patient with an enzymatic nucleic acid molecule of the invention, under conditions suitable for the treatment.","In one embodiment, the invention features a method of treatment of a patient having a condition associated with the level of PTGDS, ADORA1 and/or PTGDR, comprising contacting cells of the patient with an antisense nucleic acid molecule of the invention, under conditions suitable for the treatment.","In another embodiment, a method of treatment of a patient having a condition associated with the level of PTGDS, ADORA1 and/or PTGDR is featured, wherein the method further comprises the use of one or more drug therapies under conditions suitable for the treatment.","For example, in one embodiment, the invention features a method for treatment of asthma, allergic rhinitis, or atopic dermatitis under conditions suitable for the treatment.","In another embodiment, the invention features a method of cleaving a RNA molecule of PTGDS, ADORA1 and/or PTGDR gene comprising contacting an enzymatic nucleic acid molecule of the invention with a RNA molecule of a PTGDS, ADORA1 and/or PTGDR gene under conditions suitable for the cleavage, for example, wherein the cleavage is carried out in the presence of a divalent cation, such as Mg2+.","In one embodiment, an enzymatic nucleic acid molecule of the invention comprises a cap structure, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end of the enzymatic nucleic acid molecule.","In another embodiment, an antisense nucleic acid molecule of the invention comprises a cap structure, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end of the antisense nucleic acid molecule.","In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one enzymatic nucleic acid molecule of the invention, in a manner which allows expression of the nucleic acid molecule.","In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.","In yet another embodiment, the expression vector of the invention further comprises a sequence for an antisense nucleic acid molecule complementary to a RNA molecule of a PTGDS, ADORA1 and/or PTGDR gene.","In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more enzymatic nucleic acid molecules, which can be the same or different.","In another embodiment, the invention features a method for treatment of asthma, allergic rhinitis, or atopic dermatitis, comprising administering to a patient an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention, under conditions suitable for the treatment, including administering to the patient one or more other therapies, for example, inhalant anti-inflammatories, bronchodilators, adenosine inhibitors and adenosine A1 receptor inhibitors.","In one embodiment, the method of treatment features an enzymatic nucleic acid molecule or antisense nucleic acid molecule of the invention comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification, such as a 3′-3′ inverted abasic moiety.","In another embodiment, an enzymatic nucleic acid molecule or antisense nucleic acid molecule of the invention further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.","In another embodiment, the invention features a method of administering to a mammal, for example a human, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention, comprising contacting the mammal with the nucleic acid molecule under conditions suitable for the administration, for example, in the presence of a delivery reagent such as a lipid, cationic lipid, phospholipid, or liposome.","In yet another embodiment, the invention features a method of administering to a mammal an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention in conjunction with a therapeutic agent, comprising contacting the mammal, for example a human, with the nucleic acid molecule and the therapeutic agent under conditions suitable for the administration.","In one embodiment, the invention features the use of an enzymatic nucleic acid molecule, which can be in a hammerhead, NCH, G-cleaver, Amberzyme, Zinzyme, and/or DNAzyme motif, to down-regulate the expression of a PTGDS, an ADORA1 and/or a PTGDR gene.","The enzymatic nucleic acid molecule that cleave the specified sites in PTGDS, ADORA1 and PTGDR-specific RNAs represent a novel therapeutic approach to treat a variety of allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of PTGDS, ADORA1 and/or PTGDR expression.","In one embodiment, a nucleic acid molecule that modulates, for example, down-regulates, PTGDS replication or expression comprises between 8 and 100 bases complementary to a RNA molecule of PTGDS.","In another embodiment, a nucleic acid molecule that modulates PTGDS replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of PTGDS.","In another embodiment, a nucleic acid molecule that modulates, for example, down-regulates, PTGDR replication or expression comprises between 8 and 100 bases complementary to a RNA molecule of PTGDR.","In another embodiment, a nucleic acid molecule that modulates PTGDR replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of PTGDR.","In another embodiment, a nucleic acid molecule that modulates, for example, down-regulates, ADORA1 replication or expression comprises between 8 and 100 bases complementary to a RNA molecule of ADORA1.In another embodiment, a nucleic acid molecule that modulates ADORA1 replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of ADORA1.The invention provides a method for producing a class of nucleic acid-based gene modulating agents that exhibit a high degree of specificity for the RNA of a desired target.","For example, the enzymatic nucleic acid molecule is can be targeted to a highly conserved sequence region of target RNAs encoding PTGDS, ADORA1 and/or PTGDR (e.g., PTGDS, ADORA1 and/or PTGDR genes) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention.","Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required.","Alternatively, the nucleic acid molecules (e.g., ribozymes and antisense) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.","Nucleic acid-based inhibitors of PTGDS, ADORA1 and PTGDR expression are useful for the prevention and/or treatment of allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and any other diseases or conditions that are related to or will respond to the levels of PTGDS, ADORA1 and/or PTGDR in a cell or tissue, alone or in combination with other therapies.","The reduction of PTGDS, ADORA1 and/or PTGDR expression (specifically PTGDS, ADORA1 and/or PTGDR gene RNA levels) and thus reduction in the level of the respective protein relieves, to some extent, the symptoms of the disease or condition.","The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above.","For example, to treat a disease or condition associated with the levels of PTGDS, ADORA1 and/or PTGDR, the patient can be treated, or other appropriate cells can be treated, as is evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.","In a further embodiment, the described molecules, such as antisense or enzymatic nucleic acid molecules, can be used in combination with other known treatments to treat conditions or diseases discussed above.","For example, the described molecules can be used in combination with one or more known therapeutic agents to treat allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of PTGDS, ADORA1 and/or PTGDR expression.","In another embodiment, the invention features nucleic acid-based inhibitors (e.g., enzymatic nucleic acid molecules (e.g., ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of genes (e.g., PTGDS, ADORA1 and/or PTGDR) capable of progression and/or maintenance allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of PTGDS, ADORA1 and/or PTGDR expression.","Identification of Potential Target Sites in Human PTGDS, ADORA1 and PTGDR RNA The sequence of human PTGDS, ADORA1 and PTGDR genes are screened for accessible sites using a computer-folding algorithm.","Regions of the RNA that do not form secondary folding structures and contained potential enzymatic nucleic acid molecule and/or antisense binding/cleavage sites are identified.","The sequences of PTGDR binding/cleavage sites are shown in Tables XIX-XXIII.","Selection of Enzymatic Nucleic Acid Cleavage Sites in Human PTGDS, ADORA1 and PTGDR RNA Enzymatic nucleic acid molecule target sites are chosen by analyzing sequences of Human PTGDS (Genbank accession No: NM—000954), ADORA1 (Genbank accession No: NM—000674) and PTGDR gene (Genbank accession Nos: U31332 and U31099) and prioritizing the sites on the basis of folding.","Enzymatic nucleic acid molecules are designed that can bind each target and are individually analyzed by computer folding (Christoffersen et al., 1994 J. Mol.","Struc.","Theochem, 311, 273; Jaeger et al., 1989, Proc.","Natl.","Acad.","Sci.","USA, 86, 7706) to assess whether the enzymatic nucleic acid molecule sequences fold into the appropriate secondary structure.","Those enzymatic nucleic acid molecules with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration.","As noted below, varying binding arm lengths can be chosen to optimize activity.","Generally, at least 4 bases on each arm are able to bind to, or otherwise interact with, the target RNA.","Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient Cleavage and/or Blocking of PTGDS, ADORA1 and PTGDR RNA Enzymatic nucleic acid molecules and antisense constructs are designed to anneal to various sites in the RNA message.","The binding arms of the enzymatic nucleic acid molecules are complementary to the target site sequences described above, while the antisense constructs are fully complementary to the target site sequences described above.","The enzymatic nucleic acid molecules and antisense constructs were chemically synthesized.","The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J.","Am.","Chem.","Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.","The average stepwise coupling yields were typically ≧98%.","Enzymatic nucleic acid molecules and antisense constructs are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol.","180, 51).","Enzymatic nucleic acid molecules and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and are resuspended in water.","The sequences of the chemically synthesized enzymatic nucleic acid molecules used in this study are shown below in Tables XIX-XXIII.","The sequences of the chemically synthesized antisense constructs used in this study are complementary sequences to the Substrate sequences shown below as in Tables XIX-XXIII.","Enzymatic Nucleic Acid Molecule Cleavage of PTGDS, ADORA1 and PTGDR RNA Target In Vitro Enzymatic nucleic acid molecules targeted to the human PTGDS, ADORA1 and PTGDR RNA are designed and synthesized as described above.","These enzymatic nucleic acid molecules can be tested for cleavage activity in vitro, for example, using the following procedure.","The target sequences and the nucleotide location within the PTGDR RNA are given in Tables XIX-XXIII.","Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid molecule cleavage assay is prepared by in vitro transcription in the presence of [α-32P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification.","Alternately, substrates are 5′-32P-end labeled using T4 polynucleotide kinase enzyme.","Assays are performed by pre-warming a 2× concentration of purified enzymatic nucleic acid molecule in enzymatic nucleic acid molecule cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37° C., 10 mM MgCl2) and the cleavage reaction was initiated by adding the 2× enzymatic nucleic acid molecule mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer.","As an initial screen, assays are carried out for 1 hour at 37° C. using a final concentration of either 40 nM or 1 mM enzymatic nucleic acid molecule, i.e., enzymatic nucleic acid molecule excess.","The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95° C. for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel.","Substrate RNA and the specific RNA cleavage products generated by enzymatic nucleic acid molecule cleavage are visualized on an autoradiograph of the gel.","The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.","In Vivo Models Used to Evaluate the Down-Regulation of PTGDS, ADORA1 and PTGDR Gene Expression Animal Models Evaluating the efficacy of anti-PTGDS, ADORA-1 and/or PTGDR agents in animal models is an important prerequisite to human clinical trials.","Matsuoka et al., 2000, Science, 287, 2012-2016, describe a useful asthma animal model having generating mice deficient in the PTGDR receptor.","Sensitization and aerosol challenge of homozygous (PTGDR−/−) mice with ovalbumin was shown to induce increases in the serum concentration of immunoglobin E (IgE), an allergic mediator that activates mast cells, similar to wild-type mice subjected to the same conditions.","The concentration of TH2 cytokines and the degree of lymphocyte lung infiltration in the OVA challenged PTGDR −/− mice was shown to be greatly reduced compared to wild type mice.","In addition, the PTGDR −/− mice showed only marginal eosinophil infiltration and failed to develop airway hyperreactivity.","Similarly, this model can be used to evaluate mice that are treated with nucleic acid molecules of the invention and can furthermore be used as a positive control in determining the response of mice treated with nucleic acid molecules of the invention by using such factors as airway obstruction, lung capacity, and bronchiolar alveolar lavage (BAL) fluid in the evaluation.","Cell Culture Two human cell lines, NPE cells and NCB-20 cells are known to express PTGDR.","Cloned human PTGDR has been expressed in CHO and COS7 cells and used in various studies.","These PTGDR expressing lung cell lines can be used in cell culture assays to evaluate nucleic acid molecules of the invention.","A primary endpoint in these experiments would be the RT-PCR analysis of PTGDR mRNA expression in PTGDR expressing cells.","In addition, ligand binding assays can be developed where binding of PTGDS can be evaluated in response to treatment with nucleic acid molecules of the invention.","Indications The present body of knowledge in PTGDS, ADORA1 and PTGDR research indicates the need for methods to assay PTGDS, ADORA1 and PTGDR activity and for compounds that can regulate PTGDS, ADORA1 and PTGDR expression for research, diagnostic, and therapeutic use.","As described herein, the nucleic acid molecules of the present invention can be used in assays to diagnose disease state related of PTGDS, ADORA1 and/or PTGDR levels.","In addition, the nucleic acid molecules can be used to treat disease state related to PTGDS, ADORA1 and/or PTGDR levels.","Particular degenerative and disease states that can be associated with PTGDS, ADORA1 and PTGDR levels include, but are not limited to allergic diseases and conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and any other diseases or conditions that are related to or will respond to the levels of PTGDS, ADORA1 and/or PTGDR in a cell or tissue, alone or in combination with other therapies.","The use of anti-inflammatories, bronchodilators, adenosine inhibitors and adenosine A1 receptor inhibitors are examples of other treatments or therapies can be combined with the nucleic acid molecules of the invention.","Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g.","enzymatic nucleic acid molecules and antisense molecules) are hence within the scope of the instant invention.","Diagnostic Uses The nucleic acid molecules of this invention (e.g., enzymatic nucleic acid molecules) can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of PTGDS, ADORA1 and/or PTGDR RNA in a cell.","The close relationship between enzymatic nucleic acid molecule activity and the structure of the target RNA allows the detection of mutations in any region of the molecule that alters the base-pairing and three-dimensional structure of the target RNA.","By using multiple enzymatic nucleic acid molecules described in this invention, one can map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues.","Cleavage of target RNAs with enzymatic nucleic acid molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease.","In this manner, other genetic targets can be defined as important mediators of the disease.","These experiments can lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules and/or other chemical or biological molecules).","Other in vitro uses of enzymatic nucleic acid molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with PTGDS, ADORA1 or PTGDR-related condition.","Such RNA is detected by determining the presence of a cleavage product after treatment with an enzymatic nucleic acid molecule using standard methodology.","In a specific example, enzymatic nucleic acid molecules which cleave only wild-type or mutant forms of the target RNA are used for the assay.","The first enzymatic nucleic acid molecule is used to identify wild-type RNA present in the sample and the second enzymatic nucleic acid molecule is used to identify mutant RNA in the sample.","As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both enzymatic nucleic acid molecules to demonstrate the relative enzymatic nucleic acid molecule efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species.","The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population.","Thus each analysis requires two enzymatic nucleic acid molecules, two substrates and one unknown sample which is combined into six reactions.","The presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel.","It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells.","The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., PTGDS/PTGDR) is adequate to establish risk.","If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis.","Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.","The use of enzymatic nucleic acid molecules in diagnostic applications contemplated by the instant invention is described, for example, in George et al., U.S. Pat.","Nos.","5,834,186 and 5,741,679, Shih et al., U.S. Pat.","No.","5,589,332, Nathan et al., U.S. Pat.","No.","5,871,914, Nathan and Ellington, International PCT publication No.","WO 00/24931, Breaker et al., International PCT Publication Nos.","WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No.","WO 99/29842.Example 2 Nucleic Acid Inhibition of NOGO and NOGO Receptor Target RNA The invention features novel nucleic acid-based molecules [e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, decoy RNA, aptamers, siRNA, antisense nucleic acids containing RNA cleaving chemical groups] and methods to modulate gene expression, for example, genes encoding certain myelin proteins that inhibit or are involved in the inhibition of neurite growth, including axonal regeneration in the CNS.","In particular, the instant invention features nucleic-acid based techniques to inhibit the expression of NOGO-A (Accession No.","AJ251383), NOGO-B (Accession No.","AJ251384), and/or NOGO-C (Accession No.","AJ251385), NOGO-66 receptor (Accession No AF283463, Fournier et al., 2001, Nature, 409, 341-346), NI-35, NI-220, and/or NI-250, myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945).","In a preferred embodiment, the invention features the use of one or more of the nucleic acid-based techniques independently or in combination to inhibit the expression or function of the gene(s) encoding NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors.","Specifically, the invention features the use of nucleic acid-based techniques to specifically inhibit the expression of NOGO gene (Genbank Accession No.","AB020693) and NOGO-66 receptor (Genbank Accession No.","AF283463).","The nucleic acid molecules that interact with NOGO and NOGO receptor-specific RNAs represent a novel therapeutic approach to treat a variety of pathologic indications, including but not limited to CNS injury and cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and/or other neurodegenerative disease states which respond to the modulation of NOGO and NOGO receptor expression.","In one embodiment, a nucleic acid molecule that inhibits NOGO and/or NOGO receptor replication or expression can comprise between 12 and 100 bases complementary to a RNA molecule of NOGO or NOGO receptor.","In another embodiment, a nucleic acid molecule that inhibits NOGO or NOGO receptor replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of NOGO or NOGO receptor.","In one embodiment the invention provides a method for producing a class of nucleic acid-based gene inhibiting agents which exhibit a high degree of specificity for the RNA of a desired target.","For example, the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of target RNAs encoding NOGO-A, NOGO-B, NOGO-C and/or receptor proteins (specifically NOGO and NOGO receptor genes) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention.","Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required.","Alternatively, the nucleic acid molecules (e.g., ribozymes and antisense) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.","The nucleic acid-based inhibitors of NOGO and NOGO receptor expression are useful for the prevention and/or treatment of diseases and conditions such CNS injury, cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, muscular dystrophy and any other diseases or conditions that are related to or will respond to the levels of NOGO and/or NOGO receptor in a cell or tissue, alone or in combination with other therapies.","In addition, NOGO and/or NOGO receptor inhibition can be used as a therapeutic target for abrogating CNS neuronal growth inhibition; a situation that can selectively regenerate damaged or lesioned CNS tissue to restore specific reflex and/or locomotor functions.","In a further embodiment, the described molecules, such as antisense, siRNA or ribozymes, can be used in combination with other known treatments to treat conditions or diseases discussed above.","For example, the described molecules can be used in combination with one or more known therapeutic agents to treat CNS injury, spinal cord injury, cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and/or other neurodegenerative disease states which respond to the modulation of NOGO and/or NOGO receptor expression.","In another embodiment, the invention features nucleic acid-based inhibitors (e.g., enzymatic nucleic acid molecules (eg; ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of genes (e.g., NOGO and/or NOGO receptor) capable of progression and/or maintenance of CNS injury, spinal cord injury, cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and/or other neurodegenerative disease states which respond to the modulation of NOGO and/or NOGO receptor expression.","The lack of axon regeneration capacity in the adult CNS manifests as a limiting factor in the treatment of CNS injury, cerebrovascular accident (CVA, stroke), chemotherapy-induced neuropathy, and possibly in neurodegenerative diseases such as Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, and/or muscular dystrophy.","Neuron growth inhibition results from physical barriers imposed by glial scars, a lack of neurotrophic factors, and growth-inhibitory molecules associated with myelin.","The abrogation of neurite growth inhibition creates the potential to treat conditions for which there is currently no definitive medical intervention.","The inhibition of NOGO (Genbank Accession No AB020693) and NOGO-66 receptor (Genbank Accession No.","AF283463) is demonstrated in the following examples.","Identification of Potential Target Sites in Human NOGO RNA The sequence of human NOGO and NOGO receptor genes are screened for accessible sites using a computer-folding algorithm.","Regions of the RNA that do not form secondary folding structures and contained potential enzymatic nucleic acid molecule and/or antisense binding/cleavage sites are identified.","The sequences of these binding/cleavage sites are shown in Tables III-VII.","Selection of Enzymatic Nucleic Acid Cleavage Sites in Human NOGO and NOGO Receptor RNA Enzymatic nucleic acid molecule target sites are chosen by analyzing sequences of Human NOGO (Genbank accession No: AB020693) and prioritizing the sites on the basis of folding.","Enzymatic nucleic acid molecules are designed that can bind each target and are individually analyzed by computer folding (Christoffersen et al., 1994 J. Mol.","Struc.","Theochem, 311, 273; Jaeger et al., 1989, Proc.","Natl.","Acad.","Sci.","USA, 86, 7706) to assess whether the enzymatic nucleic acid molecule sequences fold into the appropriate secondary structure.","Those enzymatic nucleic acid molecules with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration.","As noted below, varying binding arm lengths can be chosen to optimize activity.","Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.","Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient Cleavage and/or Blocking of NOGO and NOGO Receptor RNA Enzymatic nucleic acid molecules and antisense constructs are designed to anneal to various sites in the RNA message.","The binding arms of the enzymatic nucleic acid molecules are complementary to the target site sequences described above, while the antisense constructs are fully complimentary to the target site sequences described above.","The enzymatic nucleic acid molecules and antisense constructs were chemically synthesized.","The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J.","Am.","Chem.","Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.","The average stepwise coupling yields were typically >98%.","Enzymatic nucleic acid molecules and antisense constructs are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol.","180, 51).","Enzymatic nucleic acid molecules and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and are resuspended in water.","The sequences of the chemically synthesized enzymatic nucleic acid molecules used in this study are shown below in Table III-VII.","The sequences of the chemically synthesized antisense constructs used in this study are complimentary sequences to the Substrate sequences shown below as in Table III-VII.","Enzymatic Nucleic Acid Molecule Cleavage of NOGO and NOGO Receptor RNA Target In Vitro Enzymatic nucleic acid molecules targeted to the human NOGO RNA are designed and synthesized as described above.","These enzymatic nucleic acid molecules can be tested for cleavage activity in vitro, for example, using the following procedure.","The target sequences and the nucleotide location within the NOGO receptor RNA are given in Tables III-VII.","Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid molecule cleavage assay is prepared by in vitro transcription in the presence of [α-32P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification.","Alternately, substrates are 5′-32P-end labeled using T4 polynucleotide kinase enzyme.","Assays are performed by pre-warming a 2× concentration of purified enzymatic nucleic acid molecule in enzymatic nucleic acid molecule cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37° C., 10 mM MgCl2) and the cleavage reaction was initiated by adding the 2× enzymatic nucleic acid molecule mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer.","As an initial screen, assays are carried out for 1 hour at 37° C. using a final concentration of either 40 nM or 1 mM enzymatic nucleic acid molecule, i.e., enzymatic nucleic acid molecule excess.","The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95° C. for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel.","Substrate RNA and the specific RNA cleavage products generated by enzymatic nucleic acid molecule cleavage are visualized on an autoradiograph of the gel.","The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.","Nucleic Acid Inhibition of NOGO and NOGO Receptor Target RNA In Vivo Nucleic acid molecules targeted to the human NOGO and NOGO receptor RNA are designed and synthesized as described above.","These nucleic acid molecules can be tested for cleavage activity in vivo, for example using the procedures described below.","The target sequences and the nucleotide location within the NOGO receptor RNA are given in Tables III-VII.","Cell Culture Spillmann et al., 1998, J. Biol.","Chem., 273, 19283-19293, describe the purification and biochemical characterization of a high molecular mass protein of bovine spinal cord myelin (bNI-220) which exerts potent inhibition of neurite outgrowth of NGF-primed PC12 cells and chick DRG cells.","This protein can be used to inhibit spreading of 3T3 fibroblasts and to induce collapse of chick DRG growth cones.","The monoclonal antibody, mAb IN-1, can be used to fully neutralize the inhibitory activity of bNI-220, which is a presumed NOGO gene product.","As such, nucleic acid molecules of the instant invention directed at the inhibition of NOGO expression can be used in place of mAb IN-1 in studying the inhibition of bNI-220 in cell culture experiments described in detail by Spillmann et al., supra.","Criteria used in these experiments include the evaluation of spreading behavior of 3T3 fibroblasts, the neurite outgrowth response of PC12 cells, and the growth cone motility of chick DRG growth cones.","Similarly, nucleic acid molecules of the instant invention that target NOGO or NOGO receptors can be used to evaluate inhibition of NOGO mediated activity in these cell types using the criteria described above.","Fournier et al., 2001, Nature, 409, 341 describe a mouse clone of the NOGO-66 receptor which is expressed in non-neuronal COS-7 cells.","The transfected COS-7 cell line expresses NOGO-66 receptor protein on the cell surface.","An antiserum developed to the NOGO-66 receptor can be used to specifically stain NOGO-66 receptor expressing cells by immunohistochemical staining.","As such, an assay for screening nucleic acid-based inhibitors of NOGO-66 receptor expression is provided.","Animal Models Bregman et al., 1995, Nature, 378, 498-501 and Z'Graggen et al., 1998, J. Neuroscience, 18, 4744, describe a rat based system for evaluating the role of myelin-associated neurite growth inhibitory proteins in vivo.","Young adult Lewis rats receive a mid-thoracic microsurgical spinal cord lesion or a unilateral pyramidotomy.","These animals are then treated with mAb IN-1 secreting hybridoma cell explants.","A control population receive hybridoma explants which secrete horsreradish peroxidase (HRP) antibodies.","Cyclosporin is used during the treatment period to allow hybridoma survival.","Additional control rats receive either the spinal cord lesion without any further treatment or no lesion.","After a 4-6 week recovery period, behavioral training is followed by the quantitative analysis of reflex and locomotor function.","IN-1 treated animals demonstrate growth of corticospinal axons around the lesion site and into the spinal cord which persist past the longest time point of analysis (12 weeks).","Furthermore, both reflex and locomotor function, including the functional recovery of fine motor control, is restored in IN-1 treated animals.","As such, a robust animal model as described by Bregman et al.","supra and Z'Graggen et al., supra, can be used to evaluate nucleic acid molecules of the instant invention when used in place of or in conjunction with mAb IN-1 toward use as modulators of neurite growth inhibitor function (eg.","NOGO and NOGO receptor) in vivo.","Indications The nucleic acids of the present invention can be used to treat a patient having a condition associated with the level of NOGO or NOGO receptor.","One method of treatment comprises contacting cells of a patient with a nucleic acid molecule of the present invention, under conditions suitable for said treatment.","Delivery methods and other methods of administration have been discussed herein and are commonly known in the art.","Particular degenerative and disease states that can be associated with NOGO and NOGO receptor expression modulation include, but are not limited to, CNS injury, specifically spinal cord injury, cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and/or other neurodegenerative disease states which respond to the modulation of NOGO and NOGO receptor expression.","The present body of knowledge in NOGO research indicates the need for methods to assay NOGO activity and for compounds that can regulate NOGO expression for research, diagnostic, and therapeutic use.","Other treatment methods comprise contacting cells of a patient with a nucleic acid molecule of the present invention and further comprise the use of one or more drug therapies under conditions suitable for said treatment.","The use of monoclonal antibody (eg; mAb IN-1) treatment, growth factors, antiinflammatory compounds, for example methylprednisolone, calcium blockers, apoptosis inhibiting compounds, for example GM-1 ganglioside, and physical therapies, for example treadmill therapy, are all non-limiting examples of methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g.","ribozymes and antisense molecules) of the instant invention.","Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g.","ribozymes and antisense molecules) are hence within the scope of the instant invention.","Diagnostic Uses The nucleic acid molecules of this invention (e.g., enzymatic nucleic acid molecules) can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of NOGO and/or NOGO receptor RNA in a cell.","The close relationship between enzymatic nucleic acid molecule activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA.","By using multiple enzymatic nucleic acid molecules described in this invention, one can map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues.","Cleavage of target RNAs with enzymatic nucleic acid molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease.","In this manner, other genetic targets can be defined as important mediators of the disease.","These experiments can lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules and/or other chemical or biological molecules).","Other in vitro uses of enzymatic nucleic acid molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with NOGO-related condition.","Such RNA is detected by determining the presence of a cleavage product after treatment with a enzymatic nucleic acid molecule using standard methodology.","In a specific example, enzymatic nucleic acid molecules which cleave only wild-type or mutant forms of the target RNA are used for the assay.","The first enzymatic nucleic acid molecule is used to identify wild-type RNA present in the sample and the second enzymatic nucleic acid molecule is used to identify mutant RNA in the sample.","As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both enzymatic nucleic acid molecules to demonstrate the relative enzymatic nucleic acid molecule efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species.","The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population.","Thus each analysis requires two enzymatic nucleic acid molecules, two substrates and one unknown sample which is combined into six reactions.","The presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel.","It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells.","The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., NOGO) is adequate to establish risk.","If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis.","Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.","The use of enzymatic nucleic acid molecules in diagnostic applications contemplated by the instant invention is more fully described in George et al., U.S. Pat.","Nos.","5,834,186 and 5,741,679, Shih et al., U.S. Pat.","No.","5,589,332, Nathan et al., U.S. Pat.","No.","5,871,914, Nathan and Ellington, International PCT publication No.","WO 00/24931, and Sullenger et al., International PCT publication No.","WO 99/29842.Example 3 Nucleic Acid Inhibition of IKK-Gamma and PKR Target RNA The invention features nucleic acid molecules, for example enzymatic nucleic acid molecules, antisense nucleic acid molecules, 2,5-A chimeras, decoys, siRNA, triplex oligonucleotides, siRNA and/or aptamers, and methods to modulate gene expression, for example, genes encoding a member of the IκB kinase IKK complex, such as IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKKγ) and/or a protein kinase PKR protein.","In particular, the instant invention features nucleic-acid based molecules and methods to modulate the expression of IKK-gamma (IKKγ) and protein kinase PKR.","The invention features one or more nucleic acid-based molecules and methods that independently or in combination modulate the expression of gene(s) encoding a member of the IκB kinase IKK complex or PKR.","In particular embodiments, the invention features nucleic acid-based molecules and methods that modulate the expression of a member of the IκB kinase IKK complex, for example IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKKγ) and/or a protein kinase PKR protein, such as IKK-alpha (IKK1) gene (Genbank Accession No.","NM—001278); IKK-beta (IKK2) gene, for example (Genbank Accession No.","AF080158), IKK-gamma (IKKγ) gene, for example (Genbank Accession No.","NM—003639), and protein kinase PKR gene, for example (Genbank Accession No.","NM—002759).","In one embodiment, an enzymatic nucleic acid molecule of the invention comprises a sequence selected from the group consisting of SEQ ID NOs.","7056-7249.In another embodiment, an enzymatic nucleic acid molecule of the invention comprises at least one binding arm wherein one or more of said binding arms comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.","1024-4414.In another embodiment, an antisense nucleic acid molecule or siRNA molecule of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.","1024-4414.In another embodiment, an nucleic acid molecule of the invention is adapted to treat cancer.","In yet another embodiment, an enzymatic nucleic acid molecule of the invention has an endonuclease activity to cleave RNA having IKK-gamma or PKR sequence.","In one embodiment, an enzymatic nucleic acid molecule of the invention is in an Inozyme, Zinzyme, G-cleaver, Amberzyme, DNAzyme, or Hammerhead configuration.","In another embodiment, an Inozyme of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.","1218-1721 and 3051-3549.In another embodiment, an Inozyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs.","7250-7753 and 9701-10199.In another embodiment, a Zinzyme of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.","1722-1998 and 3550-3768.In another embodiment, a Zinzyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 7754-8030 and 10200-10418.In another embodiment, an Amberzyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 8441-9069 and 11001-11547.In another embodiment, a DNAzyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 8031-8440 and 10419-11000.In another embodiment, a Hammerhead of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.","1024-1217 and 2420-3050.In another embodiment, a Hammerhead of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 7056-7249 and 9070-9700.In one embodiment, a nucleic acid molecule of the invention comprises between 12 and 100 bases complementary to RNA having sequence of IKK-gamma or PKR.","In another embodiment, a nucleic acid molecule of the invention comprises between 14 and 24 bases complementary to RNA having sequence of IKK-gamma or PKR.","In yet another embodiment, a nucleic acid molecule of the invention is chemically synthesized.","In another embodiment, a nucleic acid molecule or antisense nucleic acid molecule of the invention comprises at least one 2′-sugar modification, at least one nucleic acid base modification, or at least one phosphate backbone modification.","In one embodiment, the invention features a mammalian cell, for example a human cell, including an enzymatic nucleic acid molecule of the invention.","The present invention features method of down-regulating PKR activity in a cell, comprising contacting the cell with a nucleic acid molecule of the invention, under conditions suitable for down-regulating of PKR activity.","The present invention also features method of treatment of a patient having a condition associated with the level of PKR, comprising contacting cells of the patient with a nucleic acid molecule of the invention under conditions suitable for the treatment.","The present invention features method of down-regulating IKK-gamma activity in a cell, comprising contacting the cell with a nucleic acid molecule of the invention, under conditions suitable for down-regulating of IKK-gamma activity.","The present invention also features method of treatment of a patient having a condition associated with the level of IKK-gamma, comprising contacting cells of the patient with the nucleic acid molecule of the invention, under conditions suitable for the treatment.","In one embodiment, a method of treatment of the invention comprises the use of one or more drug therapies under conditions suitable for said treatment.","The present invention features method of cleaving RNA comprising a sequence of PKR gene comprising contacting an enzymatic nucleic acid molecule of the invention with the RNA of a PKR gene under conditions suitable for the cleavage.","The present invention also features method of cleaving RNA comprising a sequence of IKK-gamma gene comprising contacting an enzymatic nucleic acid molecule of the invention with the RNA of an IKK-gamma gene under conditions suitable for the cleavage.","In one embodiment, a method of cleavage of the invention is carried out in the presence of a divalent cation, for example Mg2+.","In another embodiment, a nucleic acid molecule of the invention comprises a cap structure, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic derivative.","The present invention also features an expression vector comprising a nucleic acid sequence encoding at least one nucleic acid molecule the invention in a manner which allows expression of the nucleic acid molecule.","In one embodiment, the invention features a mammalian cell, for example a human cell, including an expression vector contemplated by the invention.","In another embodiment, an expression vector of the invention further comprises a sequence for a nucleic acid molecule complementary to the RNA of a subunit of IKK-gamma or PKR.","In yet another embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more nucleic acid molecules, which can be the same or different.","The present invention also features a method for treatment of cancer, for example breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer, comprising administering to a patient a nucleic acid molecule of the invention under conditions suitable for said treatment.","In one embodiment, a nucleic acid molecule of the invention comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification such as a 3′-3′ inverted abasic moiety, and/or phosphorothioate linkages on at least three of the 5′ terminal nucleotides.","In another embodiment, other drug therapies contemplated by the invention include monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.","Specific chemotherapy contemplated by the invention include paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.","The invention also features a method for treatment of an inflammatory disease, for example rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury, glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, or infection, comprising the step of administering to a patient a nucleic acid molecule of the invention under conditions suitable for the treatment.","The present invention features pharmaceutical compositions comprising the nucleic acid molecules of the invention in a pharmaceutically acceptable carrier.","The invention also features a method of administering to a cell, such as mammalian cell (e.g.","human cell), where the cell can be in culture or in a mammal, such as a human, an enzymatic nucleic acid molecule or antisense molecule of the instant invention, comprising contacting the cell with the nucleic acid molecule under conditions suitable for such administration.","The method of administration can be in the presence of a delivery reagent, for example a lipid, cationic lipid, phospholipid, or liposome.","The nucleic acid molecules that target specific sites in IKK-gamma or PKR-specific RNAs represent a therapeutic approach to treat a variety of inflammatory-related diseases and conditions, including but not limited to rheumatoid arthritis, restenosis, asthma, Crohn's disease, incontinentia pigmenti, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, and any other inflammatory disease or condition which respond to the modulation of IKK-gamma or PKR function.","The enzymatic nucleic acid molecules that cleave the specified sites in IKK-gamma or PKR-specific RNAs also represent a therapeutic approach to treat a variety of cancers, including but not limited to breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, and/or other cancers which respond to the modulation of IKK-gamma or PKR function.","In one embodiment, a nucleic acid molecule that modulates, for example, down-regulates IKK-gamma or PKR expression comprises between 12 and 100 bases complementary to a RNA molecule of IKK-gamma or PKR.","In another embodiment, a nucleic acid molecule that modulates, for example IKK-gamma or PKR expression comprises between 14 and 24 bases complementary to a RNA molecule of IKK-gamma or PKR.","Nucleic acid-based inhibitors of IKK-gamma or PKR function are useful for the prevention and/or treatment of cancers and cancerous conditions such as breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, and any other diseases or conditions that are related to or will respond to the levels of IKK-gamma or PKR in a cell or tissue, alone or in combination with other therapies.","Nucleic acid-based inhibitors of IKK-gamma or PKR function are also useful for the prevention and/or treatment of inflammatory related diseases and conditions, including but not limited to rheumatoid arthritis, restenosis, asthma, Crohn's disease, incontinentia pigmenti, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, and any other inflammatory disease or condition which respond to the modulation of IKK-gamma or PKR function.","In a further embodiment, the described nucleic acid molecules, such as antisense, siRNA or enzymatic nucleic acid molecules, can be used in combination with other known treatments to treat conditions or diseases discussed above.","For example, the described molecules can be used in combination with one or more known therapeutic agents to treat breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, incontinentia pigmenti, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, and any other cancerous disease or inflammatory disease or condition which respond to the modulation of IKK-gamma or PKR expression.","Identification of Potential Target Sites in Human IKK-Gamma and PKR RNA The sequence of human IKK-gamma or PKR genes are screened for accessible sites using a computer-folding algorithm.","Regions of the RNA that do not form secondary folding structures and contained potential enzymatic nucleic acid molecule and/or antisense binding/cleavage sites are identified.","The sequences of these binding/cleavage sites are shown in Tables VIII-XVIII.","Selection of Enzymatic Nucleic Acid Cleavage Sites in Human IKK-Gamma and PKR RNA Enzymatic nucleic acid molecule target sites are chosen by analyzing sequences of Human IKK-gamma (Genbank accession No: NM—003639) and PKR (Genbank accession No: NM—002759) and prioritizing the sites on the basis of folding.","Enzymatic nucleic acid molecules are designed that can bind each target and are individually analyzed by computer folding (Christoffersen et al., 1994 J. Mol.","Struc.","Theochem, 311, 273; Jaeger et al., 1989, Proc.","Natl.","Acad.","Sci.","USA, 86, 7706) to assess whether the enzymatic nucleic acid molecule sequences fold into the appropriate secondary structure.","Those enzymatic nucleic acid molecules with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration.","As noted below, varying binding arm lengths can be chosen to optimize activity.","Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.","Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient Cleavage and/or Blocking of IKK-Gamma and PKR RNA Enzymatic nucleic acid molecules and antisense constructs are designed to anneal to various sites in the RNA message.","The binding arms of the enzymatic nucleic acid molecules are complementary to the target site sequences described above, while the antisense constructs are fully complementary to the target site sequences described above.","The enzymatic nucleic acid molecules and antisense constructs were chemically synthesized.","The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J.","Am.","Chem.","Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.","The average stepwise coupling yields were typically >98%.","Enzymatic nucleic acid molecules and antisense constructs are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol.","180, 51).","Enzymatic nucleic acid molecules and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and are resuspended in water.","The sequences of the chemically synthesized enzymatic nucleic acid molecules used in this study are shown below in Table XVIII.","The sequences of the chemically synthesized antisense constructs used in this study are complementary sequences to the Substrate sequences shown below as in Tables VIII-XVIII.","Enzymatic Nucleic Acid Molecule Cleavage of IKK-Gamma and PKR RNA Target In Vitro Enzymatic nucleic acid molecules targeted to the human IKK-gamma or PKR RNA are designed and synthesized as described above.","These enzymatic nucleic acid molecules can be tested for cleavage activity in vitro, for example, using the following procedure.","The target sequences and the nucleotide location within the IKK-gamma or PKR RNA are given in Tables VIII-XVIII.","Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid molecule cleavage assay is prepared by in vitro transcription in the presence of [a-32P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification.","Alternately, substrates are 5′-32P-end labeled using T4 polynucleotide kinase enzyme.","Assays are performed by pre-warming a 2× concentration of purified enzymatic nucleic acid molecule in enzymatic nucleic acid molecule cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37° C., 10 mM MgCl2) and the cleavage reaction was initiated by adding the 2× enzymatic nucleic acid molecule mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer.","As an initial screen, assays are carried out for 1 hour at 37° C. using a final concentration of either 40 nM or 1 mM enzymatic nucleic acid molecule, i.e., enzymatic nucleic acid molecule excess.","The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95° C. for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel.","Substrate RNA and the specific RNA cleavage products generated by enzymatic nucleic acid molecule cleavage are visualized on an autoradiograph of the gel.","The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.","Nucleic Acid Down-Regulation of IKK-Gamma and PKR Target RNA In Vivo Nucleic acid molecules targeted to the human IKK-gamma or PKR RNA are designed and synthesized as described above.","These nucleic acid molecules can be tested for cleavage activity in vivo, for example using the procedures described below.","The target sequences and the nucleotide location within the IKK-gamma or PKR RNA are given in Tables VIII-XVIII.","In Vivo Models Used to Evaluate the Down-Regulation of IKK-Gamma or PKR Gene Expression A variety of endpoints have been used in cell culture models to evaluate IKK-gamma or PKR-mediated effects after treatment with anti-IKK-gamma or PKR agents.","Phenotypic endpoints include inhibition of cell proliferation, apoptosis assays and reduction of IKK-gamma or PKR protein expression, or a decrease in NFKB expression.","Since IKK-gamma and PKR are both involved in the induction of NFKB, NFKB can be used as a surrogate marker in cell culture, animal, and clinical studies.","Because overexpression of NFKB is directly associated with increased proliferation of tumor cells, a proliferation endpoint for cell culture assays is preferably used as a primary screen.","There are several methods by which this endpoint can be measured.","Following treatment of cells with nucleic acid molecules, cells are allowed to grow (typically 5 days) after which either the cell viability, the incorporation of [3H]thymidine into cellular DNA and/or the cell density can be measured.","The assay of cell density is very straightforward and can be performed in a 96-well format using commercially available fluorescent nucleic acid stains (such as Syto® 13 or CyQuant®).","The assay using CyQuant® is described herein As a secondary, confirmatory endpoint a nucleic acid-mediated decrease in the level of IKK-gamma or PKR RNA and/or IKK-gamma or PKR protein expression can be evaluated.","Alternately, a decrease in the level of NFKB RNA can be evaluated.","Cell Culture Cell types that express/over-express NFKB include HeLa, macrophages, peripheral blood lymphocytes, hepatocytes, fibroblasts, endothelial cells and epithelial cells.","In culture, these cells can be stimulated to express/over-express NFKB by addition of TNF-alpha PMA or IL-1-beta to the culture medium.","Some of these cell types also can respond with a similar activation of NFKB following LPS treatment.","Activation of NFKB in cultured cells can be evaluated by electrophoretic mobility shift assay (EMSA).","Delineation of alterations in the subunits can be determined by Western blot.","Primary Screen A useful cell culture system in evaluating NFKB modulation is human colonic epithelial cells.","One suitable cell line is SW620 colon carcinoma cells (CCL227).","These cells respond to stimulation with TNF-alpha, LPS and/or IL-1-beta with an increase in NFKB activation.","SW620 cells are grown in MEM supplemented with 10% heat-inactivated FBS and glutamine (2 mmol/L).","TNF-alpha dose-response curves in these cells are determined by incubating cells with various concentrations of recombinant human TNF-alpha (Sigma Chemical Co.).","Maximal DNA binding activity induction can occur with 150 U/ml TNF-alpha in the culture medium.","Induction is typically evident within 10 minutes of treatment with TNF-alpha reaches a peak at one hour post-treatment and persists for up to 4 hours post-treatment.","The primary readout can be NFKB DNA activity in nuclear extracts of SW620 cells as determined by electrophoretic mobility shift assays (EMSA).","Once the appropriate TNF-alpha dose/response profile has been determined, inhibition of IKK-gamma, PKR, or NFKB activation is evaluated using specific and non-specific inhibitors of activation, sulfasalazine and steroids, respectively.","Cells are incubated with inhibitors or control media for 30 minutes prior to stimulation with TNF-alpha Nuclear extracts are prepared and evaluated for DNA binding activity by EMSA.","Once the activity of positive controls has been established, enzymatic nucleic acids targeting the IKK-gamma or PKR are evaluated in this system.","Supershift assays using polyclonal antibodies against the NFKB or PKR protein subunits can be performed to confirm down-regulation of NFKB.","Secondary Screens SW620 cells can be transfected with the 3xIg-kappa-B-Luc reporter construct 18 hours before challenge with TNF-alpha, LPS or PMA.","The readout for this assay is luciferase activity.","Test compounds are applied 17.5 hours after transfection (30 minutes before challenge).","Cells are harvested 24 hours after challenge and relative changes in luciferase activity is used as the endpoint.","Lastly, the activation of NFKB can be visualized fluorescently.","Inactive NFKB heterodimers are held in the cytoplasm by inhibitory proteins.","Once activated, the free heterodimers translocate to the nucleus.","Thus, the relative change in cytoplasmic versus nuclear fluorescence can indicate the degree of NFKB activation.","Cells can be grown on chamber slides, treated with TNF-alpha with and without test compounds), and the location of the NFKB subunit can be determined by immunofluorescence using a FITC-labeled antibody to NFKB.","Animal Models Evaluating the efficacy of anti-IKK-gamma or PKR agents in animal models is an important prerequisite to human clinical trials.","Studies have shown that human breast carcinoma cell lines express high levels of NFKB (Sovak et al., 1997, J. Clin.","Invest., 100, 2952-2960).","High levels of NFKB have also been observed in carcinogen-induced primary rat mammary tumors and in human breast cancer specimins.","Additionally, HER2/neu overexpression has been shown to activate NFKB (Pianetti et al., 2001, Oncogene, 20, 1287-1299).","As such, xenografts of cell lines that over-express NFKB can be used in animal models of tumorigenesis and/or inflammation to study the inhibition of NFKB.","Oncology Animal Model Development Tumor cell lines are characterized to establish their growth curves in mice.","These cell lines are implanted into both nude and SCID mice and primary tumor volumes are measured 3 times per week.","Growth characteristics of these tumor lines using a Matrigel implantation format can also be established.","The use of other cell lines that have been engineered to express high levels of NFKB can also be used in the described studies.","The tumor cell line(s) and implantation method that supports the most consistent and reliable tumor growth is used in animal studies testing the lead IKK-gamma or PKR nucleic acid(s).","Nucleic acids are administered by daily subcutaneous injection or by continuous subcutaneous infusion from Alzet mini osmotic pumps beginning 3 days after tumor implantation and continuing for the duration of the study.","Group sizes of at least 10 animals are employed.","Efficacy is determined by statistical comparison of tumor volume of nucleic acid-treated animals to a control group of animals treated with saline alone.","Because the growth of these tumors is generally slow (45-60 days), an initial endpoint is the time in days it takes to establish an easily measurable primary tumor (i.e.","50-100 mm3) in the presence or absence of nucleic acid treatment.","Inflammation Animal Model Development Chronic, sublethal administration of indomethacin to outbred rats produces an enteropathy characterized by thickening of the small intestine and mesentery, ulcerations, granulomatous inflammation, crypt abcesses and adhesions.","These lesions are similar to those that are characteristic findings in human patients with Crohn's disease (CD).","Thus, any beneficial therapeutic effects revealed using this model can be extrapolated to potential benefit for patients with CD.","Male Sprague-Dawley rats (200-275 g) are utilized for these studies.","Chronic intestinal inflammation is induced by two subcutaneous injections of indomethacin (7.5 mg/kg in 5% NaHCO3) administered on subsequent days (Day-0 and Day-1).","Animals are followed for four days following the first indomethacin injection.","The mortality rate associated with this model is typically less than 10%.","On the last day of the study, animals are euthanized by CO2 asphyxiation, small intestines excised and gross pathologic findings ranked according to the following criteria: 0, normal; 1, minimal abnormalities, slight thickening of the small intestine, no adhesions; 2, obvious thickening of small intestine with 1 adhesion; 3, obvious thickening of small intestine with 2 or 3 adhesions; 4, massive adhesions to the extent that the intestine cannot be separated, contents primarily fluid; 5, severe peritonitis resulting in death.","A 10-cm portion of the most affected region of the small intestine is weighed, placed in 10% neutral buffered formalin and submitted for histopathologic evaluation.","The 10 cm portion of gut from each animal is cut into five equal sections.","Transverse and longitudinal sections of each portion are cut and stained with hematoxylin and eosin.","All slides are read in a blinded fashion and each section is scored for necrosis (% area of involvement) and inflammatory response according to the following scale: Necrosis—1, 10%; 2, 10-25%; 3, 25-50%; 4, 50-75%; 5, 75-100%; Inflammation— 1=minimal in mesentery and muscle or lesion 2=mild in mesentery and muscle or lesion 3=moderate in mesentery and muscle or lesion 4=marked in lesion 5=severe in lesion The scores for each of the five sections are averaged for necrosis and for inflammation.","NFKB Levels for Patient Screening and as a Potential Endpoint Because elevated NFKB levels can be detected in cancers, cancer patients can be pre-screened for elevated NFKB prior to admission to initial clinical trials testing an anti-IKK-gamma or PKR nucleic acid.","Initial NFKB levels can be determined (by ELISA) from tumor biopsies or resected tumor samples.","During clinical trials, it can be possible to monitor circulating NFKB protein by ELISA.","Evaluation of serial blood/serum samples over the course of the anti-IKK-gamma or PKR nucleic acid treatment period could be useful in determining early indications of efficacy.","Activity of Nucleic Acid Molecules Used to Down-Regulate IKK-Gamma and PKR Gene Expression Applicant has designed and synthesized several nucleic acid molecules targeted against IKK-gamma or PKR RNA.","These nucleic acid molecules can be tested in cell proliferation and RNA reduction assays described herein.","Proliferation Assay A model proliferation assay can be done using a cell-plating density of 2,000 cells/well in 96-well plates and at least 2 cell doublings over a 5-day treatment period.","Cells used in proliferation studies can be, for example, were either lung or ovarian cancer cells (A549 and SKOV-3 cells respectively).","To calculate cell density for proliferation assays, the FIPS (fluoro-imaging processing system) method known in the art can be used.","This method allows for cell density measurements after nucleic acids are stained with CyQuant® dye, and has the advantage of accurately measuring cell densities over a very wide range 1,000-100,000 cells/well in 96-well format.","Enzymatic nucleic acid molecules (50-200 nM) are delivered in the presence of cationic lipid at 2.5-5.0 μg/mL and inhibition of proliferation can be determined on day 5 post-treatment.","RNA Assay RNA is harvested 24 hours post-treatment using the Qiagen RNeasy® 96 procedure.","Real time RT-PCR (TaqMan® assay) is performed on purified RNA samples using separate primer/probe sets specific for target IKK-gamma or PKR RNA.","Indications Particular degenerative and disease states that can be associated with IKK-gamma or PKR expression modulation include but are not limited to cancerous and/or inflammatory diseases and conditions such as breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, incontinentia pigmenti and any other diseases or conditions that are related to or respond to the levels of IKK-gamma or PKR in a cell or tissue.","The present body of knowledge in IKK-gamma and PKR research indicates the need for methods to assay IKK-gamma and PKR activity and for compounds that can regulate IKK-gamma and PKR expression for research, diagnostic, and therapeutic use.","The use of monoclonal antibodies, chemotherapy, radiation therapy, analgesics, and/or anti-inflammatory compounds, are all non-limiting examples of a methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g.","ribozymes and antisense molecules) of the instant invention.","Common chemotherapies that can be combined with nucleic acid molecules of the instant invention include various combinations of cytotoxic drugs to kill cancer cells.","These drugs include but are not limited to paclitaxel (Taxol), docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, vinorelbine etc.","Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g.","ribozymes and antisense molecules) are hence within the scope of the instant invention.","Diagnostic Uses The nucleic acid molecules of this invention (e.g., enzymatic nucleic acid molecules) can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of IKK-gamma or PKR RNA in a cell.","The close relationship between enzymatic nucleic acid molecule activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA.","By using multiple enzymatic nucleic acid molecules described in this invention, one can map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues.","Cleavage of target RNAs with enzymatic nucleic acid molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease.","In this manner, other genetic targets can be defined as important mediators of the disease.","These experiments can lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules and/or other chemical or biological molecules).","Other in vitro uses of enzymatic nucleic acid molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with IKK-gamma or PKR-related condition.","Such RNA is detected by determining the presence of a cleavage product after treatment with an enzymatic nucleic acid molecule using standard methodology.","In a specific example, enzymatic nucleic acid molecules which cleave only wild-type or mutant forms of the target RNA are used for the assay.","The first enzymatic nucleic acid molecule is used to identify wild-type RNA present in the sample and the second enzymatic nucleic acid molecule is used to identify mutant RNA in the sample.","As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both enzymatic nucleic acid molecules to demonstrate the relative enzymatic nucleic acid molecule efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species.","The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population.","Thus each analysis requires two enzymatic nucleic acid molecules, two substrates and one unknown sample which is combined into six reactions.","The presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel.","It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells.","The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., IKK-gamma or PKR) is adequate to establish risk.","If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis.","Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.","The use of enzymatic nucleic acid molecules in diagnostic applications contemplated by the instant invention is more fully described in George et al., U.S. Pat.","Nos.","5,834,186 and 5,741,679, Shih et al., U.S. Pat.","No.","5,589,332, Nathan et al., U.S. Pat.","No.","5,871,914, Nathan and Ellington, International PCT publication No.","WO 00/24931, Breaker et al., International PCT Publication Nos.","WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No.","WO 99/29842.Additional Uses Potential uses of sequence-specific enzymatic nucleic acid molecules of the instant invention can have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA (Nathans et al., 1975 Ann.","Rev.","Biochem.","44:273).","For example, the pattern of restriction fragments can be used to establish sequence relationships between two related RNAs, and large RNAs can be specifically cleaved to fragments of a size more useful for study.","The ability to engineer sequence specificity of the enzymatic nucleic acid molecule is ideal for cleavage of RNAs of unknown sequence.","Applicant has described the use of nucleic acid molecules to down-regulate gene expression of target genes in bacterial, microbial, fungal, viral, and eukaryotic systems including plant, or mammalian cells.","All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains.","All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.","One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein.","The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention.","Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.","It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.","Thus, such additional embodiments are within the scope of the present invention and the following claims.","The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that is not specifically disclosed herein.","Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” can be replaced with either of the other two terms.","The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.","Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.","In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.","Other embodiments are within the following claims.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00001 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00002 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00003 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00004 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00005 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00006 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00007 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00008 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00009 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00010 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00011 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00012 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00013 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00014 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00015 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00016 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00017 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00018 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00019 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00020 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00021 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00022 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00023 Please refer to the end of the specification for access instructions.","LENGTHY TABLE The patent application contains a lengthy table section.","A copy of the table is available in electronic form from the USPTO web site () An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3)."]],"string":"[\n [\n \"Modulation of gene expression associated with inflammation proliferation and neurite outgrowth using nucleic acid based technologies\",\n \"The present invention relates to nucleic acid molecules, including antisense, enzymatic nucleic acid molecules, and RNA interference molecules, such as hammerhead ribozymes, DNAzymes, allozymes, siRNA, decoys and antisense, which modulate the expression of prostaglandin D2 (PTGDS), prostaglandin D2 receptor (PTGDR), adenosine receptor, NOGO and NOGO receptor, and IKK genes, such as IKK-gamma, IKK-alpha, or IKK-beta, and PKR genes.\"\n ],\n [\n \"1.A nucleic acid molecule that down regulates expression or inhibits function of a receptor for a neurite growth inhibitor.\",\n \"2.A nucleic acid molecule of claim 1, wherein the receptor is a NOGO receptor.\",\n \"3.The nucleic acid of claim 1, wherein said nucleic acid molecule is adapted for use to treat conditions selected from the group consisting of CNS injury, spinal cord injury, and cerebrovascular accident.\",\n \"4.The nucleic acid molecule of claim 1 or claim 2, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule having at least one binding arm.\",\n \"5.The nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule has an endonuclease activity to cleave RNA encoded by a NOGO receptor gene.\",\n \"6.The nucleic acid of claim 4, wherein the at least one binding arm of the enzymatic nucleic acid molecule comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.\",\n \"1-1023.7.An enzymatic nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs.\",\n \"5484-7055.8.The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is an antisense nucleic acid molecule.\",\n \"9.An antisense nucleic acid molecule comprising a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.\",\n \"1-1023.10.The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is in a hammerhead (HH) motif.\",\n \"11.The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is in a hairpin, hepatitis Delta virus, group I intron, VS nucleic acid, amberzyme, zinzyme or RNAse P nucleic acid motif.\",\n \"12.The enzymatic nucleic acid molecule of claim 11, wherein said zinzyme motif comprises a sequence selected from the group consisting of SEQ ID NOs.\",\n \"6030-6272.13.The enzymatic nucleic acid molecule of claim 11, wherein said amberzyme motif comprises a sequence selected from the group consisting of SEQ ID NOs.\",\n \"6630-7055.14.The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is in a NCH motif.\",\n \"15.The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is in a G-cleaver motif.\",\n \"16.The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is a DNAzyme.\",\n \"17.The nucleic acid molecule of claim 2, wherein said nucleic acid molecule comprises between 12 and 100 bases complementary to RNA encoded by a NOGO receptor gene.\",\n \"18.The nucleic acid molecule of claim 2, wherein said nucleic acid molecule comprises between 14 and 24 bases complementary to the RNA encoded by a NOGO receptor gene.\",\n \"19.The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is chemically synthesized.\",\n \"20.The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one 2′-sugar modification.\",\n \"21.The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one nucleic acid base modification.\",\n \"22.The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one phosphate backbone modification.\",\n \"23.A mammalian cell comprising the nucleic acid molecule of claim 1.24.The mammalian cell of claim 23, wherein said mammalian cell is a human cell.\",\n \"25.A method of reducing NOGO receptor activity in a cell, comprising the step of contacting said cell with the nucleic acid molecule of claim 2, under conditions suitable for said inhibition.\",\n \"26.A method of treatment of a patient having a condition associated with levels of a NOGO receptor, comprising contacting cells of said patient with the nucleic acid molecule of claim 2, under conditions suitable for said treatment.\",\n \"27.The method of claim 26 further comprising the use of one or more drug therapies under conditions suitable for said treatment.\",\n \"28.A method of cleaving RNA encoded by a NOGO receptor gene comprising contacting the nucleic acid molecule of claim 2 with said RNA under conditions suitable for the cleavage of said RNA.\",\n \"29.The method of claim 28, wherein said cleavage is carried out in the presence of a divalent cation.\",\n \"30.The method of claim 29, wherein said divalent cation is Mg2+.\",\n \"31.The nucleic acid molecule of claim 1, wherein said nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, 3′-end, or both the 5′-end and the 3′-end.\",\n \"32.The enzymatic nucleic acid molecule of claim 10, wherein said hammerhead motif comprises a sequence selected from the group consisting of SEQ ID NOs.\",\n \"5484-5583.33.The enzymatic nucleic acid molecule of claim 14, wherein said NCH motif comprises a sequence selected from the group consisting of SEQ ID NOs.\",\n \"5584-6029.34.The enzymatic nucleic acid molecule of claim 16, wherein said DNAzyme comprises a sequence selected from the group consisting of SEQ ID NOs.\",\n \"6273-6629.35.The method of claim 25, wherein said nucleic acid molecule is in a hammerhead motif.\",\n \"36.The method of claim 25, wherein said nucleic acid molecule is a DNAzyme.\",\n \"37.An expression vector comprising at least one nucleic acid molecule of claim 1 in a manner that allows expression of the nucleic acid molecule.\",\n \"38.A mammalian cell comprising an expression vector of claim 37.39.The mammalian cell of claim 38, wherein said mammalian cell is a human cell.\",\n \"40.The expression vector of claim 37, wherein said expression vector encodes a nucleic acid molecule having a hammerhead motif.\",\n \"41.The expression vector of claim 37, wherein said expression vector further comprises a sequence for an antisense nucleic acid molecule complementary to RNA encoded by a NOGO receptor gene.\",\n \"42.The expression vector of claim 37, wherein said expression vector comprises a two or more of said nucleic acid molecules, which can be the same or different.\",\n \"43.The expression vector of claim 42, wherein said expression vector comprises a sequence encoding an antisense nucleic acid molecule complementary to RNA encoded by a NOGO receptor gene.\",\n \"44.A method for treatment of conditions selected from the group consisting of CNS injury and cerebrovascular accident comprising the step of administering to a patient the nucleic acid molecule of claim 1 under conditions suitable for said treatment.\",\n \"45.The method of claim 44, wherein said treatment of CNS injury is treatment of spinal cord injury.\",\n \"46.A method for treatment of conditions selected from the group consisting of CNS injury and cerebrovascular accident comprising the step of administering to a patient the antisense nucleic acid molecule of claim 9 under conditions suitable for said treatment.\",\n \"47.The method of claim 44, wherein said nucleic acid molecule is in a hammerhead motif.\",\n \"48.The method of claim 44, wherein said method further comprises administering to said patient one or more other therapies.\",\n \"49.The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification.\",\n \"50.The nucleic acid molecule of claim 49, wherein said nucleic acid molecule further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.\",\n \"51.The nucleic acid molecule of claim 49, wherein said 3′-end modification is 3′-3′ inverted abasic moiety.\",\n \"52.The enzymatic nucleic acid molecule of claim 16, wherein said DNAzyme comprises at least ten 2′-O-methyl modifications and a 3′-end modification.\",\n \"53.The enzymatic nucleic acid molecule of claim 52, wherein said DNAzyme further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.\",\n \"54.The enzymatic nucleic acid molecule of claim 52, wherein said 3′-end modification is 3′-3′ inverted abasic moiety.\",\n \"55.An enzymatic nucleic acid molecule that down regulates expression of a nucleic acid molecule encoding an IkappaB kinase (IKK) subunit.\",\n \"56.An enzymatic nucleic acid molecule that down regulates expression of a nucleic acid molecule encoding protein kinase PKR.\",\n \"57.An enzymatic nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs.\",\n \"7056-11665.58.An enzymatic nucleic acid molecule comprising at least one binding arm wherein one or more of said binding arms comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.\",\n \"1024-4414.59.An antisense nucleic acid molecule comprising a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.\",\n \"1024-4414.60.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule is adapted to treat cancer.\",\n \"61.The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule is adapted to treat cancer.\",\n \"62.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule has an endonuclease activity to cleave RNA encoded by a IKK-gamma gene or PKR gene.\",\n \"63.The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in an Inozyme configuration.\",\n \"64.The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.\",\n \"65.The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in a G-cleaver configuration.\",\n \"66.The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in an Amberzyme configuration.\",\n \"67.The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in a DNAzyme configuration.\",\n \"68.The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in a Hammerhead configuration.\",\n \"69.The enzymatic nucleic acid molecule of claim 63, wherein said Inozyme comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.\",\n \"1218-1721 and 3051-3549.70.The enzymatic nucleic acid molecule of claim 63, wherein said Inozyme comprises a sequence selected from the group consisting of SEQ ID NOs.\",\n \"7250-7753 and 9701-10199.71.The enzymatic nucleic acid molecule of claim 64, wherein said Zinzyme comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.\",\n \"1722-1998 and 3550-3768.72.The enzymatic nucleic acid molecule of claim 64, wherein said Zinzyme comprises a sequence selected from the group consisting of SEQ ID NOs 7754-8030 and 10200-10418.73.The enzymatic nucleic acid molecule of claim 66, wherein said Amberzyme comprises a sequence selected from the group consisting of SEQ ID NOs 8441-9069 and 11001-11547.74.The enzymatic nucleic acid molecule of claim 67, wherein said DNAzyme comprises a sequence selected from the group consisting of SEQ ID NOs 8031-8440 and 10419-11000.75.The enzymatic nucleic acid molecule of claim 68, wherein said Hammerhead comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.\",\n \"1024-1217 and 2420-3050.76.The enzymatic nucleic acid molecule of claim 68, wherein said Hammerhead comprises a sequence selected from the group consisting of SEQ ID NOs 7056-7249 and 9070-9700.77.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises between 12 and 100 bases complementary to RNA encoded by an IKK-gamma gene or PKR gene.\",\n \"78.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises between 14 and 24 bases complementary to RNA encoded by an IKK-gamma gene or PKR gene.\",\n \"79.The enzymatic nucleic acid molecule of any of claims 55-58 wherein said enzymatic nucleic acid molecule is chemically synthesized.\",\n \"80.The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule is chemically synthesized.\",\n \"81.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises at least one 2′-sugar modification.\",\n \"82.The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule comprises at least one 2′-sugar modification.\",\n \"83.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises at least one nucleic acid base modification.\",\n \"84.The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule comprises at least one nucleic acid base modification.\",\n \"85.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises at least one phosphate backbone modification.\",\n \"86.The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule comprises at least one phosphate backbone modification.\",\n \"87.A mammalian cell including the enzymatic nucleic acid molecule of any of claims 55-58.88.The mammalian cell of claim 87, wherein said mammalian cell is a human cell.\",\n \"89.A method of down-regulating PKR activity in a cell, comprising contacting said cell with the enzymatic nucleic acid molecule of claim 56, under conditions suitable for down-regulating of PKR activity.\",\n \"90.A method of treatment of a patient having a condition associated with the level of PKR, comprising contacting cells of said patient with the enzymatic nucleic acid molecule of any of claims 55-59, under conditions suitable for said treatment.\",\n \"91.A method of down-regulating IKK-gamma activity in a cell, comprising contacting said cell with the enzymatic nucleic acid molecule of any of claims 55-59, under conditions suitable for down-regulating of IKK-gamma activity.\",\n \"92.A method of treatment of a patient having a condition associated with the level of IKK-gamma, comprising contacting cells of said patient with the enzymatic nucleic acid molecule of any of claims 55-59, under conditions suitable for said treatment.\",\n \"93.The method of claim 89 further comprising the use of one or more drug therapies under conditions suitable for said treatment.\",\n \"94.The method of claim 90 further comprising the use of one or more drug therapies under conditions suitable for said treatment.\",\n \"95.The method of claim 91 further comprising the use of one or more drug therapies under conditions suitable for said treatment.\",\n \"96.The method of claim 92 further comprising the use of one or more drug therapies under conditions suitable for said treatment.\",\n \"97.A method of cleaving RNA encoded by a PKR gene comprising contacting an enzymatic nucleic acid molecule of claim 56 with said RNA under conditions suitable for the cleavage.\",\n \"98.A method of cleaving RNA encoded by an IKK-gamma gene comprising contacting an enzymatic nucleic acid molecule of claim 55 with said RNA under conditions suitable for the cleavage.\",\n \"99.The method of claim 98, wherein said cleavage is carried out in the presence of a divalent cation.\",\n \"100.The method of claim 99, wherein said cleavage is carried out in the presence of a divalent cation.\",\n \"101.The method of claim 100, wherein said divalent cation is Mg2+.\",\n \"102.The method of claim 101, wherein said divalent cation is Mg2+.\",\n \"103.The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, the 3′-end, or both the 5′-end and the 3′-end.\",\n \"104.The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, the 3′-end, or both the 5′-end and the 3′-end.\",\n \"105.The enzymatic nucleic acid molecule of claim 103, wherein the cap structure at the 5′-end, 3′-end, or both the 5′-end and the 3′-end comprises a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative.\",\n \"106.The antisense nucleic acid molecule of claim 104, wherein the cap structure at the 5′-end, 3′-end, or both the 5′-end and the 3′-end comprises a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative.\",\n \"107.The method of claim 89, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.\",\n \"108.An expression vector comprising at least one enzymatic nucleic acid molecule of claim 55 or claim 56 in a manner that allows expression of the nucleic acid molecule.\",\n \"109.A mammalian cell comprising the expression vector of claim 108.110.The mammalian cell of claim 109, wherein said mammalian cell is a human cell.\",\n \"111.The expression vector of claim 108, wherein said enzymatic nucleic acid molecule is in a hammerhead configuration.\",\n \"(DOES THIS MAKE SENSE?)\",\n \"112.The expression vector of claim 108, wherein said expression vector further comprises a sequence for an antisense nucleic acid molecule complementary to RNA encoded by an IKK-gamma subunit gene or PKR gene.\",\n \"113.The expression vector of claim 108, wherein said expression vector comprises a nucleic acid sequence encoding two or more of said enzymatic nucleic acid molecules, which can be the same or different.\",\n \"114.The expression vector of claim 108, wherein said expression vector further comprises a sequence encoding an antisense nucleic acid molecule complementary to RNA encoded by an IKK-gamma gene or PKR gene.\",\n \"115.A method for treatment of cancer comprising administering to a patient the enzymatic nucleic acid molecule of any of claims 55-58 under conditions suitable for said treatment.\",\n \"116.The method of claim 115, wherein said cancer is breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer.\",\n \"117.A method for treatment of cancer comprising administering to a patient the antisense nucleic acid molecule of claim 59 under conditions suitable for said treatment.\",\n \"118.The method of claim 117, wherein said cancer is breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer.\",\n \"119.The method of claim 115, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.\",\n \"120.The method of claim 115, wherein said method further comprises administering to said patient one or more other therapies.\",\n \"121.The method of claim 117, wherein said method further comprises administering to said patient one or more other therapies.\",\n \"122.The nucleic acid molecule of any of claims 55, 56, or 58, wherein said nucleic acid molecule comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification.\",\n \"123.The nucleic acid molecule of claim 122, wherein said nucleic acid molecule further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.\",\n \"124.The nucleic acid molecule of claim 122, wherein said 3′-end modification is a 3′-3′ inverted abasic moiety.\",\n \"125.The method of claim 93 wherein said other drug therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.\",\n \"126.The method of claim 125, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.\",\n \"127.The method of claim 94 wherein said other drug therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.\",\n \"128.The method of claim 127, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.\",\n \"129.The method of claim 95 wherein said other drug therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.\",\n \"130.The method of claim 129, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.\",\n \"131.The method of claim 96 wherein said other drug therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.\",\n \"132.The method of claim 131, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.\",\n \"133.The method of claim 120, wherein said other therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.\",\n \"134.The method of claim 133, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.\",\n \"135.The method of claim 121, wherein said other therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.\",\n \"136.The method of claim 135, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.\",\n \"137.A method for treatment of an inflammatory disease comprising the step of administering to a patient the enzymatic nucleic acid molecule of any of claims 55-58 under conditions suitable for said treatment.\",\n \"138.The method of claim 137, wherein said inflammatory disease is rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury, glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, or infection.\",\n \"139.A method for treatment of an inflammatory disease comprising the step of administering to a patient the antisense nucleic acid molecule of claim 59 under conditions suitable for said treatment.\",\n \"140.The method of claim 139, wherein said inflammatory disease is rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, or infection.\",\n \"141.The method of claim 137, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.\",\n \"142.The method of claim 137, wherein said method further comprises administering to said patient one or more other therapies.\",\n \"143.The method of claim 139, wherein said method further comprises administering to said patient one or more other therapies.\",\n \"144.A pharmaceutical composition comprising an enzymatic nucleic acid molecule of any of claims 55-58 in a pharmaceutically acceptable carrier.\",\n \"145.A pharmaceutical composition comprising an antisense nucleic acid molecule of claim 59 in a pharmaceutically acceptable carrier.\",\n \"146.The enzymatic nucleic acid molecule of claim 55, wherein said subunit of IKK-is IKK-gamma.\",\n \"147.The enzymatic nucleic acid molecule of claim 55, wherein said subunit of IKK-is IKK-alpha.\",\n \"148.The enzymatic nucleic acid molecule of claim 55, wherein said subunit of IKK-is IKK-beta.\",\n \"149.A method of administering to a cell an enzymatic nucleic acid molecule of any of claims 55-57 comprising contacting said cell with the enzymatic nucleic acid molecule under conditions suitable for said administration.\",\n \"150.The method of claim 149, wherein said cell is a mammalian cell.\",\n \"151.The method of claim 149, wherein said cell is a human cell.\",\n \"152.The method of claim 149, wherein said administration is in the presence of a delivery reagent.\",\n \"153.The method of claim 152, wherein said delivery reagent is a lipid.\",\n \"154.The method of claim 153, wherein said lipid is a cationic lipid.\",\n \"155.The method of claim 153, wherein said lipid is a phospholipid.\",\n \"156.The method of claim 152, wherein said delivery reagent is a liposome.\",\n \"157.A nucleic acid molecule that down regulates expression of a prostaglandin D2 receptor (PTGDR) gene.\",\n \"158.The nucleic acid molecule of claim 157, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule.\",\n \"159.The nucleic acid molecule of claim 157, wherein said nucleic acid molecule is an antisense nucleic acid molecule.\",\n \"160.The enzymatic nucleic acid molecule of claim 158, wherein said enzymatic nucleic acid molecule comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 11666-13262.161.The enzymatic nucleic acid molecule of claim 158, wherein said enzymatic nucleic acid molecule comprises at least one binding arm wherein the at least one binding arm comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4415-5483.162.The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4415-5483.163.The nucleic acid molecule of claim 157, wherein said nucleic acid molecule is adapted to treat asthma.\",\n \"164.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule has an endonuclease activity to cleave RNA encoded by a PTGDR gene.\",\n \"165.The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in a hammerhead configuration.\",\n \"166.The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in an Inozyme configuration.\",\n \"167.The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.\",\n \"168.The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in a DNAzyme configuration.\",\n \"169.The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in a G-cleaver configuration.\",\n \"170.The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in an Amberzyme configuration.\",\n \"171.The enzymatic nucleic acid molecule of claim 165, wherein said hammerhead configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4415-4641.172.The enzymatic nucleic acid molecule of claim 165, wherein said hammerhead configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 11666-11892.173.The enzymatic nucleic acid molecule of claim 166, wherein said Inozyme configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4642-5017.174.The enzymatic nucleic acid molecule of claim 166, wherein said Inozyme configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 11893-12268.175.The enzymatic nucleic acid molecule of claim 167, wherein said Zinzyme configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 5018-5248.176.The enzymatic nucleic acid molecule of claim 167, wherein said Zinzyme configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 12269-12499.177.The enzymatic nucleic acid molecule of claim 168, wherein said DNAzyme configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4415-5294.178.The enzymatic nucleic acid molecule of claim 168, wherein said DNAzyme configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 12500-12842.179.The enzymatic nucleic acid molecule of claim 170, wherein said Amberzyme configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 5018-5248, and 5295-5483.180.The enzymatic nucleic acid molecule of claim 170, wherein said Amberzyme configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 12843-13262.181.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises between 8 and 100 bases complementary to a RNA molecule encoded by a PTGDR gene.\",\n \"182.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises between 14 and 24 bases complementary to a RNA molecule encoded by a PTGDR gene.\",\n \"183.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule is chemically synthesized.\",\n \"184.The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule is chemically synthesized.\",\n \"185.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises at least one 2′-sugar modification.\",\n \"186.The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule comprises at least one 2′-sugar modification.\",\n \"187.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises at least one nucleic acid base modification.\",\n \"188.The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule comprises at least one nucleic acid base modification.\",\n \"189.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises at least one phosphate backbone modification.\",\n \"190.The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule comprises at least one phosphate backbone modification.\",\n \"191.A mammalian cell comprising the enzymatic nucleic acid molecule of any of claims 158, 160 or 161.192.The mammalian cell of claim 191, wherein said mammalian cell is a human cell.\",\n \"193.A method of reducing PTGDR activity in a cell, comprising contacting said cell with the enzymatic nucleic acid molecule of any of claims 158, 160 or 161, under conditions suitable for said reduction.\",\n \"194.A method of reducing PTGDR activity in a cell, comprising contacting said cell with the antisense nucleic acid molecule of claim 159 under conditions suitable for said reduction.\",\n \"195.A method of treatment of a patient having a condition associated with the level of PTGDR, comprising contacting cells of said patient with the enzymatic nucleic acid molecule of any of claims 158, 160 or 161, under conditions suitable for said treatment.\",\n \"196.A method of treatment of a patient having a condition associated with the level of PTGDR, comprising contacting cells of said patient with the antisense nucleic acid molecule of claim 159, under conditions suitable for said treatment.\",\n \"197.The method of claim 193 further comprising the use of one or more drug therapies under conditions suitable for said treatment.\",\n \"198.The method of claim 194 further comprising the use of one or more drug therapies under conditions suitable for said treatment.\",\n \"199.The method of claim 195 further comprising the use of one or more drug therapies under conditions suitable for said treatment.\",\n \"200.The method of claim 196 further comprising the use of one or more drug therapies under conditions suitable for said treatment.\",\n \"201.A method of cleaving a RNA molecule encoded by a PTGDR gene comprising contacting the enzymatic nucleic acid molecule of any of claims 158, 160 or 161 with said RNA molecule encoded by a PTGDR gene under conditions suitable for the cleavage.\",\n \"202.The method of claim 201, wherein said cleavage is carried out in the presence of a divalent cation.\",\n \"203.The method of claim 202, wherein said divalent cation is Mg2+.\",\n \"204.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, 3′-end, or both the 5′-end and the 3′-end.\",\n \"205.The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, 3′-end, or both the 5′-end and the 3′-end.\",\n \"206.The enzymatic nucleic acid molecule of claim 204, wherein the cap structure at the 5′-end, 3′-end, or both the 5′-end and the 3′-end comprises a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative.\",\n \"207.The antisense nucleic acid molecule of claim 205, wherein the cap structure at the 5′-end, 3′-end, or both the 5′-end and the 3′-end comprises a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative.\",\n \"208.The method of claim 193, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.\",\n \"209.An expression vector comprising a nucleic acid molecule encoding at least one enzymatic nucleic acid molecule of claim 158 or claim 160 in a manner that allows expression of the nucleic acid molecule.\",\n \"210.A mammalian cell comprising the expression vector of claim 209.211.The mammalian cell of claim 210, wherein said mammalian cell is a human cell.\",\n \"212.The expression vector of claim 209, wherein said enzymatic nucleic acid molecule is in a hammerhead configuration.\",\n \"213.The expression vector of claim 209, wherein said expression vector further comprises a sequence for an antisense nucleic acid molecule complementary to a RNA molecule encoded by a PTGDR gene.\",\n \"214.The expression vector of claim 209, wherein said expression vector comprises a nucleic acid sequence encoding two or more of said enzymatic nucleic acid molecules, which can be the same or different.\",\n \"215.The expression vector of claim 214, wherein said expression vector further comprises a sequence encoding an antisense nucleic acid molecule complementary to a RNA molecule encoded by a PTGDR gene.\",\n \"216.A method for treatment of an allergic condition comprising the step of administering to a patient the enzymatic nucleic acid molecule of any of claims 156-159 under conditions suitable for said treatment.\",\n \"217.The method of claim 216, wherein said allergic condition is asthma, allergic rhinitis, or atopic dermatitis.\",\n \"218.A method for treatment of an allergic condition comprising administering to a patient the antisense nucleic acid molecule of claim 159 under conditions suitable for said treatment.\",\n \"219.The method of claim 218, wherein said allergic condition is asthma, allergic rhinitis, or atopic dermatitis.\",\n \"220.The method of claim 216, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.\",\n \"221.The method of claim 216, wherein said method further comprises administering to said patient one or more other treatment therapies.\",\n \"222.The method of claim 218, wherein said method further comprises administering to said patient one or more other treatment therapies.\",\n \"223.The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification.\",\n \"224.The enzymatic nucleic acid molecule of claim 223, wherein said enzymatic nucleic acid molecule further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.\",\n \"225.The nucleic acid molecule of claim 223, wherein said 3′-end modification is a 3′-3′ inverted abasic moiety.\",\n \"226.The method of claim 197 wherein said other drug therapies are bronchodilators, anti-inflammatories, adenosine inhibitors, or adenosine A1 receptor inhibitors.\",\n \"227.A pharmaceutical composition comprising an enzymatic nucleic acid molecule of any of claims 158, 160 or 161.228.A pharmaceutical composition comprising an antisense nucleic acid molecule of claim 159.229.A method of administering to a mammal the nucleic acid molecule of claim 157, comprising contacting said mammal with the nucleic acid molecule under conditions suitable for said administration.\",\n \"230.The method of claim 229, wherein said mammal is a human.\",\n \"231.The method of claim 229 wherein said administration is in the presence of a delivery reagent.\",\n \"232.The method of claim 231, wherein said delivery reagent is a lipid.\",\n \"233.The method of claim 232, wherein said lipid is a cationic lipid.\",\n \"234.The method of claim 232, wherein said lipid is a phospholipid.\",\n \"235.The method of claim 231, wherein said delivery reagent is a liposome.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention provides compounds, compositions, and methods for the study, diagnosis, and treatment of conditions relating to the expression of NOGO and NOGO receptor genes.\",\n \"In particular, the invention provides nucleic acid molecules that are used to modulate the expression of NOGO and NOGO receptor gene products.\",\n \"The present invention further relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to IKK gamma (IKKG) and PKR levels, such as cancer, inflammatory, and autoimmune diseases and/or disorders.\",\n \"In addition, the present invention also relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to allergic response.\",\n \"Specifically, the invention provides compositions and methods for the treatment of diseases or conditions related to levels of factors involved in allergic conditions such as asthma, for example prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS) and adenosine A1 receptor (ADORA1).\",\n \"The discussions that follow are not meant to be complete and are provided only to assist understanding the invention that follows.\",\n \"The summary is not an admission that any of the work described below is prior art to the claimed invention.\",\n \"The following is a brief description of the current understanding of NOGO and NOGO receptors.\",\n \"The ceased growth of neurons following development has severe implications for lesions of the central nervous system (CNS) caused by neurodegenerative disorders and traumatic accidents.\",\n \"Although CNS neurons have the capacity to rearrange their axonal and dendritic foci in the developed brain, the regeneration of severed CNS axons spanning distance does not exist.\",\n \"Axonal growth following CNS injury is limited by the local tissue environment rather than intrinsic factors, as indicated by transplantation experiments (Richardson et al., 1980 , Nature, 284, 264-265).\",\n \"Non-neuronal glial cells of the CNS, including oligodendrocytes and astrocytes, have been shown to inhibit the axonal growth of dorsal root ganglion neurons in culture (Schwab and Thoenen, 1985 , J.\",\n \"Neurosci., 5, 2415-2423).\",\n \"Cultured dorsal root ganglion cells can extend their axons across glial cells from the peripheral nervous system, (ie; Schwann cells), but are inhibited by oligodendrocytes and myelin of the CNS (Schwab and Caroni, 1988 , J.\",\n \"Neurosci., 8, 2381-2393).\",\n \"The non-conducive properties of CNS tissue in adult vertebrates is thought to result from the existence of inhibitory factors rather than the lack of growth factors.\",\n \"The identification of proteins with neurite outgrowth inhibitory or repulsive properties include NI-35, NI-250 (Caroni and Schwab, 1988 , Neuron, 1, 85-96), myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945).\",\n \"Monoclonal antibodies (mAb IN-1) raised against NI-35/250 have been shown to partially neutralize the growth inhibitory effect of CNS myelin and oligodendrocytes.\",\n \"IN-1 treatment in vivo has resulted in long distance fiber regeneration in lesioned adult mammalian CNS tissue (Weibel et al., 1994 , Brain Res., 642, 259-266).\",\n \"Additionally, IN-1 treatment in vivo has resulted in the recovery of specific reflex and locomotor functions after spinal cord injury in adult rats (Bregman et al., 1995 , Nature, 378, 498-501).\",\n \"Recently, the cloning of NOGO-A (Genbank Accession No AJ242961), the rat complementary DNA encoding NI-220/250 has been reported (Chen et al., 2000 , Nature, 403, 434-439).\",\n \"The NOGO gene encodes at least three major protein products (NOGO-A, NOGO-B, and NOGO-C) resulting from both alternative promoter usage and alternative splicing.\",\n \"Recombinant NOGO-A inhibits neurite outgrowth from dorsal root ganglia and the spreading of 3T3 firboblasts.\",\n \"Monoclonal antibody IN-1 recognizes NOGO-A and neutralizes NOGO-A inhibition of neuronal growth in vitro.\",\n \"Evidence supports the proposal that NOGO-A is the previously described rat NI-250 since NOGO-A contains all six peptide sequences obtained from purified bNI-220, the bovine equivalent of rat NI-250 (Chen et al supra).\",\n \"Prinjha et al., 2000 , Nature, 403, 383-384, report the cloning of the human NOGO gene which encodes three different NOGO isoforms that are potent inhibitors of neurite outgrowth.\",\n \"Using oligonucleotide primers to amplify and clone overlapping regions of the open reading frame of NOGO cDNA, Phrinjha et al., supra identified three forms of cDNA clone corresponding to the three protein isoforms.\",\n \"The longest ORF of 1,192 amino acids corresponds to NOGO-A (Accession No.\",\n \"AJ251383).\",\n \"An intermediate-length splice variant that lacks residues 186-1,004 corresponds to NOGO-B (Accession No.\",\n \"AJ251384).\",\n \"The shortest splice variant, NOGO-C (Accession No.\",\n \"AJ251385), appears to be the previously described rat vp20 (Accession No.\",\n \"AF051335) and foocen-s (Accession No.\",\n \"AF132048), and also lacks residues 186-1,004.According to Prinjha et al., supra, the NOGO amino-terminal region shows no significant homology to any known protein, while the carboxy-terminal tail shares homology with neuroendocrine-specific proteins and other members of the reticulon gene family.\",\n \"In addition, the carboxy-terminal tail contains a consensus sequence that may serve as an endoplasmic-reticulum retention region.\",\n \"Based on the NOGO protein sequence, Prinjha et al., supra, postulate NOGO to be a membrane associated protein comprising a putative large extracellular domain of 1,024 residues with seven predicted N-linked glycosylation sites, two or three transmembrane domains, and a short carboxy-terminal region of 43 residues.\",\n \"Grandpre et al., 2000 , Nature , also report the identification of NOGO as a potent inhibitor of axon regeneration.\",\n \"The 4.1 kilobase NOGO human cDNA clone identified by Grandpre et al., supra, KIAA0886, is homologous to a cDNA derived from a previous effort to sequence random high molecular-weight brain derived cDNAs (Nagase et al., 1998 , DNA Res., 31, 355-364).\",\n \"This cDNA clone encodes a protein that matches all six of the peptide sequences derived from bovine NOGO.\",\n \"Grandpre et al., supra demonstrate that NOGO expression is predominantly associated with the CNS and not the peripheral nervous system (PNS).\",\n \"Cellular localization of NOGO protein appears to be predominantly reticluar in origin, however, NOGO is found on the surface of some oligodentrocytes.\",\n \"An active domain of NOGO has been identified, defined as residues 31-55 of a hydrophilic 66-residue lumenal/extracellular domain.\",\n \"A synthetic fragment corresponding to this sequence exhibits growth-cone collapsing and outgrowth inhibiting activities (Grandpre et al., supra).\",\n \"A receptor for the NOGO-A extracellular domain (NOGO-66) is described in Fournier et al., 2001, Nature, 409, 341-346.Fournier et al., have shown that isolated NOGO-66 inhibits axonal extension but does not alter non-neuronal cell morphology.\",\n \"The receptor identified has a high affinity for soluble NOGO-66, and is expressed as a glycophosphatidylinostitol-linked protein on the surface of CNS neurons.\",\n \"Furthermore, the expression of the NOGO-66 receptor in neurons that are NOGO insensitive results in NOGO dependent inhibition of axonal growth in these cells.\",\n \"Cleavage of the NOGO-66 receptor and other glycophosphatidylinostitol-linked proteins from axonal surfaces renders neurons insensitive to NOGO-66 inhibition.\",\n \"As such, disruption of the interaction between NOGO-66 and the NOGO-66 receptor provides the possibility of treating a wide variety of neurological diseases, injuries, and conditions.\",\n \"Hauswirth and Flannery, International PCT Publication No.\",\n \"WO 98/48027, describe materials and methods for the specific expression of proteins in retinal photoreceptor cells consisting of an adeno-associated viral vector contacting a rod or cone-opsin promoter.\",\n \"In addition, ribozymes which degrade mutant mRNA are described for use in the treatment of retinitis pigmentosa.\",\n \"Fechteler et al., Interanational PCT Publication No.\",\n \"WO 00/03004 describe ribozymes targeting presenilin-2 RNA for the use in treating neurodegenerative diseases such as Alzheimer's disease.\",\n \"Eldadah et al., 2000 , J.\",\n \"Neurosci., 20, 179-186, describe the protection of cerebellar granule cells from apoptosis induced by serum-potassium deprivation from ribozyme mediated inhibition of caspase-3.Seidman et al., 1999 , Antisense Nucleic Acid Drug Dev., 9, 333-340, describe in general terms, the use of antisense and ribozyme constructs for treatment of neurodegenerative diseases.\",\n \"Denman et al., 1994 , Nucleic Acids Research, 22, 2375-82, describe the ribozyme mediated degradation of beta-amyloid peptide precursor mRNA in COS-7 cells.\",\n \"Schwab and Chen, International PCT publication No.\",\n \"WO 00/31235, describe NOGO proteins and inhibitors of NOGO.\",\n \"Blatt et al., International PCT publication No.\",\n \"WO 01/59103, describe nucleic acid molecules for modulating expression of NOGO genes.\",\n \"The following is a brief description of the physiological role of nuclear factor kappa B (NFKB), IKK kinases, and protein kinase PKR.\",\n \"Nuclear factor kappa B (NFKB) is a multiunit transcription factor which regulates the expression of genes involved in a number of physiologic and pathologic processes.\",\n \"NFKB is a key component of the TNF signaling pathway.\",\n \"These processes include, but are not limited to: apoptosis, immune, inflammatory and acute phase responses.\",\n \"The REL-A gene product (a.k.a.\",\n \"RelA or p65), and p50 subunits of NFKB, have been implicated in the induction of inflammatory responses and cellular transformation.\",\n \"NFKB exists in the cytoplasm as an inactive heterodimer of the p50 and p65 subunits.\",\n \"NFKB is complexed with an inhibitory protein complex, IkappaB (IKK complex), until activated by the appropriate stimuli.\",\n \"NFKB activation can occur following stimulation of a variety of cell types by inflammatory mediators, for example TNF and IL-1, and reactive oxygen intermediates.\",\n \"In response to induction, NFKB can stimulate production of pro-inflammatory cytokines such as TNF-alpha, IL-1-beta, IL-6 and iNOS, thereby perpetuating a positive feedback loop.\",\n \"NFKB appears to play a role in a number of disease processes including: ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, arthritis, and cancer.\",\n \"The nuclear DNA-binding protein, NFKB, was first identified as a factor that binds and activates the immunoglobulin kappa light chain enhancer in B cells.\",\n \"NFKB now is known to activate transcription of a variety of other cellular genes (e.g., cytokines, adhesion proteins, oncogenes and viral proteins) in response to a variety of stimuli (e.g., phorbol esters, mitogens, cytokines and oxidative stress).\",\n \"In addition, molecular and biochemical characterization of NFKB has shown that the activity is due to a homodimer or heterodimer of a family of DNA binding subunits.\",\n \"Each subunit bears a stretch of 300 amino acids that is homologous to the oncogene, v-rel.\",\n \"The activity first described as NFKB is a heterodimer of p49 or p50 with p65.The p49 and p50 subunits of NFKB (encoded by the NF-kappa B2 or NF kappa B1 genes, respectively) are generated from the precursors NFKB1 (p105) or NFKB2 (p100).\",\n \"The p65 subunit of NFKB (now termed REL-A) is encoded by the rel-A locus.\",\n \"The roles of each specific transcription-activating complex now are being elucidated in cells (Perkins, et al., 1992 , Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci USA, 89, 1529-1533).\",\n \"For instance, the heterodimer of NFKB1 and Rel A (p50/p65) activates transcription of the promoter for the adhesion molecule, VCAM-1, while NFKB2/RelA heterodimers (p49/p65) actually inhibit transcription (Shu, et al., 1993 , Mol.\",\n \"Cell.\",\n \"Biol., 13, 6283-6289).\",\n \"Conversely, heterodimers of NFKB2/RelA (p49/p65) act with Tat-I to activate transcription of the HIV genome, while NFKB1/RelA (p50/p65) heterodimers have little effect (Liu et al., 1992 , J.\",\n \"Virol., 66, 3883-3887).\",\n \"Similarly, blocking rel A gene expression with antisense oligonucleotides specifically blocks embryonic stem cell adhesion; blocking NFKB1 gene expression with antisense oligonucleotides had no effect on cellular adhesion (Narayanan et al., 1993 , Mol.\",\n \"Cell.\",\n \"Biol., 13, 3802-3810).\",\n \"Thus, the promiscuous role initially assigned to NFKB in transcriptional activation (Lenardo, and Baltimore, 1989 , Cell, 58, 227-229) represents the sum of the activities of the rel family of DNA-binding proteins.\",\n \"This conclusion is supported by recent transgenic “knock-out” mice of individual members of the rel family.\",\n \"Such “knock-outs” show few developmental defects, suggesting that essential transcriptional activation functions can be performed by more than one member of the rel family.\",\n \"A number of specific inhibitors of NFKB function in cells exist, including treatment with phosphorothioate antisense oliogonucleotide, treatment with double-stranded NFKB binding sites, and over expression of the natural inhibitor MAD-3 (an Ikappa-B family member).\",\n \"These agents have been used to show that NFKB is required for induction of a number of molecules involved in cancer and/or inflammation, as described below.\",\n \"NFkB is required for phorbol ester-mediated induction of IL-6 (Kitajima, et al., 1992 , Science, 258, 1792-5) and IL-8 (Kunsch and Rosen, 1993 , Mol.\",\n \"Cell.\",\n \"Biol., 13, 6137-46).\",\n \"NFkB is required for induction of the adhesion molecules ICAM-1 (Eck, et al., 1993 , Mol.\",\n \"Cell.\",\n \"Biol., 13, 6530-6536), VCAM-1 (Shu et al., supra), and E-selectin (Read, et al., 1994 , J. Exp.\",\n \"Med., 179, 503-512) on endothelial cells.\",\n \"NFkB is involved in the induction of the integrin subunit, CD18, and other adhesive properties of leukocytes (Eck et al., 1993 supra).\",\n \"HER2/Neu overexpression induces NFKB via a PI3-kinase/Akt pathway involving calpain-mediated degradation of IKB-alpha.\",\n \"Breast cancer has been shown to typify the aberrant expression of NFKB/REL factors (Pianetti et al., 2001 , Oncogene, 20, 1287-1299; Sovak et al., 1999 , J. Clin.\",\n \"Invest., 100, 2952-2960).\",\n \"Inhibition of NFKB activity has been shown to induce apoptosis in murine hepatocytes (Bellas et al., 1997 , Am.\",\n \"J.\",\n \"Pathol., 151, 891-896).\",\n \"NFKB has been shown to regulate cyclooxygenase-2 expression and cell proliferation in human gastric cancer cells (Joo Weon et al., 2001 , Laboratory Investigation, 81, 349-360).\",\n \"The above studies suggest that NFKB is integrally involved in the induction of cytokines and adhesion molecules by inflammatory mediators and is involved in the transformation of cancerous cells.\",\n \"Two reported studies point to another connection between NFKB and inflammation: glucocorticoids can exert their anti-inflammatory effects by inhibiting NFKB.\",\n \"The glucocorticoid receptor and p65 both act at NFKB binding sites in the ICAM-1 promoter (van de Stolpe, et al., 1994 , J. Biol.\",\n \"Chem., 269, 6185-6192).\",\n \"Glucocorticoid receptor inhibits NFKB-mediated induction of IL-6 (Ray and Prefontaine, 1994 Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci USA, 91, 752-756).\",\n \"Conversely, overexpression of p65 inhibits glucocorticoid induction of the mouse mammary tumor virus promoter.\",\n \"Finally, protein cross-linking and co-immunoprecipitation experiments demonstrated direct physical interaction between p65 and the glucocorticoid receptor.\",\n \"The IKK complex that sequesters NFKB in the cytoplasm comprises IkappaB (IκB) proteins (IκB-alpha, IκB-beta, IκKB-epsilon, p105, and p100).\",\n \"The phosphorylation of IκB proteins results in the release of NFKB from the IκB complex which is transported to the nucleus via the unmasking of nuclear translocation signals.\",\n \"Phosphorylation marks IkB proteins for ubiquitination and degradation via the proteosome pathway.\",\n \"Most NFKB inducing stimuli initiate activation of an IκB kinase (IKK) complex that contains two catalytic subunits, IKK-alpha (IKK1) and IKK-beta (IKK2), that phosphorylate IκB-alpha and IκB-beta, with IKK-beta playing a predominant role in pro-inflammatory signaling.\",\n \"In addition to the two kinases, the IKK complex contains regulatory subunits, including IKK-gamma (NEMO/IKKAP1).\",\n \"IKK-gamma is a protein that is critical for the assembly of the IKK complex.\",\n \"IKK-gamma directly binds to IKK-beta and is required for activation of NFKB, for example by TNF-alpha, IL-1-beta, lipopolysaccharide, phorbol 12-myristate 13-acetate, the human T-cell lymphotrophic virus (HTLV-1), or double stranded RNA.\",\n \"Genomic rearrangements in IKK-gamma have been shown to impair NFKB activation and result in incontinentia pigmenti.\",\n \"Additional proteins that associate with the IKK complex include, MEK kinase (MEKK1), NFKB inducing kinase (NIK), receptor interacting protein (RIP), protein kinase CK2, and IKK-associated protein (IKAP), which appears to be associated with the IκB Kinase (IKK) complex, but does not appear to be an integral component of the tripartate IKK complex as does IKK-gamma (Krappmann et al., 2001 , J. Biol.\",\n \"Chem., 275, 29779-87).\",\n \"The RNA-dependent protein kinase PKR is a signal transducer for NFKB and IFN regulatory factor-1.PKR is required for activation of NFKB by IFN-gamma via a STAT-1 independent pathway (Amitabha et al., 2001 , J.\",\n \"Immunol., 166, 6170-6180).\",\n \"The induction of NFKB by PKR takes place though phosphorylation of IκB-alpha, and appears not to require the catalytic activity of PKR, thereby proceeding independently of the dsRNA-binding properties of PKR (Ishii et al., 2001 , Oncogene, 20, 1900-1912).\",\n \"PKR also plays an important role in the regulation of protein synthesis by modulating the activity of eukaryotic initiation factor 2 (eIF-2-alpha) through interferon induction.\",\n \"Kamiya, JP 2000253884, describes specific antisense oligonucleotides for inhibiting IκB-kinase subunit expression.\",\n \"Krappmann et al., 2001 , J. Biol.\",\n \"Chem ., describe specific antisense oligonucleotides to IKK-gamma.\",\n \"The following is a description of molecular targets involved in diseases or conditions related to allergic response.\",\n \"Asthma is a chronic inflammatory disorder of the lungs characterized by airflow obstruction, bronchial hyper-responsiveness, and airway inflammation.\",\n \"T-lymphocytes that produce TH2 cytokines and eosinophilic leukocytes infiltrate the airways.\",\n \"In the airway and in bronchial alveolar lavage (BAL) fluid of individuals with asthma, high concentrations of TH2 cytokines, interleukin-4 (14), IL-5, and IL-13, are present along with increased levels of adenosine.\",\n \"In contrast to normal individuals, asthmatics respond to adenosine challenge with marked airway obstruction.\",\n \"Upon allergen challenge, mast cells are activated by cross-linked IgE-allergen complexes.\",\n \"Large amounts of prostaglandin D2 (PGD2), the major cyclooxygenase product of arachidonic acid are released.\",\n \"PGD2 is generated from PGH2 via the activity of prostaglandin D2 synthetase (PTGDS).\",\n \"PGD2 receptors and adenosine A1 receptors are present in the lungs and airway along with various other tissues in response to allergic stimuli (Howarth, 1997 , Allergy, 52, 12).\",\n \"The significance of PGD2 as a mediator of allergic asthma has been established with the development of mice deficient in the PGD2 receptor (DP).\",\n \"DP is a heterotrimeric GTP-binding protein-coupled, rhodopsin-type receptor specific for PGD2 (Hirata et al., 1994 , PNAS USA., 91, 11192).\",\n \"These mice fail to develop airway hyperreactivity and have greatly reduced eosinophil infiltration and cytokine accumulation in response to allergens.\",\n \"Upon allergen challenge mice deficient in the prostaglandin D2 (PGD2) receptor (DP) did not develop airway hyperactivity.\",\n \"Cytokine, lymphocyte and eosinophil accumulation in the lungs were greatly reduced (Matsuoka et al., 2000 , Science, 287, 2013).\",\n \"The DP −/− mice exhibited no behavioral, anatomic, or histological abnormalities.\",\n \"Primary immune response is not affected by DP disruption.\",\n \"Asthma affects more than 100 million people worldwide and more than 17 million Americans (5% of the population).\",\n \"Since 1980 the incidence has more than doubled and deaths have tripled (5,000 deaths in 1995).\",\n \"Annual asthma-related healthcare costs in the US alone were estimated to exceed $14.5 billion in 2000.Current therapies such as inhalant anti-inflammatories and bronchodilators can be used to treat symptoms, however, these therapies do not prevent or cure asthma.\",\n \"Sandberg et al., 2001 , Prog.\",\n \"Respir.\",\n \"Res., 31, 370-373, describes ribozyme therapy for asthma and COPD.\",\n \"Sullivan et al., International U.S. Pat.\",\n \"No.\",\n \"5,616,488, describes ribozymes targeting interleukin-5 for treatment and diagnosis of asthma and other inflammatory disorders.\",\n \"Stinchcomb et al., International PCT Publication No.\",\n \"WO 95/23225, describes ribozymes and methods for inhibiting the expression of disease related genes including genes associated with asthma.\",\n \"Nyce, International PCT Publication Nos.\",\n \"WO 00/62736, WO 00/09525, WO 99/13886, WO 98/23294, WO 96/40266 and U.S. Pat.\",\n \"No.\",\n \"6,025,339 describe specific antisense oligonucleotides targeting certain mRNAs encoding particular adenosine receptors.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The invention features novel nucleic acid-based molecules, for example, enzymatic nucleic acid molecules, allozymes, antisense nucleic acids, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNA, dsRNA, siRNA, aptamers, and antisense nucleic acids containing RNA cleaving chemical groups, and methods to modulate gene expression; for example, gene(s) encoding prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), and adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3; gene(s) encoding NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors; and genes encoding an IkappaB kinase (IKK) subunit or protein kinase PKR.\",\n \"In one embodiment, the instant invention features nucleic-acid based techniques to inhibit the expression of NOGO-A (Accession No.\",\n \"AJ251383), NOGO-B (Accession No.\",\n \"AJ251384), and/or NOGO-C (Accession No.\",\n \"AJ251385), NOGO-66 receptor (Accession No AF283463, Fournier et al., 2001, Nature, 409, 341-346), NI-35, NI-220, and/or NI-250, myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945).\",\n \"The description below of the various aspects and embodiments is provided with reference to the exemplary NOGO-A and NOGO-66 receptor genes.\",\n \"However, the various aspects and embodiments are also directed to other genes which express NOGOA-like inhibitor proteins and other receptors involved in neurite outgrowth inhibition.\",\n \"Those additional genes can be analyzed for target sites using the methods described for NOGO and the NOGO-66 receptor, referred to alternatively as NOGO receptor.\",\n \"Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.\",\n \"The invention features one or more enzymatic nucleic acid-based molecules and methods that independently or in combination modulate the expression of gene(s) encoding a member of the IκB kinase IKK complex or PKR.\",\n \"In particular embodiments, the invention features nucleic acid-based molecules and methods that modulate the expression of a member of the IκB kinase IKK complex, for example IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKKγ) and/or a protein kinase PKR protein, such as IKK-alpha (IKK1) gene (Genbank Accession No.\",\n \"NM — 001278); IKK-beta (IKK2) gene, for example (Genbank Accession No.\",\n \"AF080158), IKK-gamma (IKKγ) gene, for example (Genbank Accession No.\",\n \"NM — 003639), and protein kinase PKR gene, for example (Genbank Accession No.\",\n \"NM — 002759).\",\n \"The description below of the various aspects and embodiments is provided with reference to the exemplary IKK-gamma and PKR genes.\",\n \"IKK-gamma is also known as NEMO/IKKAP1.However, the various aspects and embodiments are also directed to other genes which encode other subunits of the IKK complex, such as IKK-alpha (IKK1) or IKK-beta (IKK2).\",\n \"Those additional genes can be analyzed for target sites using the methods described for IKK-gamma or PKR.\",\n \"Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.\",\n \"In one embodiment, an enzymatic nucleic acid molecule of the invention is in a hammerhead, Inozyme, Zinzyme, DNAzyme, Amberzyme, or G-cleaver configuration.\",\n \"In another embodiment, a nucleic acid molecule of the invention comprises between 8 and 100 bases complementary to the RNA of the target gene.\",\n \"In another embodiment, a nucleic acid molecule of the invention comprises between 14 and 24 bases complementary to a RNA molecule of the target gene.\",\n \"In one embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention is chemically synthesized.\",\n \"In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one 2′-sugar modification.\",\n \"In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one nucleic acid base modification.\",\n \"In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one phosphate backbone modification.\",\n \"In one embodiment, the invention features a mammalian cell, for example a human cell, including the nucleic acid molecule of the invention.\",\n \"In another embodiment, the invention features a method of reducing target gene expression or activity in a cell, comprising contacting the cell with a nucleic acid molecule of the invention, such as an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups, under conditions suitable for the reduction.\",\n \"In yet another embodiment, the invention features a method of treatment of a patient having a condition associated with the level of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR, comprising contacting cells of the patient with an enzymatic nucleic acid molecule of the invention, under conditions suitable for the treatment.\",\n \"In another embodiment, a method of treatment of a patient having a condition associated with the level of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR is featured, wherein the method further comprises the use of one or more drug therapies under conditions suitable for the treatment.\",\n \"In another embodiment, the invention features a method of cleaving a RNA molecule of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene, comprising contacting an enzymatic nucleic acid molecule of the invention with a RNA molecule of the corresponding gene under conditions suitable for the cleavage, for example, wherein the cleavage is carried out in the presence of a divalent cation, such as Mg 2+ .\",\n \"In one embodiment, a nucleic acid molecule of the invention comprises a cap structure, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end of the enzymatic nucleic acid molecule.\",\n \"In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one nucleic acid molecule of the invention, in a manner which allows expression of the nucleic acid molecule.\",\n \"In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.\",\n \"In yet another embodiment, the expression vector of the invention further comprises a sequence for an antisense nucleic acid molecule complementary to a RNA molecule of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene.\",\n \"In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more nucleic acid molecules, such as enzymatic nucleic acid molecules, antisense, aptamers, decoys, siRNA, or 2-5A chimeras which can be the same or different.\",\n \"In one embodiment, the method of treatment features an enzymatic nucleic acid molecule of the invention comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification, such as a 3′-3′ inverted abasic moiety.\",\n \"In another embodiment, an enzymatic nucleic acid molecule or antisense nucleic acid molecule of the invention further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.\",\n \"In another embodiment, the invention features a method of administering to a mammal, for example a human, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention, comprising contacting the mammal with the nucleic acid molecule under conditions suitable for the administration, for example, in the presence of a delivery reagent such as a lipid, cationic lipid, phospholipid, or liposome.\",\n \"In yet another embodiment, the invention features a method of administering to a mammal an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention in conjunction with a therapeutic agent, comprising contacting the mammal, for example a human, with the nucleic acid molecule and the therapeutic agent under conditions suitable for the administration.\",\n \"In one embodiment, the invention features the use of an enzymatic nucleic acid molecule, which can be in a hammerhead, NCH, G-cleaver, Amberzyme, Zinzyme, and/or DNAzyme motif, to down-regulate the expression of a a target gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene.\",\n \"By “inhibit”, “down-regulate”, or “reduce”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR subunits, is reduced below that observed in the absence of the nucleic acid molecules of the invention.\",\n \"In one embodiment, inhibition, down-regulation or reduction with an enzymatic nucleic acid molecule is below that level observed in the presence of an enzymatically inactive or attenuated molecule that is able to bind to the same site on the target RNA molecule, but is unable to cleave that RNA molecule.\",\n \"In another embodiment, inhibition, down-regulation, or reduction with antisense oligonucleotides is below that level observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches.\",\n \"In another embodiment, inhibition, down-regulation, or reduction of the target gene with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.\",\n \"By “up-regulate” is meant that the expression of a gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins, protein subunits, or activity of one or more proteins or protein subunits, such as a target gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR subunits, is greater than that observed in the absence of the nucleic acid molecules of the invention.\",\n \"For example, the expression of a gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene, can be increased in order to treat, prevent, ameliorate, or modulate a pathological condition caused or exacerbated by an absence or low level of gene expression.\",\n \"By “modulate” is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more protein subunits, or activity of one or more protein subunits is up-regulated or down-regulated, such that the expression, level, or activity is greater than or less than that observed in the absence of a nucleic acid molecule of the invention.\",\n \"By “enzymatic nucleic acid molecule” it is meant a nucleic acid molecule that has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that is active to specifically cleave target a RNA molecule.\",\n \"That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave a RNA molecule and thereby inactivate a target RNA molecule.\",\n \"These complementary regions allow sufficient hybridization of an enzymatic nucleic acid molecule to a target RNA molecule and thus permit cleavage.\",\n \"One hundred percent complementarity is preferred, but complementarity as low as 50-75% can also be useful in this invention (see for example Werner and Uhlenbeck, 1995 , Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999 , Antisense and Nucleic Acid Drug Dev., 9, 25-31).\",\n \"The nucleic acids can be modified at the base, sugar, and/or phosphate groups.\",\n \"The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme.\",\n \"All of these terminologies describe nucleic acid molecules with enzymatic activity.\",\n \"The specific enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site that is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule (Cech et al., U.S. Pat.\",\n \"No.\",\n \"4,987,071; Cech et al., 1988, 260 JAMA 3030).\",\n \"By “nucleic acid molecule” as used herein is meant a molecule having nucleotides.\",\n \"The nucleic acid can be single, double, or multiple stranded and can comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.\",\n \"Exemplary nucleic acid molecules of the invention include enzymatic nucleic acid molecules, allozymes, antisense nucleic acids, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNA, dsRNA, siRNA, aptamers, and/or antisense nucleic acids containing RNA cleaving chemical groups.\",\n \"By “enzymatic portion” or “catalytic domain” is meant that portion/region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate (for example see FIGS.\",\n \"1-4 ).\",\n \"By “substrate binding arm” or “substrate binding domain” is meant that portion/region of a enzymatic nucleic acid that is able to interact, for example via complementarity (i.e., able to base-pair with), with a portion of its substrate.\",\n \"Such complementarity can be 100%, but can be less if desired.\",\n \"For example, as few as 10 bases out of 14 can be base-paired (see for example Werner and Uhlenbeck, 1995 , Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999 , Antisense and Nucleic Acid Drug Dev., 9, 25-31).\",\n \"Examples of such arms are shown generally in FIGS.\",\n \"1-4 .\",\n \"That is, these arms contain sequences within an enzymatic nucleic acid that are intended to bring enzymatic nucleic acid and target RNA together through complementary base-pairing interactions.\",\n \"The enzymatic nucleic acid of the invention can have binding arms that are contiguous or non-contiguous and can be of varying lengths.\",\n \"The length of the binding arm(s) can be greater than or equal to four nucleotides and of sufficient length to stably interact with a target RNA; in one embodiment they can be 12-100 nucleotides; in another embodiment they can be 14-24 nucleotides long (see for example Werner and Uhlenbeck, supra; Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herranze et al., 1993 , EMBO J., 12, 2567-73) or between 8 and 14 nucleotides long.\",\n \"If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., four and four, five and five nucleotides, or six and six nucleotides, or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., three and five, six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).\",\n \"By “Inozyme” or “NCH” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as NCH Rz in FIG.\",\n \"1 .\",\n \"Inozymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCH/, where N is a nucleotide, C is cytidine and H is adenosine, uridine or cytidine, and/represents the cleavage site.\",\n \"H is used interchangeably with X. Inozymes can also possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCN/, where N is a nucleotide, C is cytidine, and / represents the cleavage site.\",\n \"“I” r in FIG.\",\n \"1 represents an Inosine nucleotide, including a ribo-Inosine or xylo-Inosine nucleoside.\",\n \"By “G-cleaver” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as G-cleaver Rz in FIG.\",\n \"1 .\",\n \"G-cleavers possess endonuclease activity to cleave RNA substrates having a cleavage triplet NYN/, where N is a nucleotide, Y is uridine or cytidine and / represents the cleavage site.\",\n \"G-cleavers can be chemically modified as is generally shown in FIG.\",\n \"1 .\",\n \"By “amberzyme” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG.\",\n \"2 .\",\n \"Amberzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NG/N, where N is a nucleotide, G is guanosine, and / represents the cleavage site.\",\n \"Amberzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG.\",\n \"2 .\",\n \"In addition, differing nucleoside and/or non-nucleoside linkers can be used to substitute the 5′-gaaa-3′ loops shown in the figure.\",\n \"Amberzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2′-OH) group within its own nucleic acid sequence for activity.\",\n \"By “zinzyme” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG.\",\n \"3 .\",\n \"Zinzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet including but not limited to YG/Y, where Y is uridine or cytidine, and G is guanosine and / represents the cleavage site.\",\n \"Zinzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG.\",\n \"3 , including substituting 2′-O-methyl guanosine nucleotides for guanosine nucleotides.\",\n \"In addition, differing nucleotide and/or non-nucleotide linkers can be used to substitute the 5′-gaaa-2′ loop shown in the figure.\",\n \"Zinzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2′-OH) group within its own nucleic acid sequence for activity.\",\n \"By ‘DNAzyme’ is meant, an enzymatic nucleic acid molecule that does not require the presence of a 2′-OH group within its own nucleic acid sequence for activity.\",\n \"In particular embodiments the enzymatic nucleic acid molecule can have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups.\",\n \"DNAzymes can be synthesized chemically or expressed endogenously in vivo, by means of a single stranded DNA vector or equivalent thereof.\",\n \"An example of a DNAzyme is shown in FIG.\",\n \"4 and is generally reviewed in Usman et al., U.S. Pat.\",\n \"No.\",\n \"6,159,714; Chartrand et al., 1995 , NAR 23, 4092; Breaker et al., 1995 , Chem.\",\n \"Bio.\",\n \"2, 655; Santoro et al., 1997 , PNAS 94, 4262; Breaker, 1999 , Nature Biotechnology, 17, 422-423; and Santoro et.\",\n \"al., 2000 , J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc., 122, 2433-39.The “10-23” DNAzyme motif is one particular type of DNAzyme that was evolved using in vitro selection (see Santoro et al., supra).\",\n \"Additional DNAzyme motifs can be selected for using techniques similar to those described in these references, and hence, are within the scope of the present invention.\",\n \"By “sufficient length” is meant an oligonucleotide of greater than or equal to 3 nucleotides that is of a length great enough to provide the intended function under the expected condition.\",\n \"For example, for binding arms of enzymatic nucleic acid “sufficient length” means that the binding arm sequence is long enough to provide stable binding to a target site under the expected binding conditions.\",\n \"The binding arms are not so long as to prevent useful turnover of the nucleic acid molecule.\",\n \"By “stably interact” is meant interaction of the oligonucleotides with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions) that is sufficient to the intended purpose (e.g., cleavage of target RNA by an enzyme).\",\n \"By “equivalent” or “related” RNA to NOGO is meant to include those naturally occurring RNA molecules having homology (partial or complete) to NOGO-A, NOGO-B, NOGO-C and/or NOGO receptor proteins or encoding for proteins with similar function as NOGO or NOGO receptor proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.\",\n \"The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.\",\n \"By “equivalent” or “related” RNA to IKK-gamma is meant to include those naturally occurring RNA molecules having homology (partial or complete) to IKK-gamma proteins or encoding for proteins with similar function as IKK-gamma proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.\",\n \"The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.\",\n \"By “equivalent” or “related” RNA to PKR is meant to include those naturally occurring RNA molecules having homology (partial or complete) to PKR proteins or encoding for proteins with similar function as PKR proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.\",\n \"The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.\",\n \"By “equivalent” or “related” RNA to PTGDS is meant to include RNA molecules having homology (partial or complete) to RNA molecules encoding PTGDS proteins or encoding proteins with similar function as PTGDS proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites.\",\n \"The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.\",\n \"By “equivalent” or “related” RNA to PTGDR is meant to include RNA molecules having homology (partial or complete) to RNA molecules encoding PTGDR proteins or encoding proteins with similar function as PTGDR proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites.\",\n \"The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.\",\n \"By “equivalent” or “related” RNA to ADORA1 is meant to include RNA molecules having homology (partial or complete) to RNA molecule encoding ADORA1 proteins or encoding proteins with similar function as ADORA1 proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites.\",\n \"The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.\",\n \"By “homology” is meant the nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.\",\n \"By “antisense nucleic acid”, it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., U.S. Pat.\",\n \"No.\",\n \"5,849,902).\",\n \"Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.\",\n \"However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop.\",\n \"Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.\",\n \"For a review of current antisense strategies, see Schmajuk et al., 1999 , J. Biol.\",\n \"Chem., 274, 21783-21789, Delihas et al., 1997 , Nature, 15, 751-753, Stein et al., 1997 , Antisense N. A.\",\n \"Drug Dev., 7, 151, Crooke, 2000 , Methods Enzymol., 313, 3-45; Crooke, 1998 , Biotech.\",\n \"Genet.\",\n \"Eng.\",\n \"Rev., 15, 121-157, Crooke, 1997 , Ad.\",\n \"Pharmacol., 40, 1-49.In addition, antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.\",\n \"The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA.\",\n \"Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.\",\n \"By “RNase H activating region” is meant a region (generally greater than or equal to 4-25 nucleotides in length, and in one embodiment from 5-11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al., U.S. Pat.\",\n \"No.\",\n \"5,849,902; Arrow et al., U.S. Pat.\",\n \"No.\",\n \"5,989,912).\",\n \"The RNase H enzyme binds to the nucleic acid molecule-target RNA complex and cleaves the target RNA sequence.\",\n \"The RNase H activating region comprises, for example, phosphodiester, phosphorothioate (at least four of the nucleotides are phosphorothiote substitutions; and in another embodiment, 4-11 of the nucleotides are phosphorothiote substitutions); phosphorodithioate, 5′-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof.\",\n \"In addition to one or more backbone chemistries described above, the RNase H activating region can also comprise a variety of sugar chemistries.\",\n \"For example, the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry.\",\n \"Those skilled in the art will recognize that the foregoing are non-limiting examples and that any combination of phosphate, sugar and base chemistry of a nucleic acid that supports the activity of RNase H enzyme is within the scope of the definition of the RNase H activating region and the instant invention.\",\n \"By “2-5A antisense chimera” is meant an antisense oligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylate residue.\",\n \"These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 90, 1300; Silverman et al., 2000 , Methods Enzymol., 313, 522-533; Player and Torrence, 1998 , Pharmacol.\",\n \"Ther., 78, 55-113).\",\n \"By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that is distinct from sequence recognized by the target molecule in its natural setting.\",\n \"Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid.\",\n \"The target molecule can be any molecule of interest.\",\n \"For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein.\",\n \"This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art, see for example Gold et al., 1995 , Annu.\",\n \"Rev.\",\n \"Biochem., 64, 763; Brody and Gold, 2000 , J.\",\n \"Biotechnol., 74, 5; Sun, 2000 , Curr.\",\n \"Opin.\",\n \"Mol.\",\n \"Ther., 2, 100; Kusser, 2000 , J.\",\n \"Biotechnol., 74, 27; Hermann and Patel, 2000 , Science, 287, 820; and Jayasena, 1999 , Clinical Chemistry, 45, 1628.By “triplex forming oligonucleotides” is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix.\",\n \"Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 89, 504; Fox, 2000 , Curr.\",\n \"Med.\",\n \"Chem., 7, 17-37; Praseuth et al., 2000 , Biochim.\",\n \"Biophys.\",\n \"Acta, 1489, 181-206).\",\n \"By “gene” it is meant a nucleic acid that encodes an RNA, for example, nucleic acid sequences including but not limited to structural genes encoding a polypeptide.\",\n \"“Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another RNA molecule by either traditional Watson-Crick or other non-traditional types.\",\n \"In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., enzymatic nucleic acid cleavage, antisense or triple helix inhibition.\",\n \"Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987 , CSH Symp.\",\n \"Quant.\",\n \"Biol.\",\n \"LII pp.\",\n \"123-133; Frier et al., 1986 , Proc.\",\n \"Nat.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 83:9373-9377; Turner et al., 1987 , J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"109:3783-3785).\",\n \"A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).\",\n \"“Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.\",\n \"By “RNA” is meant a molecule comprising at least one ribonucleotide residue.\",\n \"By “ribonucleotide” or “2′-OH” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribo-furanose moiety.\",\n \"By “decoy RNA” is meant an RNA molecule or aptamer that is designed to preferentially bind to a predetermined ligand.\",\n \"Such binding can result in the inhibition or activation of a target molecule.\",\n \"The decoy RNA or aptamer can compete with a naturally occurring binding target for the binding of a specific ligand.\",\n \"For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a “decoy” and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990, Cell, 63, 601-608).\",\n \"This is but a specific example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art, see for example Gold et al., 1995 , Annu.\",\n \"Rev.\",\n \"Biochem., 64, 763; Brody and Gold, 2000 , J.\",\n \"Biotechnol., 74, 5; Sun, 2000 , Curr.\",\n \"Opin.\",\n \"Mol.\",\n \"Ther., 2, 100; Kusser, 2000 , J.\",\n \"Biotechnol., 74, 27; Hermann and Patel, 2000 , Science, 287, 820; and Jayasena, 1999 , Clinical Chemistry, 45, 1628.Similarly, a decoy RNA can be designed to bind to a D2 receptor and block the binding of PTGDS or a decoy RNA can be designed to bind to PTGDS and prevent interaction with the D2 receptor.\",\n \"The term “short interfering RNA” or “siRNA” as used herein refers to a double stranded nucleic acid molecule capable of RNA interference “RNAi”, see for example Bass, 2001 , Nature, 411, 428-429; Elbashir et al., 2001 , Nature, 411, 494-498; and Kreutzer et al., International PCT Publication No.\",\n \"WO 00/44895; Zernicka-Goetz et al., International PCT Publication No.\",\n \"WO 01/36646; Fire, International PCT Publication No.\",\n \"WO 99/32619; Plaetinck et al., International PCT Publication No.\",\n \"WO 00/01846; Mello and Fire, International PCT Publication No.\",\n \"WO 01/29058; Deschamps-Depaillette, International PCT Publication No.\",\n \"WO 99/07409; and Li et al., International PCT Publication No.\",\n \"WO 00/44914.As used herein, siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides.\",\n \"The term “allozyme” as used herein refers to an allosteric enzymatic nucleic acid molecule, see, e.g., George et al., U.S. Pat.\",\n \"Nos.\",\n \"5,834,186 and 5,741,679, Shih et al., U.S. Pat.\",\n \"No.\",\n \"5,589,332, Nathan et al., U.S. Pat.\",\n \"No.\",\n \"5,871,914, Nathan and Ellington, International PCT publication No.\",\n \"WO 00/24931, Breaker et al., International PCT Publication Nos.\",\n \"WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No.\",\n \"WO 99/29842.The term “2-5A chimera” as used herein refers to an oligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylate residue.\",\n \"These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 90, 1300; Silverman et al., 2000 , Methods Enzymol., 313, 522-533; Player and Torrence, 1998 , Pharmacol.\",\n \"Ther., 78, 55-113).\",\n \"The term “triplex forming oligonucleotides” as used herein refers to an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix.\",\n \"Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 89, 504; Fox, 2000 , Curr.\",\n \"Med.\",\n \"Chem., 7, 17-37; Praseuth et.\",\n \"al., 2000 , Biochim.\",\n \"Biophys.\",\n \"Acta, 1489, 181-206).\",\n \"Several varieties of naturally-occurring enzymatic RNAs are known presently.\",\n \"Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions.\",\n \"Table I summarizes some of the characteristics of these ribozymes.\",\n \"In general, enzymatic nucleic acids act by first binding to a target RNA.\",\n \"Such binding occurs through the target binding portion of a enzymatic nucleic acid that is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.\",\n \"Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.\",\n \"Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein.\",\n \"After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.\",\n \"Thus, a single ribozyme molecule is able to cleave many molecules of target RNA.\",\n \"In addition, the ribozyme is a highly specific inhibitor of gene expression, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage.\",\n \"Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme.\",\n \"In one embodiment of the inventions described herein, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but can also be formed in the motif of a hepatitis delta virus, group I intron, group II intron or RNase P RNA (in association with an RNA guide sequence), Neurospora VS RNA, DNAzymes, NCH cleaving motifs, or G-cleavers.\",\n \"Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al., 1992 , AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al., 1989 , Gene 82, 53, Haseloff and Gerlach, 1989 , Gene, 82, 43, and Hampel et al., 1990 Nucleic Acids Res.\",\n \"18, 299; Chowrira & McSwiggen, U.S. Pat.\",\n \"No.\",\n \"5,631,359; of the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNase P motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman, 1990 , Science 249, 783; Li and Altman, 1996 , Nucleic Acids Res.\",\n \"24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995 , EMBO.\",\n \"J.\",\n \"14, 363); Group II introns are described by Griffin et al., 1995 , Chem.\",\n \"Biol.\",\n \"2, 761; Michels and Pyle, 1995 , Biochemistry 34, 2965; Pyle et al., International PCT Publication No.\",\n \"WO 96/22689; of the Group I intron by Cech et al., U.S. Pat.\",\n \"No.\",\n \"4,987,071 and of DNAzymes by Usman et al., International PCT Publication No.\",\n \"WO 95/11304; Chartrand et al., 1995 , NAR 23, 4092; Breaker et al., 1995 , Chem.\",\n \"Bio.\",\n \"2, 655; Santoro et al., 1997 , PNAS 94, 4262, and Beigelman et al., International PCT publication No.\",\n \"WO 99/55857.NCH cleaving motifs are described in Ludwig & Sproat, International PCT Publication No.\",\n \"WO 98/58058; and G-cleavers are described in Kore et al., 1998 , Nucleic Acids Research 26, 4116-4120 and Eckstein et al., International PCT Publication No.\",\n \"WO 99/16871.Additional motifs such as the Aptazyme (Breaker et al., WO 98/43993), Amberzyme (Class I motif; FIG.\",\n \"2 ; Beigelman et al., U.S. Ser.\",\n \"No.\",\n \"09/301,511) and Zinzyme ( FIG.\",\n \"3 ) (Beigelman et al., U.S. Ser.\",\n \"No.\",\n \"09/301,511), all included by reference herein including drawings, can also be used in the present invention.\",\n \"These specific motifs or configurations are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule (Cech et al., U.S. Pat.\",\n \"No.\",\n \"4,987,071).\",\n \"In one embodiment of the present invention, a nucleic acid molecule of the instant invention can be between 12 and 100 nucleotides in length.\",\n \"Exemplary enzymatic nucleic acid molecules of the invention are shown in Tables III-XXIII.\",\n \"For example, enzymatic nucleic acid molecules of the invention can be between 15 and 50 nucleotides in length, and in another embodiment between 25 and 40 nucleotides in length, e.g., 34, 36, or 38 nucleotides in length (for example see Jarvis et al., 1996 , J. Biol.\",\n \"Chem., 271, 29107-29112).\",\n \"Exemplary DNAzymes of the invention are can between 15 and 40 nucleotides in length, and in one embodiment, between 25 and 35 nucleotides in length, e.g., 29, 30, 31, or 32 nucleotides in length (see, e.g., Santoro et al., 1998 , Biochemistry, 37, 13330-13342; Chartrand et al., 1995, Nucleic Acids Research, 23, 4092-4096).\",\n \"Exemplary antisense molecules of the invention can be between 15 and 75 nucleotides in length, and in one embodiment between 20 and 35 nucleotides in length, e.g., 25, 26, 27, or 28 nucleotides in length (see for example Woolf et al., 1992 , PNAS., 89, 7305-7309; Milner et al., 1997 , Nature Biotechnology, 15, 537-541).\",\n \"Exemplary triplex forming oligonucleotide molecules of the invention are between 10 and 40 nucleotides in length, and in one embodiment are between 12 and 25 nucleotides in length, e.g., 18, 19, 20, or 21 nucleotides in length (see for example Maher et al., 1990 , Biochemistry, 29, 8820-8826; Strobel and Dervan, 1990 , Science, 249, 73-75).\",\n \"Those skilled in the art will recognize that all that is required is for the nucleic acid molecule to be of length and conformation sufficient and suitable for the nucleic acid molecule to catalyze a reaction contemplated herein.\",\n \"The length of the nucleic acid molecules of the instant invention are not limiting within the general limits stated.\",\n \"In one embodiment, a nucleic acid molecule that modulates, for example, down-regulates, the expression of a target gene comprises between 8 and 100 bases complementary to a RNA molecule of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR.\",\n \"In another embodiment, a nucleic acid molecule that modulates the expression of a target gene comprises between 14 and 24 bases complementary to a RNA molecule of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR.\",\n \"The invention provides a method for producing a class of nucleic acid-based gene modulating agents that exhibit a high degree of specificity for the RNA of a desired target.\",\n \"For example, the enzymatic nucleic acid molecule is can be targeted to a highly conserved sequence region of target RNAs encoding prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR (e.g., prostaglandin D2 receptor (PTGDR)), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR genes) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention.\",\n \"Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required.\",\n \"Alternatively, the nucleic acid molecules (e.g., ribozymes, antisense, aptamers, and/or siRNA) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.\",\n \"As used in herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism.\",\n \"The cell can, for example, be in vitro, e.g., in cell culture, or present in a multicellular organism, including, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats.\",\n \"The cell may be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).\",\n \"By “NOGO proteins” is meant, a protein, protein receptor or a mutant protein derivative thereof, comprising neuronal inhibitor activity, preferably CNS neuronal growth inhibitor activity.\",\n \"By “IKK-gamma proteins” is meant, a peptide or protein comprising a IKK-gamma or NEMO/IKKAP1 component of the IKK complex, for example a regulatory IKK subunit involved in the assembly of the high molecular weight IKK complex and/or induction of NFKB.\",\n \"By “PKR proteins” is meant, a peptide or protein comprising a protein kinase PKR activity, for example the activation of NFKB.\",\n \"By “PTGDR proteins” is meant, a protein receptor or a mutant protein or peptide derivative thereof, having prostaglandin D2 receptor activity, for example, having the ability to bind prostaglandin D2 and/or having GTP-binding protein coupled activity.\",\n \"By “PTGDS proteins” is meant, a prostaglandin synthetase protein or a mutant protein or peptide derivative thereof, having prostaglandin D2 synthetase activity, for example, having the ability to convert PGH2 to PGD2.By “highly conserved sequence region” is meant, a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.\",\n \"The nucleic acid-based inhibitors of the invention can be added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues, for example by pulmonary delivery of an aerosol formulation with an inhaler or nebulizer.\",\n \"The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through inhalation, injection or infusion pump, with or without their incorporation in biopolymers.\",\n \"In preferred embodiments, the enzymatic nucleic acid inhibitors comprise sequences that are complementary to the substrate sequences in Tables III to XXIII.\",\n \"Examples of such enzymatic nucleic acid molecules also are shown in Tables III to XXIII.\",\n \"Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in these tables.\",\n \"In another embodiment, the invention features antisense nucleic acid molecules, siRNA and 2-5A chimeras including sequences complementary to the substrate sequences shown in Tables III to XXIII.\",\n \"Such nucleic acid molecules can include sequences as shown for the binding arms of the enzymatic nucleic acid molecules in Tables III to XXIII.\",\n \"Similarly, triplex molecules can be provided targeted to the corresponding DNA target regions, and containing the DNA equivalent of a target sequence or a sequence complementary to the specified target (substrate) sequence.\",\n \"Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.\",\n \"However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop.\",\n \"Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.\",\n \"By “consists essentially of” is meant that the active nucleic acid molecule of the invention, for example, an enzymatic nucleic acid molecule, contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind RNA such that cleavage at the target site occurs.\",\n \"Other sequences can be present that do not interfere with such cleavage.\",\n \"Thus, a core region can, for example, include one or more loop, stem-loop structure, or linker which does not prevent enzymatic activity.\",\n \"Thus, the underlined regions in the sequences in Tables III, IV, VIII, IX, XIII, XIV, XIX, and XX can be such a loop, stem-loop, nucleotide linker, and/or non-nucleotide linker and can be represented generally as sequence “X”.\",\n \"For example, a core sequence for a hammerhead enzymatic nucleic acid can comprise a conserved sequence, such as 5′-CUGAUGAG-3′ and 5′-CGAA-3′ connected by “X”, where X is 5′- GCCGUUAGGC -3′ (SEQ ID NO: 13274), or any other Stem II region known in the art, or a nucleotide and/or non-nucleotide linker.\",\n \"Similarly, for other nucleic acid molecules of the instant invention, such as Inozyme, G-cleaver, amberzyme, zinzyme, DNAzyme, antisense, 2-5A antisense, triplex forming nucleic acid, siRNA and decoy nucleic acids, other sequences or non-nucleotide linkers can be present that do not interfere with the function of the nucleic acid molecule.\",\n \"Sequence X can be a linker of ≧2 nucleotides in length, including 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the nucleotides can be internally base-paired to form a stem of ≧2 base pairs.\",\n \"Alternatively or in addition, sequence X can be a non-nucleotide linker.\",\n \"In yet another embodiment, the nucleotide linker X can be a nucleic acid aptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others (for a review see Gold et al., 1995 , Annu.\",\n \"Rev.\",\n \"Biochem., 64, 763; and Szostak & Ellington, 1993, in The RNA World , ed.\",\n \"Gesteland and Atkins, pp.\",\n \"511, CSH Laboratory Press).\",\n \"A “nucleic acid aptamer” as used herein is meant to indicate a nucleic acid sequence capable of interacting with a ligand.\",\n \"The ligand can be any natural or a synthetic molecule, including but not limited to a resin, metabolites, nucleosides, nucleotides, drugs, toxins, transition state analogs, peptides, lipids, proteins, amino acids, nucleic acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria and others.\",\n \"In yet another embodiment, the non-nucleotide linker X is as defined herein.\",\n \"The term “non-nucleotide” as used herein include either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds.\",\n \"Specific examples include those described by Seela and Kaiser, Nucleic Acids Res.\",\n \"1990, 18:6353 and Nucleic Acids Res.\",\n \"1987, 15:3113; Cload and Schepartz, J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"1991, 113:6324; Richardson and Schepartz, J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"1991, 113:5109; Ma et al., Nucleic Acids Res.\",\n \"1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res.\",\n \"1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett.\",\n \"1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No.\",\n \"WO 89/02439; Usman et al., International Publication No.\",\n \"WO 95/06731; Dudycz et al., International Publication No.\",\n \"WO 95/11910 and Ferentz and Verdine, J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"1991, 113:4000, all hereby incorporated by reference herein.\",\n \"A “non-nucleotide” further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.\",\n \"The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.\",\n \"Thus, in a preferred embodiment, the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties, and having enzymatic activity to cleave an RNA or DNA molecule.\",\n \"In another aspect of the invention, nucleic acid molecules that interact with target RNA molecules and down-regulate target genes (e.g., prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene) activity are expressed from transcription units inserted into DNA or RNA vectors.\",\n \"The recombinant vectors can be DNA plasmids or viral vectors.\",\n \"Enzymatic nucleic acid molecule or antisense expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.\",\n \"The recombinant vectors capable of expressing the enzymatic nucleic acid molecules or antisense can be delivered as described above, and persist in target cells.\",\n \"Alternatively, viral vectors can be used that provide for transient expression of enzymatic nucleic acid molecules or antisense.\",\n \"Such vectors can be repeatedly administered as necessary.\",\n \"Once expressed, the enzymatic nucleic acid molecules or antisense bind to the target RNA and down-regulate its function or expression.\",\n \"Delivery of enzymatic nucleic acid molecule or antisense expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell.\",\n \"Antisense DNA can be expressed via the use of a single stranded DNA intracellular expression vector.\",\n \"By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.\",\n \"By “patient” is meant an organism, which is a donor or recipient of explanted cells, or the cells themselves.\",\n \"“Patient” also refers to an organism to which the nucleic acid molecules of the invention can be administered.\",\n \"A patient can be a mammal or mammalian cells.\",\n \"In one embodiment, a patient is a human or human cells.\",\n \"By “enhanced enzymatic activity” is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both the catalytic activity and the stability of the nucleic acid molecules of the invention.\",\n \"In this invention, the product of these properties can be increased in vivo compared to an all RNA enzymatic nucleic acid or all DNA enzyme.\",\n \"In some cases, the activity or stability of the nucleic acid molecule can be decreased (i.e., less than ten-fold), but the overall activity of the nucleic acid molecule is enhanced, in vivo.\",\n \"The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above.\",\n \"For example, to treat a disease or condition associated with the levels of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR, the patient can be treated, or other appropriate cells can be treated, as is evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.\",\n \"In a further embodiment, the described molecules, such as antisense or enzymatic nucleic acid molecules, can be used in combination with other known treatments to treat conditions or diseases discussed above.\",\n \"For example, the described molecules can be used in combination with one or more known therapeutic agents to treat allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR expression.\",\n \"By “comprising” is meant including, but not limited to, whatever follows the word “comprising”.\",\n \"Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.\",\n \"By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”.\",\n \"Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.\",\n \"Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.\"\n ],\n [\n \"This patent application claims priority from U.S. Ser.\",\n \"No.\",\n \"09/827,395, filed Apr.\",\n \"5, 2001, entitled “METHOD AND REAGENT FOR THE INHIBITION OF NOGO AND NOGO RECEPTOR GENES”, which is a continuation-in-part of Blatt, U.S. Ser.\",\n \"No.\",\n \"09/780,533 filed Feb. 9, 2001, entitled “METHOD AND REAGENT FOR THE INHIBITION OF NOGO GENE” which claims priority from Blatt, U.S. Ser.\",\n \"No.\",\n \"60/181,797, filed Feb. 11, 2000, entitled “METHOD AND REAGENT FOR THE INHIBITION OF NOGO GENE”.\",\n \"This patent application also claims priority from U.S. Ser.\",\n \"No.\",\n \"60/294,412, filed May 29, 2001, entitled “ENZYMATIC NUCLEIC ACID TREATMENT OF DISEASES OR CONDITIONS RELATED TO LEVELS OF IKK-GAMMA AND PKR”, and U.S. Ser.\",\n \"No.\",\n \"60/315,315, filed Aug. 28, 2001, entitled “METHOD AND REAGENT FOR THE TREATMENT OF ASTHMA AND ALLERGIC CONDITIONS”.\",\n \"These applications are hereby incorporated by reference herein in their entirety including the drawings.\",\n \"BACKGROUND OF THE INVENTION The present invention provides compounds, compositions, and methods for the study, diagnosis, and treatment of conditions relating to the expression of NOGO and NOGO receptor genes.\",\n \"In particular, the invention provides nucleic acid molecules that are used to modulate the expression of NOGO and NOGO receptor gene products.\",\n \"The present invention further relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to IKK gamma (IKKG) and PKR levels, such as cancer, inflammatory, and autoimmune diseases and/or disorders.\",\n \"In addition, the present invention also relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to allergic response.\",\n \"Specifically, the invention provides compositions and methods for the treatment of diseases or conditions related to levels of factors involved in allergic conditions such as asthma, for example prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS) and adenosine A1 receptor (ADORA1).\",\n \"The discussions that follow are not meant to be complete and are provided only to assist understanding the invention that follows.\",\n \"The summary is not an admission that any of the work described below is prior art to the claimed invention.\",\n \"The following is a brief description of the current understanding of NOGO and NOGO receptors.\",\n \"The ceased growth of neurons following development has severe implications for lesions of the central nervous system (CNS) caused by neurodegenerative disorders and traumatic accidents.\",\n \"Although CNS neurons have the capacity to rearrange their axonal and dendritic foci in the developed brain, the regeneration of severed CNS axons spanning distance does not exist.\",\n \"Axonal growth following CNS injury is limited by the local tissue environment rather than intrinsic factors, as indicated by transplantation experiments (Richardson et al., 1980, Nature, 284, 264-265).\",\n \"Non-neuronal glial cells of the CNS, including oligodendrocytes and astrocytes, have been shown to inhibit the axonal growth of dorsal root ganglion neurons in culture (Schwab and Thoenen, 1985, J.\",\n \"Neurosci., 5, 2415-2423).\",\n \"Cultured dorsal root ganglion cells can extend their axons across glial cells from the peripheral nervous system, (ie; Schwann cells), but are inhibited by oligodendrocytes and myelin of the CNS (Schwab and Caroni, 1988, J.\",\n \"Neurosci., 8, 2381-2393).\",\n \"The non-conducive properties of CNS tissue in adult vertebrates is thought to result from the existence of inhibitory factors rather than the lack of growth factors.\",\n \"The identification of proteins with neurite outgrowth inhibitory or repulsive properties include NI-35, NI-250 (Caroni and Schwab, 1988, Neuron, 1, 85-96), myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945).\",\n \"Monoclonal antibodies (mAb IN-1) raised against NI-35/250 have been shown to partially neutralize the growth inhibitory effect of CNS myelin and oligodendrocytes.\",\n \"IN-1 treatment in vivo has resulted in long distance fiber regeneration in lesioned adult mammalian CNS tissue (Weibel et al., 1994, Brain Res., 642, 259-266).\",\n \"Additionally, IN-1 treatment in vivo has resulted in the recovery of specific reflex and locomotor functions after spinal cord injury in adult rats (Bregman et al., 1995, Nature, 378, 498-501).\",\n \"Recently, the cloning of NOGO-A (Genbank Accession No AJ242961), the rat complementary DNA encoding NI-220/250 has been reported (Chen et al., 2000, Nature, 403, 434-439).\",\n \"The NOGO gene encodes at least three major protein products (NOGO-A, NOGO-B, and NOGO-C) resulting from both alternative promoter usage and alternative splicing.\",\n \"Recombinant NOGO-A inhibits neurite outgrowth from dorsal root ganglia and the spreading of 3T3 firboblasts.\",\n \"Monoclonal antibody IN-1 recognizes NOGO-A and neutralizes NOGO-A inhibition of neuronal growth in vitro.\",\n \"Evidence supports the proposal that NOGO-A is the previously described rat NI-250 since NOGO-A contains all six peptide sequences obtained from purified bNI-220, the bovine equivalent of rat NI-250 (Chen et al supra).\",\n \"Prinjha et al., 2000, Nature, 403, 383-384, report the cloning of the human NOGO gene which encodes three different NOGO isoforms that are potent inhibitors of neurite outgrowth.\",\n \"Using oligonucleotide primers to amplify and clone overlapping regions of the open reading frame of NOGO cDNA, Phrinjha et al., supra identified three forms of cDNA clone corresponding to the three protein isoforms.\",\n \"The longest ORF of 1,192 amino acids corresponds to NOGO-A (Accession No.\",\n \"AJ251383).\",\n \"An intermediate-length splice variant that lacks residues 186-1,004 corresponds to NOGO-B (Accession No.\",\n \"AJ251384).\",\n \"The shortest splice variant, NOGO-C (Accession No.\",\n \"AJ251385), appears to be the previously described rat vp20 (Accession No.\",\n \"AF051335) and foocen-s (Accession No.\",\n \"AF132048), and also lacks residues 186-1,004.According to Prinjha et al., supra, the NOGO amino-terminal region shows no significant homology to any known protein, while the carboxy-terminal tail shares homology with neuroendocrine-specific proteins and other members of the reticulon gene family.\",\n \"In addition, the carboxy-terminal tail contains a consensus sequence that may serve as an endoplasmic-reticulum retention region.\",\n \"Based on the NOGO protein sequence, Prinjha et al., supra, postulate NOGO to be a membrane associated protein comprising a putative large extracellular domain of 1,024 residues with seven predicted N-linked glycosylation sites, two or three transmembrane domains, and a short carboxy-terminal region of 43 residues.\",\n \"Grandpre et al., 2000, Nature, also report the identification of NOGO as a potent inhibitor of axon regeneration.\",\n \"The 4.1 kilobase NOGO human cDNA clone identified by Grandpre et al., supra, KIAA0886, is homologous to a cDNA derived from a previous effort to sequence random high molecular-weight brain derived cDNAs (Nagase et al., 1998, DNA Res., 31, 355-364).\",\n \"This cDNA clone encodes a protein that matches all six of the peptide sequences derived from bovine NOGO.\",\n \"Grandpre et al., supra demonstrate that NOGO expression is predominantly associated with the CNS and not the peripheral nervous system (PNS).\",\n \"Cellular localization of NOGO protein appears to be predominantly reticluar in origin, however, NOGO is found on the surface of some oligodentrocytes.\",\n \"An active domain of NOGO has been identified, defined as residues 31-55 of a hydrophilic 66-residue lumenal/extracellular domain.\",\n \"A synthetic fragment corresponding to this sequence exhibits growth-cone collapsing and outgrowth inhibiting activities (Grandpre et al., supra).\",\n \"A receptor for the NOGO-A extracellular domain (NOGO-66) is described in Fournier et al., 2001, Nature, 409, 341-346.Fournier et al., have shown that isolated NOGO-66 inhibits axonal extension but does not alter non-neuronal cell morphology.\",\n \"The receptor identified has a high affinity for soluble NOGO-66, and is expressed as a glycophosphatidylinostitol-linked protein on the surface of CNS neurons.\",\n \"Furthermore, the expression of the NOGO-66 receptor in neurons that are NOGO insensitive results in NOGO dependent inhibition of axonal growth in these cells.\",\n \"Cleavage of the NOGO-66 receptor and other glycophosphatidylinostitol-linked proteins from axonal surfaces renders neurons insensitive to NOGO-66 inhibition.\",\n \"As such, disruption of the interaction between NOGO-66 and the NOGO-66 receptor provides the possibility of treating a wide variety of neurological diseases, injuries, and conditions.\",\n \"Hauswirth and Flannery, International PCT Publication No.\",\n \"WO 98/48027, describe materials and methods for the specific expression of proteins in retinal photoreceptor cells consisting of an adeno-associated viral vector contacting a rod or cone-opsin promoter.\",\n \"In addition, ribozymes which degrade mutant mRNA are described for use in the treatment of retinitis pigmentosa.\",\n \"Fechteler et al., Interanational PCT Publication No.\",\n \"WO 00/03004 describe ribozymes targeting presenilin-2 RNA for the use in treating neurodegenerative diseases such as Alzheimer's disease.\",\n \"Eldadah et al., 2000, J.\",\n \"Neurosci., 20, 179-186, describe the protection of cerebellar granule cells from apoptosis induced by serum-potassium deprivation from ribozyme mediated inhibition of caspase-3.Seidman et al., 1999, Antisense Nucleic Acid Drug Dev., 9, 333-340, describe in general terms, the use of antisense and ribozyme constructs for treatment of neurodegenerative diseases.\",\n \"Denman et al., 1994, Nucleic Acids Research, 22, 2375-82, describe the ribozyme mediated degradation of beta-amyloid peptide precursor mRNA in COS-7 cells.\",\n \"Schwab and Chen, International PCT publication No.\",\n \"WO 00/31235, describe NOGO proteins and inhibitors of NOGO.\",\n \"Blatt et al., International PCT publication No.\",\n \"WO 01/59103, describe nucleic acid molecules for modulating expression of NOGO genes.\",\n \"The following is a brief description of the physiological role of nuclear factor kappa B (NFKB), IKK kinases, and protein kinase PKR.\",\n \"Nuclear factor kappa B (NFKB) is a multiunit transcription factor which regulates the expression of genes involved in a number of physiologic and pathologic processes.\",\n \"NFKB is a key component of the TNF signaling pathway.\",\n \"These processes include, but are not limited to: apoptosis, immune, inflammatory and acute phase responses.\",\n \"The REL-A gene product (a.k.a.\",\n \"RelA or p65), and p50 subunits of NFKB, have been implicated in the induction of inflammatory responses and cellular transformation.\",\n \"NFKB exists in the cytoplasm as an inactive heterodimer of the p50 and p65 subunits.\",\n \"NFKB is complexed with an inhibitory protein complex, IkappaB (IKK complex), until activated by the appropriate stimuli.\",\n \"NFKB activation can occur following stimulation of a variety of cell types by inflammatory mediators, for example TNF and IL-1, and reactive oxygen intermediates.\",\n \"In response to induction, NFKB can stimulate production of pro-inflammatory cytokines such as TNF-alpha, IL-1-beta, IL-6 and iNOS, thereby perpetuating a positive feedback loop.\",\n \"NFKB appears to play a role in a number of disease processes including: ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, arthritis, and cancer.\",\n \"The nuclear DNA-binding protein, NFKB, was first identified as a factor that binds and activates the immunoglobulin kappa light chain enhancer in B cells.\",\n \"NFKB now is known to activate transcription of a variety of other cellular genes (e.g., cytokines, adhesion proteins, oncogenes and viral proteins) in response to a variety of stimuli (e.g., phorbol esters, mitogens, cytokines and oxidative stress).\",\n \"In addition, molecular and biochemical characterization of NFKB has shown that the activity is due to a homodimer or heterodimer of a family of DNA binding subunits.\",\n \"Each subunit bears a stretch of 300 amino acids that is homologous to the oncogene, v-rel.\",\n \"The activity first described as NFKB is a heterodimer of p49 or p50 with p65.The p49 and p50 subunits of NFKB (encoded by the NF-kappa B2 or NF kappa B1 genes, respectively) are generated from the precursors NFKB1 (p105) or NFKB2 (p100).\",\n \"The p65 subunit of NFKB (now termed REL-A) is encoded by the rel-A locus.\",\n \"The roles of each specific transcription-activating complex now are being elucidated in cells (Perkins, et al., 1992, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci USA, 89, 1529-1533).\",\n \"For instance, the heterodimer of NFKB1 and Rel A (p50/p65) activates transcription of the promoter for the adhesion molecule, VCAM-1, while NFKB2/RelA heterodimers (p49/p65) actually inhibit transcription (Shu, et al., 1993, Mol.\",\n \"Cell.\",\n \"Biol., 13, 6283-6289).\",\n \"Conversely, heterodimers of NFKB2/RelA (p49/p65) act with Tat-I to activate transcription of the HIV genome, while NFKB1/RelA (p50/p65) heterodimers have little effect (Liu et al., 1992, J.\",\n \"Virol., 66, 3883-3887).\",\n \"Similarly, blocking rel A gene expression with antisense oligonucleotides specifically blocks embryonic stem cell adhesion; blocking NFKB1 gene expression with antisense oligonucleotides had no effect on cellular adhesion (Narayanan et al., 1993, Mol.\",\n \"Cell.\",\n \"Biol., 13, 3802-3810).\",\n \"Thus, the promiscuous role initially assigned to NFKB in transcriptional activation (Lenardo, and Baltimore, 1989, Cell, 58, 227-229) represents the sum of the activities of the rel family of DNA-binding proteins.\",\n \"This conclusion is supported by recent transgenic “knock-out” mice of individual members of the rel family.\",\n \"Such “knock-outs” show few developmental defects, suggesting that essential transcriptional activation functions can be performed by more than one member of the rel family.\",\n \"A number of specific inhibitors of NFKB function in cells exist, including treatment with phosphorothioate antisense oliogonucleotide, treatment with double-stranded NFKB binding sites, and over expression of the natural inhibitor MAD-3 (an Ikappa-B family member).\",\n \"These agents have been used to show that NFKB is required for induction of a number of molecules involved in cancer and/or inflammation, as described below.\",\n \"NFkB is required for phorbol ester-mediated induction of IL-6 (Kitajima, et al., 1992, Science, 258, 1792-5) and IL-8 (Kunsch and Rosen, 1993, Mol.\",\n \"Cell.\",\n \"Biol., 13, 6137-46).\",\n \"NFkB is required for induction of the adhesion molecules ICAM-1 (Eck, et al., 1993, Mol.\",\n \"Cell.\",\n \"Biol., 13, 6530-6536), VCAM-1 (Shu et al., supra), and E-selectin (Read, et al., 1994, J. Exp.\",\n \"Med., 179, 503-512) on endothelial cells.\",\n \"NFkB is involved in the induction of the integrin subunit, CD18, and other adhesive properties of leukocytes (Eck et al., 1993 supra).\",\n \"HER2/Neu overexpression induces NFKB via a PI3-kinase/Akt pathway involving calpain-mediated degradation of IKB-alpha.\",\n \"Breast cancer has been shown to typify the aberrant expression of NFKB/REL factors (Pianetti et al., 2001, Oncogene, 20, 1287-1299; Sovak et al., 1999, J. Clin.\",\n \"Invest., 100, 2952-2960).\",\n \"Inhibition of NFKB activity has been shown to induce apoptosis in murine hepatocytes (Bellas et al., 1997, Am.\",\n \"J.\",\n \"Pathol., 151, 891-896).\",\n \"NFKB has been shown to regulate cyclooxygenase-2 expression and cell proliferation in human gastric cancer cells (Joo Weon et al., 2001, Laboratory Investigation, 81, 349-360).\",\n \"The above studies suggest that NFKB is integrally involved in the induction of cytokines and adhesion molecules by inflammatory mediators and is involved in the transformation of cancerous cells.\",\n \"Two reported studies point to another connection between NFKB and inflammation: glucocorticoids can exert their anti-inflammatory effects by inhibiting NFKB.\",\n \"The glucocorticoid receptor and p65 both act at NFKB binding sites in the ICAM-1 promoter (van de Stolpe, et al., 1994, J. Biol.\",\n \"Chem., 269, 6185-6192).\",\n \"Glucocorticoid receptor inhibits NFKB-mediated induction of IL-6 (Ray and Prefontaine, 1994 Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci USA, 91, 752-756).\",\n \"Conversely, overexpression of p65 inhibits glucocorticoid induction of the mouse mammary tumor virus promoter.\",\n \"Finally, protein cross-linking and co-immunoprecipitation experiments demonstrated direct physical interaction between p65 and the glucocorticoid receptor.\",\n \"The IKK complex that sequesters NFKB in the cytoplasm comprises IkappaB (IκB) proteins (IκB-alpha, IκB-beta, IκKB-epsilon, p105, and p100).\",\n \"The phosphorylation of IκB proteins results in the release of NFKB from the IκB complex which is transported to the nucleus via the unmasking of nuclear translocation signals.\",\n \"Phosphorylation marks IkB proteins for ubiquitination and degradation via the proteosome pathway.\",\n \"Most NFKB inducing stimuli initiate activation of an IκB kinase (IKK) complex that contains two catalytic subunits, IKK-alpha (IKK1) and IKK-beta (IKK2), that phosphorylate IκB-alpha and IκB-beta, with IKK-beta playing a predominant role in pro-inflammatory signaling.\",\n \"In addition to the two kinases, the IKK complex contains regulatory subunits, including IKK-gamma (NEMO/IKKAP1).\",\n \"IKK-gamma is a protein that is critical for the assembly of the IKK complex.\",\n \"IKK-gamma directly binds to IKK-beta and is required for activation of NFKB, for example by TNF-alpha, IL-1-beta, lipopolysaccharide, phorbol 12-myristate 13-acetate, the human T-cell lymphotrophic virus (HTLV-1), or double stranded RNA.\",\n \"Genomic rearrangements in IKK-gamma have been shown to impair NFKB activation and result in incontinentia pigmenti.\",\n \"Additional proteins that associate with the IKK complex include, MEK kinase (MEKK1), NFKB inducing kinase (NIK), receptor interacting protein (RIP), protein kinase CK2, and IKK-associated protein (IKAP), which appears to be associated with the IκB Kinase (IKK) complex, but does not appear to be an integral component of the tripartate IKK complex as does IKK-gamma (Krappmann et al., 2001, J. Biol.\",\n \"Chem., 275, 29779-87).\",\n \"The RNA-dependent protein kinase PKR is a signal transducer for NFKB and IFN regulatory factor-1.PKR is required for activation of NFKB by IFN-gamma via a STAT-1 independent pathway (Amitabha et al., 2001, J.\",\n \"Immunol., 166, 6170-6180).\",\n \"The induction of NFKB by PKR takes place though phosphorylation of IκB-alpha, and appears not to require the catalytic activity of PKR, thereby proceeding independently of the dsRNA-binding properties of PKR (Ishii et al., 2001, Oncogene, 20, 1900-1912).\",\n \"PKR also plays an important role in the regulation of protein synthesis by modulating the activity of eukaryotic initiation factor 2 (eIF-2-alpha) through interferon induction.\",\n \"Kamiya, JP 2000253884, describes specific antisense oligonucleotides for inhibiting IκB-kinase subunit expression.\",\n \"Krappmann et al., 2001, J. Biol.\",\n \"Chem., describe specific antisense oligonucleotides to IKK-gamma.\",\n \"The following is a description of molecular targets involved in diseases or conditions related to allergic response.\",\n \"Asthma is a chronic inflammatory disorder of the lungs characterized by airflow obstruction, bronchial hyper-responsiveness, and airway inflammation.\",\n \"T-lymphocytes that produce TH2 cytokines and eosinophilic leukocytes infiltrate the airways.\",\n \"In the airway and in bronchial alveolar lavage (BAL) fluid of individuals with asthma, high concentrations of TH2 cytokines, interleukin-4 (14), IL-5, and IL-13, are present along with increased levels of adenosine.\",\n \"In contrast to normal individuals, asthmatics respond to adenosine challenge with marked airway obstruction.\",\n \"Upon allergen challenge, mast cells are activated by cross-linked IgE-allergen complexes.\",\n \"Large amounts of prostaglandin D2 (PGD2), the major cyclooxygenase product of arachidonic acid are released.\",\n \"PGD2 is generated from PGH2 via the activity of prostaglandin D2 synthetase (PTGDS).\",\n \"PGD2 receptors and adenosine A1 receptors are present in the lungs and airway along with various other tissues in response to allergic stimuli (Howarth, 1997, Allergy, 52, 12).\",\n \"The significance of PGD2 as a mediator of allergic asthma has been established with the development of mice deficient in the PGD2 receptor (DP).\",\n \"DP is a heterotrimeric GTP-binding protein-coupled, rhodopsin-type receptor specific for PGD2 (Hirata et al., 1994, PNAS USA., 91, 11192).\",\n \"These mice fail to develop airway hyperreactivity and have greatly reduced eosinophil infiltration and cytokine accumulation in response to allergens.\",\n \"Upon allergen challenge mice deficient in the prostaglandin D2 (PGD2) receptor (DP) did not develop airway hyperactivity.\",\n \"Cytokine, lymphocyte and eosinophil accumulation in the lungs were greatly reduced (Matsuoka et al., 2000, Science, 287, 2013).\",\n \"The DP −/− mice exhibited no behavioral, anatomic, or histological abnormalities.\",\n \"Primary immune response is not affected by DP disruption.\",\n \"Asthma affects more than 100 million people worldwide and more than 17 million Americans (5% of the population).\",\n \"Since 1980 the incidence has more than doubled and deaths have tripled (5,000 deaths in 1995).\",\n \"Annual asthma-related healthcare costs in the US alone were estimated to exceed $14.5 billion in 2000.Current therapies such as inhalant anti-inflammatories and bronchodilators can be used to treat symptoms, however, these therapies do not prevent or cure asthma.\",\n \"Sandberg et al., 2001, Prog.\",\n \"Respir.\",\n \"Res., 31, 370-373, describes ribozyme therapy for asthma and COPD.\",\n \"Sullivan et al., International U.S. Pat.\",\n \"No.\",\n \"5,616,488, describes ribozymes targeting interleukin-5 for treatment and diagnosis of asthma and other inflammatory disorders.\",\n \"Stinchcomb et al., International PCT Publication No.\",\n \"WO 95/23225, describes ribozymes and methods for inhibiting the expression of disease related genes including genes associated with asthma.\",\n \"Nyce, International PCT Publication Nos.\",\n \"WO 00/62736, WO 00/09525, WO 99/13886, WO 98/23294, WO 96/40266 and U.S. Pat.\",\n \"No.\",\n \"6,025,339 describe specific antisense oligonucleotides targeting certain mRNAs encoding particular adenosine receptors.\",\n \"SUMMARY OF THE INVENTION The invention features novel nucleic acid-based molecules, for example, enzymatic nucleic acid molecules, allozymes, antisense nucleic acids, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNA, dsRNA, siRNA, aptamers, and antisense nucleic acids containing RNA cleaving chemical groups, and methods to modulate gene expression; for example, gene(s) encoding prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), and adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3; gene(s) encoding NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors; and genes encoding an IkappaB kinase (IKK) subunit or protein kinase PKR.\",\n \"In one embodiment, the instant invention features nucleic-acid based techniques to inhibit the expression of NOGO-A (Accession No.\",\n \"AJ251383), NOGO-B (Accession No.\",\n \"AJ251384), and/or NOGO-C (Accession No.\",\n \"AJ251385), NOGO-66 receptor (Accession No AF283463, Fournier et al., 2001, Nature, 409, 341-346), NI-35, NI-220, and/or NI-250, myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945).\",\n \"The description below of the various aspects and embodiments is provided with reference to the exemplary NOGO-A and NOGO-66 receptor genes.\",\n \"However, the various aspects and embodiments are also directed to other genes which express NOGOA-like inhibitor proteins and other receptors involved in neurite outgrowth inhibition.\",\n \"Those additional genes can be analyzed for target sites using the methods described for NOGO and the NOGO-66 receptor, referred to alternatively as NOGO receptor.\",\n \"Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.\",\n \"The invention features one or more enzymatic nucleic acid-based molecules and methods that independently or in combination modulate the expression of gene(s) encoding a member of the IκB kinase IKK complex or PKR.\",\n \"In particular embodiments, the invention features nucleic acid-based molecules and methods that modulate the expression of a member of the IκB kinase IKK complex, for example IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKKγ) and/or a protein kinase PKR protein, such as IKK-alpha (IKK1) gene (Genbank Accession No.\",\n \"NM—001278); IKK-beta (IKK2) gene, for example (Genbank Accession No.\",\n \"AF080158), IKK-gamma (IKKγ) gene, for example (Genbank Accession No.\",\n \"NM—003639), and protein kinase PKR gene, for example (Genbank Accession No.\",\n \"NM—002759).\",\n \"The description below of the various aspects and embodiments is provided with reference to the exemplary IKK-gamma and PKR genes.\",\n \"IKK-gamma is also known as NEMO/IKKAP1.However, the various aspects and embodiments are also directed to other genes which encode other subunits of the IKK complex, such as IKK-alpha (IKK1) or IKK-beta (IKK2).\",\n \"Those additional genes can be analyzed for target sites using the methods described for IKK-gamma or PKR.\",\n \"Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.\",\n \"In one embodiment, an enzymatic nucleic acid molecule of the invention is in a hammerhead, Inozyme, Zinzyme, DNAzyme, Amberzyme, or G-cleaver configuration.\",\n \"In another embodiment, a nucleic acid molecule of the invention comprises between 8 and 100 bases complementary to the RNA of the target gene.\",\n \"In another embodiment, a nucleic acid molecule of the invention comprises between 14 and 24 bases complementary to a RNA molecule of the target gene.\",\n \"In one embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention is chemically synthesized.\",\n \"In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one 2′-sugar modification.\",\n \"In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one nucleic acid base modification.\",\n \"In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one phosphate backbone modification.\",\n \"In one embodiment, the invention features a mammalian cell, for example a human cell, including the nucleic acid molecule of the invention.\",\n \"In another embodiment, the invention features a method of reducing target gene expression or activity in a cell, comprising contacting the cell with a nucleic acid molecule of the invention, such as an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups, under conditions suitable for the reduction.\",\n \"In yet another embodiment, the invention features a method of treatment of a patient having a condition associated with the level of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR, comprising contacting cells of the patient with an enzymatic nucleic acid molecule of the invention, under conditions suitable for the treatment.\",\n \"In another embodiment, a method of treatment of a patient having a condition associated with the level of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR is featured, wherein the method further comprises the use of one or more drug therapies under conditions suitable for the treatment.\",\n \"In another embodiment, the invention features a method of cleaving a RNA molecule of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene, comprising contacting an enzymatic nucleic acid molecule of the invention with a RNA molecule of the corresponding gene under conditions suitable for the cleavage, for example, wherein the cleavage is carried out in the presence of a divalent cation, such as Mg2+.\",\n \"In one embodiment, a nucleic acid molecule of the invention comprises a cap structure, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end of the enzymatic nucleic acid molecule.\",\n \"In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one nucleic acid molecule of the invention, in a manner which allows expression of the nucleic acid molecule.\",\n \"In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.\",\n \"In yet another embodiment, the expression vector of the invention further comprises a sequence for an antisense nucleic acid molecule complementary to a RNA molecule of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene.\",\n \"In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more nucleic acid molecules, such as enzymatic nucleic acid molecules, antisense, aptamers, decoys, siRNA, or 2-5A chimeras which can be the same or different.\",\n \"In one embodiment, the method of treatment features an enzymatic nucleic acid molecule of the invention comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification, such as a 3′-3′ inverted abasic moiety.\",\n \"In another embodiment, an enzymatic nucleic acid molecule or antisense nucleic acid molecule of the invention further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.\",\n \"In another embodiment, the invention features a method of administering to a mammal, for example a human, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention, comprising contacting the mammal with the nucleic acid molecule under conditions suitable for the administration, for example, in the presence of a delivery reagent such as a lipid, cationic lipid, phospholipid, or liposome.\",\n \"In yet another embodiment, the invention features a method of administering to a mammal an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention in conjunction with a therapeutic agent, comprising contacting the mammal, for example a human, with the nucleic acid molecule and the therapeutic agent under conditions suitable for the administration.\",\n \"In one embodiment, the invention features the use of an enzymatic nucleic acid molecule, which can be in a hammerhead, NCH, G-cleaver, Amberzyme, Zinzyme, and/or DNAzyme motif, to down-regulate the expression of a a target gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene.\",\n \"By “inhibit”, “down-regulate”, or “reduce”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR subunits, is reduced below that observed in the absence of the nucleic acid molecules of the invention.\",\n \"In one embodiment, inhibition, down-regulation or reduction with an enzymatic nucleic acid molecule is below that level observed in the presence of an enzymatically inactive or attenuated molecule that is able to bind to the same site on the target RNA molecule, but is unable to cleave that RNA molecule.\",\n \"In another embodiment, inhibition, down-regulation, or reduction with antisense oligonucleotides is below that level observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches.\",\n \"In another embodiment, inhibition, down-regulation, or reduction of the target gene with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.\",\n \"By “up-regulate” is meant that the expression of a gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins, protein subunits, or activity of one or more proteins or protein subunits, such as a target gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR subunits, is greater than that observed in the absence of the nucleic acid molecules of the invention.\",\n \"For example, the expression of a gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene, can be increased in order to treat, prevent, ameliorate, or modulate a pathological condition caused or exacerbated by an absence or low level of gene expression.\",\n \"By “modulate” is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more protein subunits, or activity of one or more protein subunits is up-regulated or down-regulated, such that the expression, level, or activity is greater than or less than that observed in the absence of a nucleic acid molecule of the invention.\",\n \"By “enzymatic nucleic acid molecule” it is meant a nucleic acid molecule that has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that is active to specifically cleave target a RNA molecule.\",\n \"That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave a RNA molecule and thereby inactivate a target RNA molecule.\",\n \"These complementary regions allow sufficient hybridization of an enzymatic nucleic acid molecule to a target RNA molecule and thus permit cleavage.\",\n \"One hundred percent complementarity is preferred, but complementarity as low as 50-75% can also be useful in this invention (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31).\",\n \"The nucleic acids can be modified at the base, sugar, and/or phosphate groups.\",\n \"The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme.\",\n \"All of these terminologies describe nucleic acid molecules with enzymatic activity.\",\n \"The specific enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site that is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule (Cech et al., U.S. Pat.\",\n \"No.\",\n \"4,987,071; Cech et al., 1988, 260 JAMA 3030).\",\n \"By “nucleic acid molecule” as used herein is meant a molecule having nucleotides.\",\n \"The nucleic acid can be single, double, or multiple stranded and can comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.\",\n \"Exemplary nucleic acid molecules of the invention include enzymatic nucleic acid molecules, allozymes, antisense nucleic acids, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNA, dsRNA, siRNA, aptamers, and/or antisense nucleic acids containing RNA cleaving chemical groups.\",\n \"By “enzymatic portion” or “catalytic domain” is meant that portion/region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate (for example see FIGS.\",\n \"1-4).\",\n \"By “substrate binding arm” or “substrate binding domain” is meant that portion/region of a enzymatic nucleic acid that is able to interact, for example via complementarity (i.e., able to base-pair with), with a portion of its substrate.\",\n \"Such complementarity can be 100%, but can be less if desired.\",\n \"For example, as few as 10 bases out of 14 can be base-paired (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31).\",\n \"Examples of such arms are shown generally in FIGS.\",\n \"1-4.That is, these arms contain sequences within an enzymatic nucleic acid that are intended to bring enzymatic nucleic acid and target RNA together through complementary base-pairing interactions.\",\n \"The enzymatic nucleic acid of the invention can have binding arms that are contiguous or non-contiguous and can be of varying lengths.\",\n \"The length of the binding arm(s) can be greater than or equal to four nucleotides and of sufficient length to stably interact with a target RNA; in one embodiment they can be 12-100 nucleotides; in another embodiment they can be 14-24 nucleotides long (see for example Werner and Uhlenbeck, supra; Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herranze et al., 1993, EMBO J., 12, 2567-73) or between 8 and 14 nucleotides long.\",\n \"If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., four and four, five and five nucleotides, or six and six nucleotides, or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., three and five, six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).\",\n \"By “Inozyme” or “NCH” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as NCH Rz in FIG.\",\n \"1.Inozymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCH/, where N is a nucleotide, C is cytidine and H is adenosine, uridine or cytidine, and/represents the cleavage site.\",\n \"H is used interchangeably with X. Inozymes can also possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCN/, where N is a nucleotide, C is cytidine, and / represents the cleavage site.\",\n \"“I” r in FIG.\",\n \"1 represents an Inosine nucleotide, including a ribo-Inosine or xylo-Inosine nucleoside.\",\n \"By “G-cleaver” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as G-cleaver Rz in FIG.\",\n \"1.G-cleavers possess endonuclease activity to cleave RNA substrates having a cleavage triplet NYN/, where N is a nucleotide, Y is uridine or cytidine and / represents the cleavage site.\",\n \"G-cleavers can be chemically modified as is generally shown in FIG.\",\n \"1.By “amberzyme” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG.\",\n \"2.Amberzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NG/N, where N is a nucleotide, G is guanosine, and / represents the cleavage site.\",\n \"Amberzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG.\",\n \"2.In addition, differing nucleoside and/or non-nucleoside linkers can be used to substitute the 5′-gaaa-3′ loops shown in the figure.\",\n \"Amberzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2′-OH) group within its own nucleic acid sequence for activity.\",\n \"By “zinzyme” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG.\",\n \"3.Zinzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet including but not limited to YG/Y, where Y is uridine or cytidine, and G is guanosine and / represents the cleavage site.\",\n \"Zinzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG.\",\n \"3, including substituting 2′-O-methyl guanosine nucleotides for guanosine nucleotides.\",\n \"In addition, differing nucleotide and/or non-nucleotide linkers can be used to substitute the 5′-gaaa-2′ loop shown in the figure.\",\n \"Zinzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2′-OH) group within its own nucleic acid sequence for activity.\",\n \"By ‘DNAzyme’ is meant, an enzymatic nucleic acid molecule that does not require the presence of a 2′-OH group within its own nucleic acid sequence for activity.\",\n \"In particular embodiments the enzymatic nucleic acid molecule can have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups.\",\n \"DNAzymes can be synthesized chemically or expressed endogenously in vivo, by means of a single stranded DNA vector or equivalent thereof.\",\n \"An example of a DNAzyme is shown in FIG.\",\n \"4 and is generally reviewed in Usman et al., U.S. Pat.\",\n \"No.\",\n \"6,159,714; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem.\",\n \"Bio.\",\n \"2, 655; Santoro et al., 1997, PNAS 94, 4262; Breaker, 1999, Nature Biotechnology, 17, 422-423; and Santoro et.\",\n \"al., 2000, J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc., 122, 2433-39.The “10-23” DNAzyme motif is one particular type of DNAzyme that was evolved using in vitro selection (see Santoro et al., supra).\",\n \"Additional DNAzyme motifs can be selected for using techniques similar to those described in these references, and hence, are within the scope of the present invention.\",\n \"By “sufficient length” is meant an oligonucleotide of greater than or equal to 3 nucleotides that is of a length great enough to provide the intended function under the expected condition.\",\n \"For example, for binding arms of enzymatic nucleic acid “sufficient length” means that the binding arm sequence is long enough to provide stable binding to a target site under the expected binding conditions.\",\n \"The binding arms are not so long as to prevent useful turnover of the nucleic acid molecule.\",\n \"By “stably interact” is meant interaction of the oligonucleotides with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions) that is sufficient to the intended purpose (e.g., cleavage of target RNA by an enzyme).\",\n \"By “equivalent” or “related” RNA to NOGO is meant to include those naturally occurring RNA molecules having homology (partial or complete) to NOGO-A, NOGO-B, NOGO-C and/or NOGO receptor proteins or encoding for proteins with similar function as NOGO or NOGO receptor proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.\",\n \"The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.\",\n \"By “equivalent” or “related” RNA to IKK-gamma is meant to include those naturally occurring RNA molecules having homology (partial or complete) to IKK-gamma proteins or encoding for proteins with similar function as IKK-gamma proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.\",\n \"The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.\",\n \"By “equivalent” or “related” RNA to PKR is meant to include those naturally occurring RNA molecules having homology (partial or complete) to PKR proteins or encoding for proteins with similar function as PKR proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.\",\n \"The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.\",\n \"By “equivalent” or “related” RNA to PTGDS is meant to include RNA molecules having homology (partial or complete) to RNA molecules encoding PTGDS proteins or encoding proteins with similar function as PTGDS proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites.\",\n \"The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.\",\n \"By “equivalent” or “related” RNA to PTGDR is meant to include RNA molecules having homology (partial or complete) to RNA molecules encoding PTGDR proteins or encoding proteins with similar function as PTGDR proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites.\",\n \"The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.\",\n \"By “equivalent” or “related” RNA to ADORA1 is meant to include RNA molecules having homology (partial or complete) to RNA molecule encoding ADORA1 proteins or encoding proteins with similar function as ADORA1 proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites.\",\n \"The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.\",\n \"By “homology” is meant the nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.\",\n \"By “antisense nucleic acid”, it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., U.S. Pat.\",\n \"No.\",\n \"5,849,902).\",\n \"Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.\",\n \"However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop.\",\n \"Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.\",\n \"For a review of current antisense strategies, see Schmajuk et al., 1999, J. Biol.\",\n \"Chem., 274, 21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et al., 1997, Antisense N. A.\",\n \"Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol., 313, 3-45; Crooke, 1998, Biotech.\",\n \"Genet.\",\n \"Eng.\",\n \"Rev., 15, 121-157, Crooke, 1997, Ad.\",\n \"Pharmacol., 40, 1-49.In addition, antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.\",\n \"The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA.\",\n \"Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.\",\n \"By “RNase H activating region” is meant a region (generally greater than or equal to 4-25 nucleotides in length, and in one embodiment from 5-11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al., U.S. Pat.\",\n \"No.\",\n \"5,849,902; Arrow et al., U.S. Pat.\",\n \"No.\",\n \"5,989,912).\",\n \"The RNase H enzyme binds to the nucleic acid molecule-target RNA complex and cleaves the target RNA sequence.\",\n \"The RNase H activating region comprises, for example, phosphodiester, phosphorothioate (at least four of the nucleotides are phosphorothiote substitutions; and in another embodiment, 4-11 of the nucleotides are phosphorothiote substitutions); phosphorodithioate, 5′-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof.\",\n \"In addition to one or more backbone chemistries described above, the RNase H activating region can also comprise a variety of sugar chemistries.\",\n \"For example, the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry.\",\n \"Those skilled in the art will recognize that the foregoing are non-limiting examples and that any combination of phosphate, sugar and base chemistry of a nucleic acid that supports the activity of RNase H enzyme is within the scope of the definition of the RNase H activating region and the instant invention.\",\n \"By “2-5A antisense chimera” is meant an antisense oligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylate residue.\",\n \"These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and Torrence, 1998, Pharmacol.\",\n \"Ther., 78, 55-113).\",\n \"By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that is distinct from sequence recognized by the target molecule in its natural setting.\",\n \"Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid.\",\n \"The target molecule can be any molecule of interest.\",\n \"For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein.\",\n \"This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art, see for example Gold et al., 1995, Annu.\",\n \"Rev.\",\n \"Biochem., 64, 763; Brody and Gold, 2000, J.\",\n \"Biotechnol., 74, 5; Sun, 2000, Curr.\",\n \"Opin.\",\n \"Mol.\",\n \"Ther., 2, 100; Kusser, 2000, J.\",\n \"Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.By “triplex forming oligonucleotides” is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix.\",\n \"Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 89, 504; Fox, 2000, Curr.\",\n \"Med.\",\n \"Chem., 7, 17-37; Praseuth et al., 2000, Biochim.\",\n \"Biophys.\",\n \"Acta, 1489, 181-206).\",\n \"By “gene” it is meant a nucleic acid that encodes an RNA, for example, nucleic acid sequences including but not limited to structural genes encoding a polypeptide.\",\n \"“Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another RNA molecule by either traditional Watson-Crick or other non-traditional types.\",\n \"In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., enzymatic nucleic acid cleavage, antisense or triple helix inhibition.\",\n \"Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp.\",\n \"Quant.\",\n \"Biol.\",\n \"LII pp.\",\n \"123-133; Frier et al., 1986, Proc.\",\n \"Nat.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 83:9373-9377; Turner et al., 1987, J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"109:3783-3785).\",\n \"A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).\",\n \"“Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.\",\n \"By “RNA” is meant a molecule comprising at least one ribonucleotide residue.\",\n \"By “ribonucleotide” or “2′-OH” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribo-furanose moiety.\",\n \"By “decoy RNA” is meant an RNA molecule or aptamer that is designed to preferentially bind to a predetermined ligand.\",\n \"Such binding can result in the inhibition or activation of a target molecule.\",\n \"The decoy RNA or aptamer can compete with a naturally occurring binding target for the binding of a specific ligand.\",\n \"For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a “decoy” and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990, Cell, 63, 601-608).\",\n \"This is but a specific example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art, see for example Gold et al., 1995, Annu.\",\n \"Rev.\",\n \"Biochem., 64, 763; Brody and Gold, 2000, J.\",\n \"Biotechnol., 74, 5; Sun, 2000, Curr.\",\n \"Opin.\",\n \"Mol.\",\n \"Ther., 2, 100; Kusser, 2000, J.\",\n \"Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.Similarly, a decoy RNA can be designed to bind to a D2 receptor and block the binding of PTGDS or a decoy RNA can be designed to bind to PTGDS and prevent interaction with the D2 receptor.\",\n \"The term “short interfering RNA” or “siRNA” as used herein refers to a double stranded nucleic acid molecule capable of RNA interference “RNAi”, see for example Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., International PCT Publication No.\",\n \"WO 00/44895; Zernicka-Goetz et al., International PCT Publication No.\",\n \"WO 01/36646; Fire, International PCT Publication No.\",\n \"WO 99/32619; Plaetinck et al., International PCT Publication No.\",\n \"WO 00/01846; Mello and Fire, International PCT Publication No.\",\n \"WO 01/29058; Deschamps-Depaillette, International PCT Publication No.\",\n \"WO 99/07409; and Li et al., International PCT Publication No.\",\n \"WO 00/44914.As used herein, siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides.\",\n \"The term “allozyme” as used herein refers to an allosteric enzymatic nucleic acid molecule, see, e.g., George et al., U.S. Pat.\",\n \"Nos.\",\n \"5,834,186 and 5,741,679, Shih et al., U.S. Pat.\",\n \"No.\",\n \"5,589,332, Nathan et al., U.S. Pat.\",\n \"No.\",\n \"5,871,914, Nathan and Ellington, International PCT publication No.\",\n \"WO 00/24931, Breaker et al., International PCT Publication Nos.\",\n \"WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No.\",\n \"WO 99/29842.The term “2-5A chimera” as used herein refers to an oligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylate residue.\",\n \"These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and Torrence, 1998, Pharmacol.\",\n \"Ther., 78, 55-113).\",\n \"The term “triplex forming oligonucleotides” as used herein refers to an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix.\",\n \"Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 89, 504; Fox, 2000, Curr.\",\n \"Med.\",\n \"Chem., 7, 17-37; Praseuth et.\",\n \"al., 2000, Biochim.\",\n \"Biophys.\",\n \"Acta, 1489, 181-206).\",\n \"Several varieties of naturally-occurring enzymatic RNAs are known presently.\",\n \"Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions.\",\n \"Table I summarizes some of the characteristics of these ribozymes.\",\n \"In general, enzymatic nucleic acids act by first binding to a target RNA.\",\n \"Such binding occurs through the target binding portion of a enzymatic nucleic acid that is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.\",\n \"Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.\",\n \"Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein.\",\n \"After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.\",\n \"Thus, a single ribozyme molecule is able to cleave many molecules of target RNA.\",\n \"In addition, the ribozyme is a highly specific inhibitor of gene expression, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage.\",\n \"Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme.\",\n \"In one embodiment of the inventions described herein, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but can also be formed in the motif of a hepatitis delta virus, group I intron, group II intron or RNase P RNA (in association with an RNA guide sequence), Neurospora VS RNA, DNAzymes, NCH cleaving motifs, or G-cleavers.\",\n \"Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al., 1992, AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al., 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al., 1990 Nucleic Acids Res.\",\n \"18, 299; Chowrira & McSwiggen, U.S. Pat.\",\n \"No.\",\n \"5,631,359; of the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNase P motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res.\",\n \"24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO.\",\n \"J.\",\n \"14, 363); Group II introns are described by Griffin et al., 1995, Chem.\",\n \"Biol.\",\n \"2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al., International PCT Publication No.\",\n \"WO 96/22689; of the Group I intron by Cech et al., U.S. Pat.\",\n \"No.\",\n \"4,987,071 and of DNAzymes by Usman et al., International PCT Publication No.\",\n \"WO 95/11304; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem.\",\n \"Bio.\",\n \"2, 655; Santoro et al., 1997, PNAS 94, 4262, and Beigelman et al., International PCT publication No.\",\n \"WO 99/55857.NCH cleaving motifs are described in Ludwig & Sproat, International PCT Publication No.\",\n \"WO 98/58058; and G-cleavers are described in Kore et al., 1998, Nucleic Acids Research 26, 4116-4120 and Eckstein et al., International PCT Publication No.\",\n \"WO 99/16871.Additional motifs such as the Aptazyme (Breaker et al., WO 98/43993), Amberzyme (Class I motif; FIG.\",\n \"2; Beigelman et al., U.S. Ser.\",\n \"No.\",\n \"09/301,511) and Zinzyme (FIG.\",\n \"3) (Beigelman et al., U.S. Ser.\",\n \"No.\",\n \"09/301,511), all included by reference herein including drawings, can also be used in the present invention.\",\n \"These specific motifs or configurations are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule (Cech et al., U.S. Pat.\",\n \"No.\",\n \"4,987,071).\",\n \"In one embodiment of the present invention, a nucleic acid molecule of the instant invention can be between 12 and 100 nucleotides in length.\",\n \"Exemplary enzymatic nucleic acid molecules of the invention are shown in Tables III-XXIII.\",\n \"For example, enzymatic nucleic acid molecules of the invention can be between 15 and 50 nucleotides in length, and in another embodiment between 25 and 40 nucleotides in length, e.g., 34, 36, or 38 nucleotides in length (for example see Jarvis et al., 1996, J. Biol.\",\n \"Chem., 271, 29107-29112).\",\n \"Exemplary DNAzymes of the invention are can between 15 and 40 nucleotides in length, and in one embodiment, between 25 and 35 nucleotides in length, e.g., 29, 30, 31, or 32 nucleotides in length (see, e.g., Santoro et al., 1998, Biochemistry, 37, 13330-13342; Chartrand et al., 1995, Nucleic Acids Research, 23, 4092-4096).\",\n \"Exemplary antisense molecules of the invention can be between 15 and 75 nucleotides in length, and in one embodiment between 20 and 35 nucleotides in length, e.g., 25, 26, 27, or 28 nucleotides in length (see for example Woolf et al., 1992, PNAS., 89, 7305-7309; Milner et al., 1997, Nature Biotechnology, 15, 537-541).\",\n \"Exemplary triplex forming oligonucleotide molecules of the invention are between 10 and 40 nucleotides in length, and in one embodiment are between 12 and 25 nucleotides in length, e.g., 18, 19, 20, or 21 nucleotides in length (see for example Maher et al., 1990, Biochemistry, 29, 8820-8826; Strobel and Dervan, 1990, Science, 249, 73-75).\",\n \"Those skilled in the art will recognize that all that is required is for the nucleic acid molecule to be of length and conformation sufficient and suitable for the nucleic acid molecule to catalyze a reaction contemplated herein.\",\n \"The length of the nucleic acid molecules of the instant invention are not limiting within the general limits stated.\",\n \"In one embodiment, a nucleic acid molecule that modulates, for example, down-regulates, the expression of a target gene comprises between 8 and 100 bases complementary to a RNA molecule of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR.\",\n \"In another embodiment, a nucleic acid molecule that modulates the expression of a target gene comprises between 14 and 24 bases complementary to a RNA molecule of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR.\",\n \"The invention provides a method for producing a class of nucleic acid-based gene modulating agents that exhibit a high degree of specificity for the RNA of a desired target.\",\n \"For example, the enzymatic nucleic acid molecule is can be targeted to a highly conserved sequence region of target RNAs encoding prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR (e.g., prostaglandin D2 receptor (PTGDR)), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR genes) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention.\",\n \"Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required.\",\n \"Alternatively, the nucleic acid molecules (e.g., ribozymes, antisense, aptamers, and/or siRNA) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.\",\n \"As used in herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism.\",\n \"The cell can, for example, be in vitro, e.g., in cell culture, or present in a multicellular organism, including, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats.\",\n \"The cell may be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).\",\n \"By “NOGO proteins” is meant, a protein, protein receptor or a mutant protein derivative thereof, comprising neuronal inhibitor activity, preferably CNS neuronal growth inhibitor activity.\",\n \"By “IKK-gamma proteins” is meant, a peptide or protein comprising a IKK-gamma or NEMO/IKKAP1 component of the IKK complex, for example a regulatory IKK subunit involved in the assembly of the high molecular weight IKK complex and/or induction of NFKB.\",\n \"By “PKR proteins” is meant, a peptide or protein comprising a protein kinase PKR activity, for example the activation of NFKB.\",\n \"By “PTGDR proteins” is meant, a protein receptor or a mutant protein or peptide derivative thereof, having prostaglandin D2 receptor activity, for example, having the ability to bind prostaglandin D2 and/or having GTP-binding protein coupled activity.\",\n \"By “PTGDS proteins” is meant, a prostaglandin synthetase protein or a mutant protein or peptide derivative thereof, having prostaglandin D2 synthetase activity, for example, having the ability to convert PGH2 to PGD2.By “highly conserved sequence region” is meant, a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.\",\n \"The nucleic acid-based inhibitors of the invention can be added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues, for example by pulmonary delivery of an aerosol formulation with an inhaler or nebulizer.\",\n \"The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through inhalation, injection or infusion pump, with or without their incorporation in biopolymers.\",\n \"In preferred embodiments, the enzymatic nucleic acid inhibitors comprise sequences that are complementary to the substrate sequences in Tables III to XXIII.\",\n \"Examples of such enzymatic nucleic acid molecules also are shown in Tables III to XXIII.\",\n \"Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in these tables.\",\n \"In another embodiment, the invention features antisense nucleic acid molecules, siRNA and 2-5A chimeras including sequences complementary to the substrate sequences shown in Tables III to XXIII.\",\n \"Such nucleic acid molecules can include sequences as shown for the binding arms of the enzymatic nucleic acid molecules in Tables III to XXIII.\",\n \"Similarly, triplex molecules can be provided targeted to the corresponding DNA target regions, and containing the DNA equivalent of a target sequence or a sequence complementary to the specified target (substrate) sequence.\",\n \"Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.\",\n \"However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop.\",\n \"Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.\",\n \"By “consists essentially of” is meant that the active nucleic acid molecule of the invention, for example, an enzymatic nucleic acid molecule, contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind RNA such that cleavage at the target site occurs.\",\n \"Other sequences can be present that do not interfere with such cleavage.\",\n \"Thus, a core region can, for example, include one or more loop, stem-loop structure, or linker which does not prevent enzymatic activity.\",\n \"Thus, the underlined regions in the sequences in Tables III, IV, VIII, IX, XIII, XIV, XIX, and XX can be such a loop, stem-loop, nucleotide linker, and/or non-nucleotide linker and can be represented generally as sequence “X”.\",\n \"For example, a core sequence for a hammerhead enzymatic nucleic acid can comprise a conserved sequence, such as 5′-CUGAUGAG-3′ and 5′-CGAA-3′ connected by “X”, where X is 5′-GCCGUUAGGC-3′ (SEQ ID NO: 13274), or any other Stem II region known in the art, or a nucleotide and/or non-nucleotide linker.\",\n \"Similarly, for other nucleic acid molecules of the instant invention, such as Inozyme, G-cleaver, amberzyme, zinzyme, DNAzyme, antisense, 2-5A antisense, triplex forming nucleic acid, siRNA and decoy nucleic acids, other sequences or non-nucleotide linkers can be present that do not interfere with the function of the nucleic acid molecule.\",\n \"Sequence X can be a linker of ≧2 nucleotides in length, including 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the nucleotides can be internally base-paired to form a stem of ≧2 base pairs.\",\n \"Alternatively or in addition, sequence X can be a non-nucleotide linker.\",\n \"In yet another embodiment, the nucleotide linker X can be a nucleic acid aptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others (for a review see Gold et al., 1995, Annu.\",\n \"Rev.\",\n \"Biochem., 64, 763; and Szostak & Ellington, 1993, in The RNA World, ed.\",\n \"Gesteland and Atkins, pp.\",\n \"511, CSH Laboratory Press).\",\n \"A “nucleic acid aptamer” as used herein is meant to indicate a nucleic acid sequence capable of interacting with a ligand.\",\n \"The ligand can be any natural or a synthetic molecule, including but not limited to a resin, metabolites, nucleosides, nucleotides, drugs, toxins, transition state analogs, peptides, lipids, proteins, amino acids, nucleic acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria and others.\",\n \"In yet another embodiment, the non-nucleotide linker X is as defined herein.\",\n \"The term “non-nucleotide” as used herein include either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds.\",\n \"Specific examples include those described by Seela and Kaiser, Nucleic Acids Res.\",\n \"1990, 18:6353 and Nucleic Acids Res.\",\n \"1987, 15:3113; Cload and Schepartz, J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"1991, 113:6324; Richardson and Schepartz, J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"1991, 113:5109; Ma et al., Nucleic Acids Res.\",\n \"1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res.\",\n \"1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett.\",\n \"1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No.\",\n \"WO 89/02439; Usman et al., International Publication No.\",\n \"WO 95/06731; Dudycz et al., International Publication No.\",\n \"WO 95/11910 and Ferentz and Verdine, J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"1991, 113:4000, all hereby incorporated by reference herein.\",\n \"A “non-nucleotide” further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.\",\n \"The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.\",\n \"Thus, in a preferred embodiment, the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties, and having enzymatic activity to cleave an RNA or DNA molecule.\",\n \"In another aspect of the invention, nucleic acid molecules that interact with target RNA molecules and down-regulate target genes (e.g., prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene) activity are expressed from transcription units inserted into DNA or RNA vectors.\",\n \"The recombinant vectors can be DNA plasmids or viral vectors.\",\n \"Enzymatic nucleic acid molecule or antisense expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.\",\n \"The recombinant vectors capable of expressing the enzymatic nucleic acid molecules or antisense can be delivered as described above, and persist in target cells.\",\n \"Alternatively, viral vectors can be used that provide for transient expression of enzymatic nucleic acid molecules or antisense.\",\n \"Such vectors can be repeatedly administered as necessary.\",\n \"Once expressed, the enzymatic nucleic acid molecules or antisense bind to the target RNA and down-regulate its function or expression.\",\n \"Delivery of enzymatic nucleic acid molecule or antisense expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell.\",\n \"Antisense DNA can be expressed via the use of a single stranded DNA intracellular expression vector.\",\n \"By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.\",\n \"By “patient” is meant an organism, which is a donor or recipient of explanted cells, or the cells themselves.\",\n \"“Patient” also refers to an organism to which the nucleic acid molecules of the invention can be administered.\",\n \"A patient can be a mammal or mammalian cells.\",\n \"In one embodiment, a patient is a human or human cells.\",\n \"By “enhanced enzymatic activity” is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both the catalytic activity and the stability of the nucleic acid molecules of the invention.\",\n \"In this invention, the product of these properties can be increased in vivo compared to an all RNA enzymatic nucleic acid or all DNA enzyme.\",\n \"In some cases, the activity or stability of the nucleic acid molecule can be decreased (i.e., less than ten-fold), but the overall activity of the nucleic acid molecule is enhanced, in vivo.\",\n \"The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above.\",\n \"For example, to treat a disease or condition associated with the levels of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR, the patient can be treated, or other appropriate cells can be treated, as is evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.\",\n \"In a further embodiment, the described molecules, such as antisense or enzymatic nucleic acid molecules, can be used in combination with other known treatments to treat conditions or diseases discussed above.\",\n \"For example, the described molecules can be used in combination with one or more known therapeutic agents to treat allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR expression.\",\n \"By “comprising” is meant including, but not limited to, whatever follows the word “comprising”.\",\n \"Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.\",\n \"By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”.\",\n \"Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.\",\n \"Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 shows examples of chemically stabilized ribozyme motifs.\",\n \"HH Rz, represents hammerhead ribozyme motif (Usman et al., 1996, Curr.\",\n \"Op.\",\n \"Struct.\",\n \"Bio., 1, 527); NCH Rz represents the NCH ribozyme motif (Ludwig & Sproat, International PCT Publication No.\",\n \"WO 98/58058); G-Cleaver, represents G-cleaver ribozyme motif (Kore et al., 1998, Nucleic Acids Research 26, 4116-4120, Eckstein et al., International PCT publication No.\",\n \"WO 99/16871).\",\n \"N or n, represent independently a nucleotide that can be same or different and have complementarity to each other; rI, represents ribo-Inosine nucleotide; arrow indicates the site of cleavage within the target.\",\n \"Position 4 of the HH Rz and the NCH Rz is shown as having 2′-C-allyl modification, but those skilled in the art will recognize that this position can be modified with other modifications well known in the art, so long as such modifications do not significantly inhibit the activity of the ribozyme.\",\n \"FIG.\",\n \"2 shows an example of the Amberzyme ribozyme motif that is chemically stabilized (see for example Beigelman et al., International PCT publication No.\",\n \"WO 99/55857).\",\n \"FIG.\",\n \"3 shows an example of the Zinzyme A ribozyme motif that is chemically stabilized (see for example Beigelman et al., Beigelman et al., International PCT publication No.\",\n \"WO 99/55857).\",\n \"FIG.\",\n \"4 shows an example of a specific DNAzyme motif, commonly referred to as the “10-23 motif”, as described by Santoro et al., 1997, PNAS, 94, 4262.DESCRIPTION OF THE PREFERRED EMBODIMENTS Nucleic Acid Molecules and Mechanism of Action Antisense: Antisense molecules can be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides and primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, November 1994, BioPharm, 20-33).\",\n \"The antisense oligonucleotide binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme.\",\n \"Antisense molecules can also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit.\",\n \"Rev.\",\n \"in Oncogenesis 7, 151-190).\",\n \"In addition, binding of single stranded DNA to RNA can result in nuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke, supra).\",\n \"To date, the only backbone modified DNA chemistry which act as substrates for RNase H are phosphorothioates, phosphorodithioates, and borontrifluoridates.\",\n \"Recently it has been reported that 2′-arabino and 2′-fluoro arabino-containing oligos can also activate RNase H activity.\",\n \"A number of antisense molecules have been described that utilize novel configurations of chemically modified nucleotides, secondary structure, and/or RNase H substrate domains (Woolf et al., International PCT Publication No.\",\n \"WO 98/13526; Thompson et al., International PCT Publication No.\",\n \"WO 99/54459; Hartmann et al., U.S. Ser.\",\n \"No.\",\n \"60/101,174, filed on Sep. 21, 1998) all of these are incorporated by reference herein in their entirety.\",\n \"In addition, antisense deoxyoligoribonucleotides can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.\",\n \"Antisense DNA can be expressed via the use of a single stranded DNA intracellular expression vector or equivalents and variations thereof.\",\n \"Triplex Forming Oligonucleotides (TFO): Single stranded DNA can be designed to bind to genomic DNA in a sequence specific manner.\",\n \"TFOs are comprised of pyrimidine-rich oligonucleotides which bind DNA helices through Hoogsteen Base-pairing (Wu-Pong, supra).\",\n \"The resulting triple helix composed of the DNA sense, DNA antisense, and TFO disrupts RNA synthesis by RNA polymerase.\",\n \"The TFO mechanism can result in gene expression or cell death since binding can be irreversible (Mukhopadhyay & Roth, supra).\",\n \"2-5A Antisense Chimera: The 2-5A system is an interferon mediated mechanism for RNA degradation found in higher vertebrates (Mitra et al., 1996, Proc Nat Acad Sci USA 93, 6780-6785).\",\n \"Two types of enzymes, 2-5A synthetase and RNase L, are required for RNA cleavage.\",\n \"The 2-5A synthetases require double stranded RNA to form 2′-5′ oligoadenylates (2-5A).\",\n \"2-5A then acts as an allosteric effector for utilizing RNase L, which has the ability to cleave single stranded RNA.\",\n \"The ability to form 2-5A structures with double stranded RNA makes this system particularly useful for inhibition of viral replication.\",\n \"(2′-5′) oligoadenylate structures can be covalently linked to antisense molecules to form chimeric oligonucleotides capable of RNA cleavage (Torrence, supra).\",\n \"These molecules putatively bind and activate a 2-5A dependent RNase, the oligonucleotide/enzyme complex then binds to a target RNA molecule which can then be cleaved by the RNase enzyme.\",\n \"Aptamer: Nucleic acid aptamers can be selected to specifically bind to a particular ligand of interest (see for example Gold et al., U.S. Pat.\",\n \"No.\",\n \"5,567,588 and U.S. Pat.\",\n \"No.\",\n \"5,475,096, Gold et al., 1995, Annu.\",\n \"Rev.\",\n \"Biochem., 64, 763; Brody and Gold, 2000, J.\",\n \"Biotechnol., 74, 5; Sun, 2000, Curr.\",\n \"Opin.\",\n \"Mol.\",\n \"Ther., 2, 100; Kusser, 2000, J.\",\n \"Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628).\",\n \"For example, the use of in vitro selection can be applied to evolve nucleic acid aptamers with binding specificity for the NOGO receptor, prostaglandin D2 receptor (PTGDR), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors.\",\n \"Nucleic acid aptamers can include chemical modifications and linkers as described herein.\",\n \"Aptamer molecules of the invention that bind to a cellular receptor, such as prostaglandin D2 receptor (PTGDR), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, or protein kinase PKR receptor, and modulate the activity of the receptor or ligand having specificity for the receptor.\",\n \"RNAi: RNA interference refers to the process of sequence specific post transcriptional gene silencing in animals mediated by short interfering RNAs (siRNA) (Fire et al., 1998, Nature, 391, 806).\",\n \"The corresponding process in plants is commonly referred to as post transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi.\",\n \"The process of post transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358).\",\n \"Such protection from foreign gene expression may have evolved in response to the production of double stranded RNAs (dsRNA) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single stranded RNA or viral genomic RNA.\",\n \"The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized.\",\n \"This mechanism appears to be different from the interferon response that results from dsRNA mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L. The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer.\",\n \"Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363).\",\n \"Short interfering RNAs derived from dicer activity are typically about 21-23 nucleotides in length and comprise about 19 base pair duplexes.\",\n \"Dicer has also been implicated in the excision of 21 and 22 nucleotide small temporal RNAs (stRNA) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834).\",\n \"The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single stranded RNA having sequence homologous to the siRNA.\",\n \"Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).\",\n \"Short interfering RNA mediated RNAi has been studied in a variety of systems.\",\n \"Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. Elegans.\",\n \"Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describes RNAi mediated by dsRNA in mouse embryos.\",\n \"Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA.\",\n \"Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells.\",\n \"Recent work in Drosophila embryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity.\",\n \"These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two nucleotide 3′-overhangs.\",\n \"Furthermore, substitution of one or both siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3′-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated.\",\n \"Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity.\",\n \"In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J., 20, 6877).\",\n \"Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309), however siRNA molecules lacking a 5′-phosphate are active when introduced exogenously, suggesting that 5′-phosphorylation of siRNA constructs may occur in vivo.\",\n \"Enzymatic Nucleic Acid: Several varieties of naturally-occurring enzymatic RNAs are presently known.\",\n \"In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc.\",\n \"R. Soc.\",\n \"London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing cleavage and ligation of phosphodiester linkages (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr.\",\n \"Op.\",\n \"Biotech., 7, 442; Santoro et al., 1997, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci., 94, 4262; Tang et al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra; Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish et al., 1997, Biochemistry 36, 6495; all of these are incorporated by reference herein).\",\n \"Each can catalyze a series of reactions including the hydrolysis of phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions.\",\n \"The enzymatic nature of an enzymatic nucleic acid molecule has significant advantages, one advantage being that the concentration of enzymatic nucleic acid molecule necessary to affect a therapeutic treatment is lower.\",\n \"This advantage reflects the ability of the enzymatic nucleic acid molecule to act enzymatically.\",\n \"Thus, a single enzymatic nucleic acid molecule is able to cleave many molecules of target RNA.\",\n \"In addition, the enzymatic nucleic acid molecule is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage.\",\n \"Single mismatches, or base-substitutions, near the site of cleavage can be chosen to completely eliminate catalytic activity of a enzymatic nucleic acid molecule.\",\n \"Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner.\",\n \"With the proper design, such enzymatic nucleic acid molecules can be targeted to RNA transcripts, and achieve efficient cleavage in vitro (Zaug et al., 324, Nature 429 1986; Uhlenbeck, 1987 Nature 328, 596; Kim et al., 84 Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 8788, 1987; Dreyfus, 1988, Einstein Quart.\",\n \"J. Bio.\",\n \"Med., 6, 92; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989; Santoro et al., 1997 supra).\",\n \"Because of their sequence specificity, trans-cleaving enzymatic nucleic acid molecules can be used as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann.\",\n \"Rep. Med.\",\n \"Chem.\",\n \"30, 285-294; Christoffersen and Marr, 1995 J. Med.\",\n \"Chem.\",\n \"38, 2023-2037).\",\n \"Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA.\",\n \"Such a cleavage event renders the RNA non-functional and abrogates protein expression from that RNA.\",\n \"In this manner, synthesis of a protein associated with a disease state can be selectively inhibited (Warashina et al., 1999, Chemistry and Biology, 6, 237-250).\",\n \"Enzymatic nucleic acid molecules of the invention that are allosterically regulated (“allozymes”) can be used to down-regulate prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR expression.\",\n \"These allosteric enzymatic nucleic acids or allozymes (see for example George et al., U.S. Pat.\",\n \"Nos.\",\n \"5,834,186 and 5,741,679, Shih et al., U.S. Pat.\",\n \"No.\",\n \"5,589,332, Nathan et al., U.S. Pat.\",\n \"No.\",\n \"5,871,914, Nathan and Ellington, International PCT publication No.\",\n \"WO 00/24931, Breaker et al., International PCT Publication Nos.\",\n \"WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No.\",\n \"WO 99/29842) are designed to respond to a signaling agent, for example, mutant and/or wild type protein, other proteins and/or RNAs involved in target gene signal transduction, compounds, metals, polymers, molecules and/or drugs that are targeted to target gene expressing cells etc., which in turn modulates the activity of the enzymatic nucleic acid molecule.\",\n \"In response to interaction with a predetermined signaling agent, the allosteric enzymatic nucleic acid molecule's activity is activated or inhibited such that the expression of a particular target is selectively down-regulated.\",\n \"The target can comprise wild-type protein, mutant protein, and/or a predetermined component of the protein's signal transduction pathway.\",\n \"In a specific example, allosteric enzymatic nucleic acid molecules that are activated by interaction with a RNA encoding a PTGDR protein are used as therapeutic agents in vivo.\",\n \"The presence of RNA encoding the PTGDS protein activates the allosteric enzymatic nucleic acid molecule that subsequently cleaves the RNA encoding a PTGDR protein resulting in the inhibition of PTGDR protein expression.\",\n \"In this manner, cells that express both PTGDS and PTGDR protein are selectively targeted.\",\n \"In another non-limiting example, an allozyme can be activated by a prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR protein, peptide, or mutant polypeptide that causes the allozyme to inhibit the expression of a target gene, by, for example, cleaving RNA encoded by the target gene.\",\n \"In this non-limiting example, the allozyme acts as a decoy to inhibit the function of the target protein and also inhibit the expression of the protein once activated by the prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR protein.\",\n \"Target Sites Targets for useful enzymatic nucleic acid molecules and antisense nucleic acids can be determined as disclosed in Draper et al., WO 93/23569; Sullivan et al., WO 93/23057; Thompson et al., WO 94/02595; Draper et al., WO 95/04818; McSwiggen et al., U.S. Pat.\",\n \"No.\",\n \"5,525,468, and hereby incorporated by reference herein in totality.\",\n \"Other examples include the following PCT applications, which concern inactivation of expression of disease-related genes: WO 95/23225, WO 95/13380, WO 94/02595, incorporated by reference herein.\",\n \"Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art.\",\n \"Enzymatic nucleic acid molecules and antisense to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described.\",\n \"The sequences of human prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR RNAs were screened for optimal enzymatic nucleic acid and antisense target sites using a computer-folding algorithm.\",\n \"Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme, or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified.\",\n \"These sites are shown in Tables III to XXIII (all sequences are 5′ to 3′ in the tables; underlined regions can be any sequence “X” or linker X, the actual sequence is not relevant here).\",\n \"The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of enzymatic nucleic acid molecule.\",\n \"While human sequences can be screened and enzymatic nucleic acid molecule and/or antisense thereafter designed, as discussed in Stinchcomb et al., WO 95/23225, mouse targeted enzymatic nucleic acid molecules can be useful to test efficacy of action of the enzymatic nucleic acid molecule and/or antisense prior to testing in humans.\",\n \"Antisense, siRNA, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified.\",\n \"The nucleic acid molecules are individually analyzed by computer folding (Jaeger et al., 1989 Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 86, 7706) to assess whether the sequences fold into the appropriate secondary structure.\",\n \"Those nucleic acid molecules with unfavorable intramolecular interactions such as between the binding arms and the catalytic core are eliminated from consideration.\",\n \"Varying binding arm lengths can be chosen to optimize activity.\",\n \"Antisense, siRNA, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified and were designed to anneal to various sites in the RNA target.\",\n \"The binding arms are complementary to the target site sequences described above.\",\n \"The nucleic acid molecules were chemically synthesized.\",\n \"The method of synthesis used follows the procedure for normal DNA/RNA synthesis as described below and in Usman et al., 1987 J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc., 109, 7845; Scaringe et al., 1990 Nucleic Acids Res., 18, 5433; and Wincott et al., 1995 Nucleic Acids Res.\",\n \"23, 2677-2684; Caruthers et al., 1992, Methods in Enzymology 211, 3-19.Synthesis of Nucleic Acid Molecules Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive.\",\n \"In this invention, small nucleic acid motifs (“small refers to nucleic acid motifs less than about 100 nucleotides in length, and in one embodiment less than about 80 nucleotides in length, and in another embodiment less than about 50 nucleotides in length; e.g., antisense oligonucleotides, hammerhead or the NCH ribozymes) can be used for exogenous delivery.\",\n \"The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of RNA structure.\",\n \"Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.\",\n \"Oligonucleotides (e.g., antisense GeneBlocs) are synthesized using protocols known in the art as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No.\",\n \"WO 99/54459, Wincott et al., 1995, Nucleic Acids Res.\",\n \"23, 2677-2684, Wincott et al., 1997, Methods Mol.\",\n \"Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat.\",\n \"No.\",\n \"6,001,311.All of these references are incorporated herein by reference.\",\n \"The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.\",\n \"In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45 sec coupling step for 2′-deoxy nucleotides.\",\n \"Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle.\",\n \"Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle.\",\n \"A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl.\",\n \"A 22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl.\",\n \"Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%.\",\n \"Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM I2, 49 mM pyridine, 9% water in THF (PERSEPTIVE™).\",\n \"Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle.\",\n \"S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.\",\n \"Deprotection of the antisense oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq.\",\n \"methylamine (1 mL) at 65° C. for 10 min.\",\n \"After cooling to −20° C., the supernatant is removed from the polymer support.\",\n \"The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant.\",\n \"The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.\",\n \"The method of synthesis used for normal RNA including certain enzymatic nucleic acid molecules follows the procedure as described in Usman et al., 1987, J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res.\",\n \"23, 2677-2684 Wincott et al., 1997, Methods Mol.\",\n \"Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.\",\n \"In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2′-O-methylated nucleotides.\",\n \"Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle.\",\n \"Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle.\",\n \"A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl.\",\n \"A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.\",\n \"Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%.\",\n \"Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I2, 49 mM pyridine, 9% water in THF (PERSEPTIVE™).\",\n \"Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle.\",\n \"S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used.\",\n \"Deprotection of the RNA is performed using either a two-pot or one-pot protocol.\",\n \"For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq.\",\n \"methylamine (1 mL) at 65° C. for 10 min.\",\n \"After cooling to −20° C., the supernatant is removed from the polymer support.\",\n \"The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant.\",\n \"The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.\",\n \"The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA•3HF to provide a 1.4 M HF concentration) and heated to 65° C. After 1.5 h, the oligomer is quenched with 1.5 M NH4HCO3.Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65° C. for 15 min.\",\n \"The vial is brought to r.t. TEA•3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 min.\",\n \"The sample is cooled at −20° C. and then quenched with 1.5 M NH4HCO3.For purification of the trityl-on oligomers, the quenched NH4HCO3 solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA.\",\n \"After washing, the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 min.\",\n \"The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again.\",\n \"The oligonucleotide is then eluted with 30% acetonitrile.\",\n \"Inactive hammerhead ribozymes or binding attenuated control (BAC) oligonucleotides are synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel, K. J., et al., 1992, Nucleic Acids Res., 20, 3252).\",\n \"Similarly, one or more nucleotide substitutions can be introduced in other enzymatic nucleic acid molecules to inactivate the molecule and such molecules can serve as a negative control.\",\n \"The average stepwise coupling yields are typically ≧98% (Wincott et al., 1995 Nucleic Acids Res.\",\n \"23, 2677-2684).\",\n \"Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96 well format, all that is important is the ratio of chemicals used in the reaction.\",\n \"Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No.\",\n \"WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem.\",\n \"8, 204).\",\n \"The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp.\",\n \"Ser.\",\n \"31, 163).\",\n \"Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., Supra, the totality of which is hereby incorporated herein by reference) and are re-suspended in water.\",\n \"The sequences of the nucleic acid molecules, including enzymatic nucleic acid molecules and antisense, that are chemically synthesized, are shown in Tables III-XXIII.\",\n \"The sequences of the enzymatic nucleic acid constructs that are chemically synthesized are complementary to the Substrate sequences shown in Tables III-XXIII.\",\n \"Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the enzymatic nucleic acid (all but the binding arms) is altered to affect activity.\",\n \"The enzymatic nucleic acid construct sequences listed in Tables III-XXIII can be formed of ribonucleotides or other nucleotides or non-nucleotides.\",\n \"Such enzymatic nucleic acid molecules with enzymatic activity are equivalent to the enzymatic nucleic acid molecules described specifically in the Tables.\",\n \"Optimizing Activity of the Nucleic Acid Molecule of the Invention Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases can increase their potency (see e.g., Eckstein et al., International Publication No.\",\n \"WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem.\",\n \"Sci.\",\n \"17, 334; Usman et al., International Publication No.\",\n \"WO 93/15187; and Rossi et al., International Publication No.\",\n \"WO 91/03162; Sproat, U.S. Pat.\",\n \"No.\",\n \"5,334,711; and Burgin et al., supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules herein).\",\n \"Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.\",\n \"(All these publications are hereby incorporated by reference herein).\",\n \"There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy.\",\n \"For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS.\",\n \"17, 34; Usman et al., 1994, Nucleic Acids Symp.\",\n \"Ser.\",\n \"31, 163; Burgin et al., 1996, Biochemistry, 35, 14090).\",\n \"Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No.\",\n \"WO 92/07065; Perrault et al.\",\n \"Nature, 1990, 344, 565-568; Pieken et al.\",\n \"Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem.\",\n \"Sci., 1992, 17, 334-339; Usman et al.\",\n \"International Publication PCT No.\",\n \"WO 93/15187; Sproat, U.S. Pat.\",\n \"No.\",\n \"5,334,711 and Beigelman et al., 1995, J. Biol.\",\n \"Chem., 270, 25702; Beigelman et al., International PCT publication No.\",\n \"WO 97/26270; Beigelman et al., U.S. Pat.\",\n \"No.\",\n \"5,716,824; Usman et al., U.S. Pat.\",\n \"No.\",\n \"5,627,053; Woolf et al., International PCT Publication No.\",\n \"WO 98/13526; Thompson et al., U.S. Ser.\",\n \"No.\",\n \"60/082,404 which was filed on Apr.\",\n \"20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu.\",\n \"Rev.\",\n \"Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg.\",\n \"Med.\",\n \"Chem., 5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein).\",\n \"Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into ribozymes without inhibiting catalysis, and are incorporated by reference herein.\",\n \"In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid molecules of the instant invention.\",\n \"While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorothioate, and/or 5′-methylphosphonate linkages improves stability, too many of these modifications can cause some toxicity.\",\n \"Therefore when designing nucleic acid molecules the amount of these internucleotide linkages should be minimized.\",\n \"The reduction in the concentration of these linkages should lower toxicity resulting in increased efficacy and higher specificity of these molecules.\",\n \"Nucleic acid molecules having chemical modifications that maintain or enhance activity are provided.\",\n \"Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid.\",\n \"Thus, in a cell and/or in vivo the activity may not be significantly lowered.\",\n \"Therapeutic nucleic acid molecules delivered exogenously are optimally stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein.\",\n \"This period of time varies between hours to days depending upon the disease state.\",\n \"Clearly, nucleic acid molecules must be resistant to nucleases in order to function as effective intracellular therapeutic agents.\",\n \"Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995 Nucleic Acids Res.\",\n \"23, 2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19 (incorporated by reference herein) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.\",\n \"In one embodiment, nucleic acid molecules of the invention include one or more G-clamp nucleotides.\",\n \"A G-clamp nucleotide is a modified cytosine analog wherein modifications result in the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc., 120, 8531-8532.A single G-clamp analog substation within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides.\",\n \"The inclusion of such nucleotides in nucleic acid molecules of the invention can enable both enhanced affinity and specificity to nucleic acid targets.\",\n \"Therapeutic nucleic acid molecules (e.g., enzymatic nucleic acid molecules and antisense nucleic acid molecules) delivered exogenously are optimally stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein.\",\n \"This period of time varies between hours to days depending upon the disease state.\",\n \"These nucleic acid molecules should be resistant to nucleases in order to function as effective intracellular therapeutic agents.\",\n \"Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.\",\n \"In another embodiment, the invention features conjugates and/or complexes of nucleic acid molecules targeting prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR.\",\n \"Compositions and conjugates are used to facilitate delivery of molecules into a biological system, such as cells.\",\n \"The conjugates provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention.\",\n \"The present invention encompasses the design and synthesis of novel agents for the delivery of molecules, including but not limited to small molecules, lipids, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes.\",\n \"In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers.\",\n \"These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat.\",\n \"No.\",\n \"5,854,038).\",\n \"Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.\",\n \"The term “biodegradable nucleic acid linker molecule” as used herein, refers to a nucleic acid molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule.\",\n \"The stability of the biodegradable nucleic acid linker molecule can be modulated by using various combinations of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, for example 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides.\",\n \"The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus based linkage, for example a phosphoramidate or phosphodiester linkage.\",\n \"The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.\",\n \"The term “biodegradable” as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.\",\n \"The term “biologically active molecule” as used herein, refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system.\",\n \"Non-limiting examples of biologically active molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siRNA, dsRNA, allozymes, aptamers, decoys and analogs thereof.\",\n \"Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.\",\n \"The term “phospholipid” as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group.\",\n \"For example, a phospholipid can comprise a phosphorus containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.\",\n \"In another embodiment, nucleic acid catalysts having chemical modifications that maintain or enhance enzymatic activity are provided.\",\n \"Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids.\",\n \"Thus, in a cell and/or in vivo the activity of the nucleic acid may not be significantly lowered.\",\n \"As exemplified herein such enzymatic nucleic acids are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al., 1996, Biochemistry, 35, 14090).\",\n \"Such enzymatic nucleic acids herein are said to “maintain” the enzymatic activity of an all RNA ribozyme or all DNA DNAzyme.\",\n \"In another aspect the nucleic acid molecules comprise a 5′ and/or a 3′-cap structure.\",\n \"By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see for example Wincott et al., WO 97/26270, incorporated by reference herein).\",\n \"These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and can help in delivery and/or localization within a cell.\",\n \"The cap can be present at the 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can be present on both terminus.\",\n \"In non-limiting examples, the 5′-cap includes inverted abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Wincott et al., International PCT publication No.\",\n \"WO 97/26270, incorporated by reference herein).\",\n \"In another embodiment the 3′-cap includes, for example 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).\",\n \"By the term “non-nucleotide” is meant any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.\",\n \"The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.\",\n \"An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups.\",\n \"The alkyl group can have, for example, 1 to 12 carbons.\",\n \"In one embodiment of the invention, the alkyl group is a lower alkyl of from 1 to 7 carbons.\",\n \"In another embodiment the alkyl group is 1 to 4 carbons.\",\n \"The alkyl group can be substituted or unsubstituted.\",\n \"When substituted the substituted group(s) can be hydroxyl, cyano, alkoxy, ═O, ═S, NO2 or N(CH3)2, amino, or SH.\",\n \"The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups.\",\n \"The alkenyl group can have, for example, 1 to 12 carbons.\",\n \"In one embodiment of the invention the alkenyl group can be a lower alkenyl of from 1 to 7 carbons.\",\n \"In another embodiment the alkenyl group can be 1 to 4 carbons.\",\n \"The alkenyl group can be substituted or unsubstituted.\",\n \"When substituted the substituted group(s) can be, for example, hydroxyl, cyano, alkoxy, ═O, ═S, NO2, halogen, N(CH3)2, amino, or SH.\",\n \"The term “alkyl” also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups.\",\n \"The alkynyl group can have, for example, 1 to 12 carbons.\",\n \"In one embodiment of the invention, the alkynyl group is a lower alkynyl of from 1 to 7 carbons.\",\n \"In another embodiment of the invention, the alkynyl group is 1 to 4 carbons.\",\n \"The alkynyl group can be substituted or unsubstituted.\",\n \"When substituted the substituted group(s) can be, for example, hydroxyl, cyano, alkoxy, ═O, ═S, NO2 or N(CH3)2, amino or SH.\",\n \"Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups.\",\n \"An “aryl” group refers to an aromatic group which has at least one ring having a conjugated p electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which can be optionally substituted.\",\n \"The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups.\",\n \"An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above).\",\n \"Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms.\",\n \"The carbon atoms are optionally substituted.\",\n \"Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms.\",\n \"Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.\",\n \"An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen.\",\n \"An “ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylaryl or hydrogen.\",\n \"By “nucleotide” is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar.\",\n \"Nucleotides are recognized in the art to include natural bases (standard), and modified bases well known in the art.\",\n \"Such bases are generally located at the 1′ position of a nucleotide sugar moiety.\",\n \"Nucleotides generally comprise a base, sugar and a phosphate group.\",\n \"The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No.\",\n \"WO 92/07065; Usman et al., International PCT Publication No.\",\n \"WO 93/15187; Uhlman & Peyman, supra all are hereby incorporated by reference herein).\",\n \"There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res.\",\n \"22, 2183.Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, for example, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.\",\n \"6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N-6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).\",\n \"By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.\",\n \"By “nucleoside” is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar.\",\n \"Nucleosides are recognized in the art to include natural bases (standard), and modified bases well known in the art.\",\n \"Such bases are generally located at the 1′ position of a nucleoside sugar moiety.\",\n \"Nucleosides generally comprise a base and sugar group.\",\n \"The nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, modified nucleosides, non-natural nucleosides, non-standard nucleosides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No.\",\n \"WO 92/07065; Usman et al., International PCT Publication No.\",\n \"WO 93/15187; Uhlman & Peyman, supra all are hereby incorporated by reference herein).\",\n \"There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res.\",\n \"22, 2183.Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.\",\n \"6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N-6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).\",\n \"By “modified bases” in this aspect is meant nucleoside bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.\",\n \"In one embodiment, the invention features modified enzymatic nucleic acid molecules with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.\",\n \"For a review of oligonucleotide backbone modifications see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39.These references are hereby incorporated by reference herein.\",\n \"By “abasic” is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1′ position, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative (for more details see Wincott et al., International PCT publication No.\",\n \"WO 97/26270).\",\n \"By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1′ carbon of β-D-ribo-furanose.\",\n \"By “modified nucleoside” is meant any nucleotide base that contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.\",\n \"In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′-NH2 or 2′-O—NH2, which can be modified or unmodified.\",\n \"Such modified groups are described, for example, in Eckstein et al., U.S. Pat.\",\n \"No.\",\n \"5,672,695 and Matulic-Adamic et al., WO 98/28317, respectively, which are both incorporated by reference in their entireties.\",\n \"Various modifications to nucleic acid (e.g., antisense and ribozyme) structure can be made to enhance the utility of these molecules.\",\n \"For example, such modifications can enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, including e.g., enhancing penetration of cellular membranes and conferring the ability to recognize and bind to targeted cells.\",\n \"Use of the nucleic acid-based molecules of the invention can lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules (including different enzymatic nucleic acid molecule motifs) and/or other chemical or biological molecules).\",\n \"The treatment of patients with nucleic acid molecules can also include combinations of different types of nucleic acid molecules.\",\n \"Therapies can be devised which include a mixture of enzymatic nucleic acid molecules (including different enzymatic nucleic acid molecule motifs), antisense and/or 2-5A chimera molecules to one or more targets to alleviate symptoms of a disease.\",\n \"Administration of Nucleic Acid Molecules Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed.\",\n \"Akhtar, 1995 which are both incorporated herein by reference.\",\n \"Sullivan et al., PCT WO 94/02595, further describes the general methods for delivery of enzymatic RNA molecules.\",\n \"These protocols can be utilized for the delivery of virtually any nucleic acid molecule.\",\n \"Nucleic acid molecules can be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.\",\n \"The nucleic acid molecules or the invention are administered via pulmonary delivery, such as by inhalation of an aerosol or spray dried formulation administered by an inhalation device or nebulizer.\",\n \"Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump.\",\n \"Other routes of delivery include, but are not limited to oral (tablet or pill form) and/or intrathecal delivery (Gold, 1997, Neuroscience, 76, 1153-1158).\",\n \"Other approaches include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers.\",\n \"For a comprehensive review on drug delivery strategies including CNS delivery, see Ho et al., 1999, Curr.\",\n \"Opin.\",\n \"Mol.\",\n \"Ther., 1, 336-343 and Jain, Drug Delivery Systems: Technologies and Commercial Opportunities, Decision Resources, 1998 and Groothuis et al., 1997, J.\",\n \"NeuroVirol., 3, 387-400.More detailed descriptions of nucleic acid delivery and administration are provided in Sullivan et al., supra, Draper et al., PCT WO93/23569, Beigelman et al., PCT WO99/05094, and Klimuk et al., PCT WO99/04819 all of which have been incorporated by reference herein.\",\n \"Experiments have demonstrated the efficient in vivo uptake of nucleic acids by neurons.\",\n \"As an example of local administration of nucleic acids to nerve cells, Sommer et al., 1998, Antisense Nuc.\",\n \"Acid Drug Dev., 8, 75, describe a study in which a 15mer phosphorothioate antisense nucleic acid molecule to c-fos is administered to rats via microinjection into the brain.\",\n \"Antisense molecules labeled with tetramethylrhodamine-isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by exclusively by neurons thirty minutes post-injection.\",\n \"A diffuse cytoplasmic staining and nuclear staining was observed in these cells.\",\n \"As an example of systemic administration of nucleic acid to nerve cells, Epa et al., 2000, Antisense Nuc.\",\n \"Acid Drug Dev., 10, 469, describe an in vivo mouse study in which beta-cyclodextrin-adamantane-oligonucleotide conjugates were used to target the p75 neurotrophin receptor in neuronally differentiated PC12 cells.\",\n \"Following a two week course of IP administration, pronounced uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells.\",\n \"In addition, a marked and consistent down-regulation of p75 was observed in DRG neurons.\",\n \"Additional approaches to the targeting of nucleic acid to neurons are described in Broaddus et al., 1998, J.\",\n \"Neurosurg., 88(4), 734; Karle et al., 1997, Eur.\",\n \"J.\",\n \"Pharmocol., 340(2/3), 153; Bannai et al., 1998, Brain Research, 784(1,2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199; Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, Brain Res.\",\n \"Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39.Nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells that express NOGO and NOGO receptors for modulation of NOGO and/or NOGO receptor expression.\",\n \"The delivery of nucleic acid molecules of the invention, targeting NOGO and NOGO receptors is provided by a variety of different strategies.\",\n \"Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier.\",\n \"Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers.\",\n \"Furthermore, gene therapy approaches, for example as described in Kaplitt et al., U.S. Pat.\",\n \"No.\",\n \"6,180,613, can be used to express nucleic acid molecules in the CNS.\",\n \"The molecules of the instant invention can be used as pharmaceutical agents.\",\n \"Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, or all of the symptoms) of a disease state in a patient.\",\n \"The negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition.\",\n \"When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed.\",\n \"The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the other compositions known in the art.\",\n \"The present invention also includes pharmaceutically acceptable formulations of the compounds described.\",\n \"These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.\",\n \"A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., local administration or systemic administration, into a cell or patient, including, for example, a human.\",\n \"Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection.\",\n \"Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to).\",\n \"For example, pharmacological compositions injected into the blood stream should be soluble.\",\n \"Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect.\",\n \"By “local administration” is meant in vivo local absorption or accumulation of drugs in the specific tissue, organ, or compartment of the body.\",\n \"Administration routes that can lead to local absorption include, without limitations: inhalation, direct injection, or dermatological applications.\",\n \"By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body.\",\n \"Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.\",\n \"Each of these administration routes expose the desired compound, e.g., nucleic acids, to an accessible diseased tissue.\",\n \"The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size.\",\n \"The use of a liposome or other drug carrier comprising the compounds of the instant invention, for example PEG or phospholipids conjugates, can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES).\",\n \"A nucleic acid formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful.\",\n \"This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells.\",\n \"Both local and systemic administration approaches can be used to administer nucleic acid molecules of the invention for the treatment of asthma or related conditions.\",\n \"In one embodiment, the nucleic acid molecule or formulation comprising the nucleic acid molecule is administered to a patient with an inhaler or nebulizer, providing rapid local uptake of the nucleic acid molecules into relevant pulmonary tissues.\",\n \"In another embodiment, the nucleic acid molecule or formulation comprising the nucleic acid molecule is administered to a patient systemically, for example by intravenous or subcutaneous injection, providing sustained uptake of the nucleic acid molecules into relevant bodily tissues.\",\n \"By pharmaceutically acceptable formulation is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity.\",\n \"Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: PEG conjugated nucleic acids, phospholipid conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into various tissues, for exaple the CNS (Jolliet-Riant and Tillement, 1999, Fundam.\",\n \"Clin.\",\n \"Pharmacol., 13, 16-26); biodegradable polymers, such as poly(DL-lactide-coglycolide) microspheres for sustained release delivery after implantation (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass.\",\n \"; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).\",\n \"Other non-limiting examples of delivery strategies, including CNS delivery of the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm.\",\n \"Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv.\",\n \"Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.All these references are hereby incorporated herein by reference.\",\n \"The invention also features the use of the composition comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes).\",\n \"Nucleic acid molecules of the invention can also comprise covalently attached PEG molecules of various molecular weights.\",\n \"These formulations offer a method for increasing the accumulation of drugs in target tissues.\",\n \"This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al.\",\n \"Chem.\",\n \"Rev.\",\n \"1995, 95, 2601-2627; Ishiwata et al., Chem.\",\n \"Pharm.\",\n \"Bull 1995, 43, 1005-1011).\",\n \"Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim.\",\n \"Biophys.\",\n \"Acta, 1238, 86-90).\",\n \"The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol.\",\n \"Chem.\",\n \"1995, 42, 24864-24870; Choi et al., International PCT Publication No.\",\n \"WO 96/10391; Ansell et al., International PCT Publication No.\",\n \"WO 96/10390; Holland et al., International PCT Publication No.\",\n \"WO 96/10392; all of which are incorporated by reference herein).\",\n \"Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.\",\n \"All of these references are incorporated by reference herein.\",\n \"The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent.\",\n \"Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R.\",\n \"Gennaro edit.\",\n \"1985) hereby incorporated by reference herein.\",\n \"For example, preservatives, stabilizers, dyes and flavoring agents can be provided.\",\n \"These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.\",\n \"In addition, antioxidants and suspending agents can be used.\",\n \"A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, or all of the symptoms) of a disease state.\",\n \"The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize.\",\n \"Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.\",\n \"The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.\",\n \"The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.\",\n \"In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier.\",\n \"One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients.\",\n \"The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.\",\n \"Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations.\",\n \"Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets.\",\n \"These excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.\",\n \"The tablets can be uncoated or they can be coated by known techniques.\",\n \"In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.\",\n \"For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.\",\n \"Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.\",\n \"Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.\",\n \"Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.\",\n \"The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.\",\n \"Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.\",\n \"The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.\",\n \"Sweetening agents and flavoring agents can be added to provide palatable oral preparations.\",\n \"These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.\",\n \"Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.\",\n \"Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above.\",\n \"Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.\",\n \"Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions.\",\n \"The oily phase can be a vegetable oil or a mineral oil or mixtures of these.\",\n \"Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.\",\n \"The emulsions can also contain sweetening and flavoring agents.\",\n \"Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose.\",\n \"Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents.\",\n \"The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension.\",\n \"This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.\",\n \"The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.\",\n \"Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution.\",\n \"In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.\",\n \"For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides.\",\n \"In addition, fatty acids such as oleic acid find use in the preparation of injectables.\",\n \"The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug.\",\n \"These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.\",\n \"Such materials include cocoa butter and polyethylene glycols.\",\n \"Nucleic acid molecules of the invention can be administered parenterally in a sterile medium.\",\n \"The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.\",\n \"Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.\",\n \"Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day).\",\n \"The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration.\",\n \"Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.\",\n \"It is understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.\",\n \"For administration to non-human animals, the composition can also be added to the animal feed or drinking water.\",\n \"It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet.\",\n \"It can also be convenient to present the composition as a premix for addition to the feed or drinking water.\",\n \"The nucleic acid molecules of the present invention can also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect.\",\n \"The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.\",\n \"Alternatively, certain of the nucleic acid molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci., USA 83, 399; Scanlon et al., 1991, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res.\",\n \"Dev., 2, 3-15; Dropulic et al., 1992, J.\",\n \"Virol., 66, 1432-41; Weerasinghe et al., 1991, J.\",\n \"Virol., 65, 5531-4; Ojwang et al., 1992, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45; all of these references are hereby incorporated in their totalities by reference herein).\",\n \"Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector.\",\n \"The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp.\",\n \"Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol.\",\n \"Chem., 269, 25856; all of these references are hereby incorporated in their totalities by reference herein).\",\n \"Gene therapy approaches specific to the CNS are described by Blesch et al., 2000, Drug News Perspect., 13, 269-280; Peterson et al., 2000, Cent.\",\n \"Nerv.\",\n \"Syst.\",\n \"Dis., 485-508; Peel and Klein, 2000, J. Neurosci.\",\n \"Methods, 98, 95-104; Hagihara et al., 2000, Gene Ther., 7, 759-763; and Herrlinger et al., 2000, Methods Mol.\",\n \"Med., 35, 287-312.AAV-mediated delivery of nucleic acid to cells of the nervous system is further described by Kaplitt et al., U.S. Pat.\",\n \"No.\",\n \"6,180,613.In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.\",\n \"The recombinant vectors can be DNA plasmids or viral vectors.\",\n \"Ribozyme expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.\",\n \"The recombinant vectors capable of expressing the nucleic acid molecules can be delivered as described above, and persist in target cells.\",\n \"Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules.\",\n \"Such vectors can be repeatedly administered as necessary.\",\n \"Once expressed, the nucleic acid molecule binds to the target mRNA.\",\n \"Delivery of nucleic acid molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).\",\n \"In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the instant invention is disclosed.\",\n \"The nucleic acid sequence encoding the nucleic acid molecule of the instant invention is operably linked in a manner that allows expression of that nucleic acid molecule.\",\n \"In another aspect the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); c) a nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner that allows expression and/or delivery of said nucleic acid molecule.\",\n \"The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the nucleic acid catalyst of the invention; and/or an intron (intervening sequences).\",\n \"Transcription of the nucleic acid molecule sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III).\",\n \"Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.)\",\n \"present nearby.\",\n \"Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol.\",\n \"Cell.\",\n \"Biol., 10, 4529-37).\",\n \"All of these references are incorporated by reference herein.\",\n \"Several investigators have demonstrated that nucleic acid molecules, such as ribozymes expressed from such promoters can function in mammalian cells (e.g.\",\n \"Kashani-Sabet et al., 1992, Antisense Res.\",\n \"Dev., 2, 3-15; Ojwang et al., 1992, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"U.S.A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566).\",\n \"More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat.\",\n \"No.\",\n \"5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al., International PCT Publication No.\",\n \"WO 96/18736; all of these publications are incorporated by reference herein).\",\n \"The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).\",\n \"In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention, in a manner that allows expression of that nucleic acid molecule.\",\n \"The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner that allows expression and/or delivery of said nucleic acid molecule.\",\n \"In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3′-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said open reading frame and said termination region, in a manner that allows expression and/or delivery of said nucleic acid molecule.\",\n \"In yet another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.\",\n \"In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3′-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.\",\n \"EXAMPLES The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.\",\n \"Example 1 Nucleic Acid Molecules Targeting PTGDS, ADORA1 and PTGDR RNA The invention features novel nucleic acid-based molecules, for example, enzymatic nucleic acid molecules, allozymes, antisense nucleic acids, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNA, dsRNA, siRNA, aptamers, and antisense nucleic acids containing RNA cleaving chemical groups, and methods to modulate gene expression, for example, genes encoding prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), and adenosine receptors (AR) such as adenosine receptor A1, A2a, A2b, and A3.In particular, the instant invention features nucleic-acid based molecules and methods to modulate the expression of PTGDR, PTGDS, and adenosine A1 receptor (ADORA1).\",\n \"In one embodiment, the invention features one or more nucleic acid-based molecules and methods that independently or in combination modulate the expression of gene(s) encoding prostaglandin D2 receptors (PTGDR), prostaglandin D2 synthetase (PTGDS) and adenosine receptors such as ADORA1.Specifically, the present invention features nucleic acid molecules that modulate the expression of prostaglandin D2 receptor (PTGDR) gene, for example Genbank Accession Nos.\",\n \"U31332 and U31099, prostaglandin D2 synthetase (PTGDS) gene, for example Genbank Accession No.\",\n \"NM—000954, and Adenosine A1 receptor (ADORA1), for example Genbank Accession No.\",\n \"NM—000674.In another embodiment, the invention features an enzymatic nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 11666-13262.In yet another embodiment, the invention features an enzymatic nucleic acid molecule comprising at least one binding arm wherein one or more of said binding arms comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs: 4415-5483.In one embodiment, the invention features an antisense nucleic acid molecule comprising a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs: 4415-5483.In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention is adapted to treat asthma.\",\n \"In one embodiment, an enzymatic nucleic acid molecule of the invention has an endonuclease activity to cleave RNA encoded by a PTGDS and/or PTGDR gene.\",\n \"In another embodiment, an enzymatic nucleic acid molecule of the invention is in a hammerhead, Inozyme, Zinzyme, DNAzyme, Amberzyme, or G-cleaver configuration.\",\n \"In another embodiment, an enzymatic nucleic acid molecule of the invention having a hammerhead configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 4415-4641.In yet another embodiment, an enzymatic nucleic acid molecule of invention having a hammerhead configuration comprises a sequence having SEQ ID NOs: 11666-11892.In another embodiment, an enzymatic nucleic acid molecule of the invention having an Inozyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 4642-5017.In yet another embodiment, an enzymatic nucleic acid molecule of invention having an Inozyme configuration comprises a sequence having SEQ ID NOs: 11893-12268.In another embodiment, an enzymatic nucleic acid molecule of the invention having a Zinzyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 5018-5248.In yet another embodiment, an enzymatic nucleic acid molecule of invention having a Zinzyme configuration comprises a sequence having SEQ ID NOs: 12269-12499.In another embodiment, an enzymatic nucleic acid molecule of the invention having a DNAzyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 4415-5294.In yet another embodiment, an enzymatic nucleic acid molecule of invention having a DNAzyme configuration comprises a sequence having SEQ ID NOs: 12500-12842.In another embodiment, an enzymatic nucleic acid molecule of the invention having an Amberzyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 5018-5248, and 5295-5483.In yet another embodiment, an enzymatic nucleic acid molecule of invention having an Amberzyme configuration comprises a sequence having SEQ ID NOs: 12843-13262.In one embodiment, an enzymatic nucleic acid molecule of the invention comprises between 8 and 100 bases complementary to the RNA of PTGDS, ADORA1 and/or PTGDR gene.\",\n \"In another embodiment, an enzymatic nucleic acid molecule of the invention comprises between 14 and 24 bases complementary to a RNA molecule of a PTGDS or PTGDR gene.\",\n \"In one embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention is chemically synthesized.\",\n \"In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one 2′-sugar modification.\",\n \"In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one nucleic acid base modification.\",\n \"In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one phosphate backbone modification.\",\n \"In one embodiment, the invention features a mammalian cell, for example a human cell, including the enzymatic nucleic acid molecule of the invention.\",\n \"In another embodiment, the invention features a method of reducing PTGDS, ADORA1 and/or PTGDR expression or activity in a cell, comprising contacting the cell with an enzymatic nucleic acid molecule of the invention, under conditions suitable for the reduction.\",\n \"In another embodiment, the invention features a method of reducing PTGDS, ADORA1 and/or PTGDR expression or activity in a cell, comprising the step of contacting the cell with an antisense nucleic acid molecule of the invention under conditions suitable for the reduction.\",\n \"In yet another embodiment, the invention features a method of treatment of a patient having a condition associated with the level of PTGDS, ADORA1 and/or PTGDR, comprising contacting cells of the patient with an enzymatic nucleic acid molecule of the invention, under conditions suitable for the treatment.\",\n \"In one embodiment, the invention features a method of treatment of a patient having a condition associated with the level of PTGDS, ADORA1 and/or PTGDR, comprising contacting cells of the patient with an antisense nucleic acid molecule of the invention, under conditions suitable for the treatment.\",\n \"In another embodiment, a method of treatment of a patient having a condition associated with the level of PTGDS, ADORA1 and/or PTGDR is featured, wherein the method further comprises the use of one or more drug therapies under conditions suitable for the treatment.\",\n \"For example, in one embodiment, the invention features a method for treatment of asthma, allergic rhinitis, or atopic dermatitis under conditions suitable for the treatment.\",\n \"In another embodiment, the invention features a method of cleaving a RNA molecule of PTGDS, ADORA1 and/or PTGDR gene comprising contacting an enzymatic nucleic acid molecule of the invention with a RNA molecule of a PTGDS, ADORA1 and/or PTGDR gene under conditions suitable for the cleavage, for example, wherein the cleavage is carried out in the presence of a divalent cation, such as Mg2+.\",\n \"In one embodiment, an enzymatic nucleic acid molecule of the invention comprises a cap structure, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end of the enzymatic nucleic acid molecule.\",\n \"In another embodiment, an antisense nucleic acid molecule of the invention comprises a cap structure, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end of the antisense nucleic acid molecule.\",\n \"In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one enzymatic nucleic acid molecule of the invention, in a manner which allows expression of the nucleic acid molecule.\",\n \"In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.\",\n \"In yet another embodiment, the expression vector of the invention further comprises a sequence for an antisense nucleic acid molecule complementary to a RNA molecule of a PTGDS, ADORA1 and/or PTGDR gene.\",\n \"In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more enzymatic nucleic acid molecules, which can be the same or different.\",\n \"In another embodiment, the invention features a method for treatment of asthma, allergic rhinitis, or atopic dermatitis, comprising administering to a patient an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention, under conditions suitable for the treatment, including administering to the patient one or more other therapies, for example, inhalant anti-inflammatories, bronchodilators, adenosine inhibitors and adenosine A1 receptor inhibitors.\",\n \"In one embodiment, the method of treatment features an enzymatic nucleic acid molecule or antisense nucleic acid molecule of the invention comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification, such as a 3′-3′ inverted abasic moiety.\",\n \"In another embodiment, an enzymatic nucleic acid molecule or antisense nucleic acid molecule of the invention further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.\",\n \"In another embodiment, the invention features a method of administering to a mammal, for example a human, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention, comprising contacting the mammal with the nucleic acid molecule under conditions suitable for the administration, for example, in the presence of a delivery reagent such as a lipid, cationic lipid, phospholipid, or liposome.\",\n \"In yet another embodiment, the invention features a method of administering to a mammal an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention in conjunction with a therapeutic agent, comprising contacting the mammal, for example a human, with the nucleic acid molecule and the therapeutic agent under conditions suitable for the administration.\",\n \"In one embodiment, the invention features the use of an enzymatic nucleic acid molecule, which can be in a hammerhead, NCH, G-cleaver, Amberzyme, Zinzyme, and/or DNAzyme motif, to down-regulate the expression of a PTGDS, an ADORA1 and/or a PTGDR gene.\",\n \"The enzymatic nucleic acid molecule that cleave the specified sites in PTGDS, ADORA1 and PTGDR-specific RNAs represent a novel therapeutic approach to treat a variety of allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of PTGDS, ADORA1 and/or PTGDR expression.\",\n \"In one embodiment, a nucleic acid molecule that modulates, for example, down-regulates, PTGDS replication or expression comprises between 8 and 100 bases complementary to a RNA molecule of PTGDS.\",\n \"In another embodiment, a nucleic acid molecule that modulates PTGDS replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of PTGDS.\",\n \"In another embodiment, a nucleic acid molecule that modulates, for example, down-regulates, PTGDR replication or expression comprises between 8 and 100 bases complementary to a RNA molecule of PTGDR.\",\n \"In another embodiment, a nucleic acid molecule that modulates PTGDR replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of PTGDR.\",\n \"In another embodiment, a nucleic acid molecule that modulates, for example, down-regulates, ADORA1 replication or expression comprises between 8 and 100 bases complementary to a RNA molecule of ADORA1.In another embodiment, a nucleic acid molecule that modulates ADORA1 replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of ADORA1.The invention provides a method for producing a class of nucleic acid-based gene modulating agents that exhibit a high degree of specificity for the RNA of a desired target.\",\n \"For example, the enzymatic nucleic acid molecule is can be targeted to a highly conserved sequence region of target RNAs encoding PTGDS, ADORA1 and/or PTGDR (e.g., PTGDS, ADORA1 and/or PTGDR genes) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention.\",\n \"Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required.\",\n \"Alternatively, the nucleic acid molecules (e.g., ribozymes and antisense) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.\",\n \"Nucleic acid-based inhibitors of PTGDS, ADORA1 and PTGDR expression are useful for the prevention and/or treatment of allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and any other diseases or conditions that are related to or will respond to the levels of PTGDS, ADORA1 and/or PTGDR in a cell or tissue, alone or in combination with other therapies.\",\n \"The reduction of PTGDS, ADORA1 and/or PTGDR expression (specifically PTGDS, ADORA1 and/or PTGDR gene RNA levels) and thus reduction in the level of the respective protein relieves, to some extent, the symptoms of the disease or condition.\",\n \"The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above.\",\n \"For example, to treat a disease or condition associated with the levels of PTGDS, ADORA1 and/or PTGDR, the patient can be treated, or other appropriate cells can be treated, as is evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.\",\n \"In a further embodiment, the described molecules, such as antisense or enzymatic nucleic acid molecules, can be used in combination with other known treatments to treat conditions or diseases discussed above.\",\n \"For example, the described molecules can be used in combination with one or more known therapeutic agents to treat allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of PTGDS, ADORA1 and/or PTGDR expression.\",\n \"In another embodiment, the invention features nucleic acid-based inhibitors (e.g., enzymatic nucleic acid molecules (e.g., ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of genes (e.g., PTGDS, ADORA1 and/or PTGDR) capable of progression and/or maintenance allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of PTGDS, ADORA1 and/or PTGDR expression.\",\n \"Identification of Potential Target Sites in Human PTGDS, ADORA1 and PTGDR RNA The sequence of human PTGDS, ADORA1 and PTGDR genes are screened for accessible sites using a computer-folding algorithm.\",\n \"Regions of the RNA that do not form secondary folding structures and contained potential enzymatic nucleic acid molecule and/or antisense binding/cleavage sites are identified.\",\n \"The sequences of PTGDR binding/cleavage sites are shown in Tables XIX-XXIII.\",\n \"Selection of Enzymatic Nucleic Acid Cleavage Sites in Human PTGDS, ADORA1 and PTGDR RNA Enzymatic nucleic acid molecule target sites are chosen by analyzing sequences of Human PTGDS (Genbank accession No: NM—000954), ADORA1 (Genbank accession No: NM—000674) and PTGDR gene (Genbank accession Nos: U31332 and U31099) and prioritizing the sites on the basis of folding.\",\n \"Enzymatic nucleic acid molecules are designed that can bind each target and are individually analyzed by computer folding (Christoffersen et al., 1994 J. Mol.\",\n \"Struc.\",\n \"Theochem, 311, 273; Jaeger et al., 1989, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 86, 7706) to assess whether the enzymatic nucleic acid molecule sequences fold into the appropriate secondary structure.\",\n \"Those enzymatic nucleic acid molecules with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration.\",\n \"As noted below, varying binding arm lengths can be chosen to optimize activity.\",\n \"Generally, at least 4 bases on each arm are able to bind to, or otherwise interact with, the target RNA.\",\n \"Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient Cleavage and/or Blocking of PTGDS, ADORA1 and PTGDR RNA Enzymatic nucleic acid molecules and antisense constructs are designed to anneal to various sites in the RNA message.\",\n \"The binding arms of the enzymatic nucleic acid molecules are complementary to the target site sequences described above, while the antisense constructs are fully complementary to the target site sequences described above.\",\n \"The enzymatic nucleic acid molecules and antisense constructs were chemically synthesized.\",\n \"The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.\",\n \"The average stepwise coupling yields were typically ≧98%.\",\n \"Enzymatic nucleic acid molecules and antisense constructs are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol.\",\n \"180, 51).\",\n \"Enzymatic nucleic acid molecules and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and are resuspended in water.\",\n \"The sequences of the chemically synthesized enzymatic nucleic acid molecules used in this study are shown below in Tables XIX-XXIII.\",\n \"The sequences of the chemically synthesized antisense constructs used in this study are complementary sequences to the Substrate sequences shown below as in Tables XIX-XXIII.\",\n \"Enzymatic Nucleic Acid Molecule Cleavage of PTGDS, ADORA1 and PTGDR RNA Target In Vitro Enzymatic nucleic acid molecules targeted to the human PTGDS, ADORA1 and PTGDR RNA are designed and synthesized as described above.\",\n \"These enzymatic nucleic acid molecules can be tested for cleavage activity in vitro, for example, using the following procedure.\",\n \"The target sequences and the nucleotide location within the PTGDR RNA are given in Tables XIX-XXIII.\",\n \"Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid molecule cleavage assay is prepared by in vitro transcription in the presence of [α-32P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification.\",\n \"Alternately, substrates are 5′-32P-end labeled using T4 polynucleotide kinase enzyme.\",\n \"Assays are performed by pre-warming a 2× concentration of purified enzymatic nucleic acid molecule in enzymatic nucleic acid molecule cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37° C., 10 mM MgCl2) and the cleavage reaction was initiated by adding the 2× enzymatic nucleic acid molecule mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer.\",\n \"As an initial screen, assays are carried out for 1 hour at 37° C. using a final concentration of either 40 nM or 1 mM enzymatic nucleic acid molecule, i.e., enzymatic nucleic acid molecule excess.\",\n \"The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95° C. for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel.\",\n \"Substrate RNA and the specific RNA cleavage products generated by enzymatic nucleic acid molecule cleavage are visualized on an autoradiograph of the gel.\",\n \"The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.\",\n \"In Vivo Models Used to Evaluate the Down-Regulation of PTGDS, ADORA1 and PTGDR Gene Expression Animal Models Evaluating the efficacy of anti-PTGDS, ADORA-1 and/or PTGDR agents in animal models is an important prerequisite to human clinical trials.\",\n \"Matsuoka et al., 2000, Science, 287, 2012-2016, describe a useful asthma animal model having generating mice deficient in the PTGDR receptor.\",\n \"Sensitization and aerosol challenge of homozygous (PTGDR−/−) mice with ovalbumin was shown to induce increases in the serum concentration of immunoglobin E (IgE), an allergic mediator that activates mast cells, similar to wild-type mice subjected to the same conditions.\",\n \"The concentration of TH2 cytokines and the degree of lymphocyte lung infiltration in the OVA challenged PTGDR −/− mice was shown to be greatly reduced compared to wild type mice.\",\n \"In addition, the PTGDR −/− mice showed only marginal eosinophil infiltration and failed to develop airway hyperreactivity.\",\n \"Similarly, this model can be used to evaluate mice that are treated with nucleic acid molecules of the invention and can furthermore be used as a positive control in determining the response of mice treated with nucleic acid molecules of the invention by using such factors as airway obstruction, lung capacity, and bronchiolar alveolar lavage (BAL) fluid in the evaluation.\",\n \"Cell Culture Two human cell lines, NPE cells and NCB-20 cells are known to express PTGDR.\",\n \"Cloned human PTGDR has been expressed in CHO and COS7 cells and used in various studies.\",\n \"These PTGDR expressing lung cell lines can be used in cell culture assays to evaluate nucleic acid molecules of the invention.\",\n \"A primary endpoint in these experiments would be the RT-PCR analysis of PTGDR mRNA expression in PTGDR expressing cells.\",\n \"In addition, ligand binding assays can be developed where binding of PTGDS can be evaluated in response to treatment with nucleic acid molecules of the invention.\",\n \"Indications The present body of knowledge in PTGDS, ADORA1 and PTGDR research indicates the need for methods to assay PTGDS, ADORA1 and PTGDR activity and for compounds that can regulate PTGDS, ADORA1 and PTGDR expression for research, diagnostic, and therapeutic use.\",\n \"As described herein, the nucleic acid molecules of the present invention can be used in assays to diagnose disease state related of PTGDS, ADORA1 and/or PTGDR levels.\",\n \"In addition, the nucleic acid molecules can be used to treat disease state related to PTGDS, ADORA1 and/or PTGDR levels.\",\n \"Particular degenerative and disease states that can be associated with PTGDS, ADORA1 and PTGDR levels include, but are not limited to allergic diseases and conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and any other diseases or conditions that are related to or will respond to the levels of PTGDS, ADORA1 and/or PTGDR in a cell or tissue, alone or in combination with other therapies.\",\n \"The use of anti-inflammatories, bronchodilators, adenosine inhibitors and adenosine A1 receptor inhibitors are examples of other treatments or therapies can be combined with the nucleic acid molecules of the invention.\",\n \"Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g.\",\n \"enzymatic nucleic acid molecules and antisense molecules) are hence within the scope of the instant invention.\",\n \"Diagnostic Uses The nucleic acid molecules of this invention (e.g., enzymatic nucleic acid molecules) can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of PTGDS, ADORA1 and/or PTGDR RNA in a cell.\",\n \"The close relationship between enzymatic nucleic acid molecule activity and the structure of the target RNA allows the detection of mutations in any region of the molecule that alters the base-pairing and three-dimensional structure of the target RNA.\",\n \"By using multiple enzymatic nucleic acid molecules described in this invention, one can map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues.\",\n \"Cleavage of target RNAs with enzymatic nucleic acid molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease.\",\n \"In this manner, other genetic targets can be defined as important mediators of the disease.\",\n \"These experiments can lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules and/or other chemical or biological molecules).\",\n \"Other in vitro uses of enzymatic nucleic acid molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with PTGDS, ADORA1 or PTGDR-related condition.\",\n \"Such RNA is detected by determining the presence of a cleavage product after treatment with an enzymatic nucleic acid molecule using standard methodology.\",\n \"In a specific example, enzymatic nucleic acid molecules which cleave only wild-type or mutant forms of the target RNA are used for the assay.\",\n \"The first enzymatic nucleic acid molecule is used to identify wild-type RNA present in the sample and the second enzymatic nucleic acid molecule is used to identify mutant RNA in the sample.\",\n \"As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both enzymatic nucleic acid molecules to demonstrate the relative enzymatic nucleic acid molecule efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species.\",\n \"The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population.\",\n \"Thus each analysis requires two enzymatic nucleic acid molecules, two substrates and one unknown sample which is combined into six reactions.\",\n \"The presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel.\",\n \"It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells.\",\n \"The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., PTGDS/PTGDR) is adequate to establish risk.\",\n \"If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis.\",\n \"Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.\",\n \"The use of enzymatic nucleic acid molecules in diagnostic applications contemplated by the instant invention is described, for example, in George et al., U.S. Pat.\",\n \"Nos.\",\n \"5,834,186 and 5,741,679, Shih et al., U.S. Pat.\",\n \"No.\",\n \"5,589,332, Nathan et al., U.S. Pat.\",\n \"No.\",\n \"5,871,914, Nathan and Ellington, International PCT publication No.\",\n \"WO 00/24931, Breaker et al., International PCT Publication Nos.\",\n \"WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No.\",\n \"WO 99/29842.Example 2 Nucleic Acid Inhibition of NOGO and NOGO Receptor Target RNA The invention features novel nucleic acid-based molecules [e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, decoy RNA, aptamers, siRNA, antisense nucleic acids containing RNA cleaving chemical groups] and methods to modulate gene expression, for example, genes encoding certain myelin proteins that inhibit or are involved in the inhibition of neurite growth, including axonal regeneration in the CNS.\",\n \"In particular, the instant invention features nucleic-acid based techniques to inhibit the expression of NOGO-A (Accession No.\",\n \"AJ251383), NOGO-B (Accession No.\",\n \"AJ251384), and/or NOGO-C (Accession No.\",\n \"AJ251385), NOGO-66 receptor (Accession No AF283463, Fournier et al., 2001, Nature, 409, 341-346), NI-35, NI-220, and/or NI-250, myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945).\",\n \"In a preferred embodiment, the invention features the use of one or more of the nucleic acid-based techniques independently or in combination to inhibit the expression or function of the gene(s) encoding NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors.\",\n \"Specifically, the invention features the use of nucleic acid-based techniques to specifically inhibit the expression of NOGO gene (Genbank Accession No.\",\n \"AB020693) and NOGO-66 receptor (Genbank Accession No.\",\n \"AF283463).\",\n \"The nucleic acid molecules that interact with NOGO and NOGO receptor-specific RNAs represent a novel therapeutic approach to treat a variety of pathologic indications, including but not limited to CNS injury and cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and/or other neurodegenerative disease states which respond to the modulation of NOGO and NOGO receptor expression.\",\n \"In one embodiment, a nucleic acid molecule that inhibits NOGO and/or NOGO receptor replication or expression can comprise between 12 and 100 bases complementary to a RNA molecule of NOGO or NOGO receptor.\",\n \"In another embodiment, a nucleic acid molecule that inhibits NOGO or NOGO receptor replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of NOGO or NOGO receptor.\",\n \"In one embodiment the invention provides a method for producing a class of nucleic acid-based gene inhibiting agents which exhibit a high degree of specificity for the RNA of a desired target.\",\n \"For example, the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of target RNAs encoding NOGO-A, NOGO-B, NOGO-C and/or receptor proteins (specifically NOGO and NOGO receptor genes) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention.\",\n \"Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required.\",\n \"Alternatively, the nucleic acid molecules (e.g., ribozymes and antisense) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.\",\n \"The nucleic acid-based inhibitors of NOGO and NOGO receptor expression are useful for the prevention and/or treatment of diseases and conditions such CNS injury, cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, muscular dystrophy and any other diseases or conditions that are related to or will respond to the levels of NOGO and/or NOGO receptor in a cell or tissue, alone or in combination with other therapies.\",\n \"In addition, NOGO and/or NOGO receptor inhibition can be used as a therapeutic target for abrogating CNS neuronal growth inhibition; a situation that can selectively regenerate damaged or lesioned CNS tissue to restore specific reflex and/or locomotor functions.\",\n \"In a further embodiment, the described molecules, such as antisense, siRNA or ribozymes, can be used in combination with other known treatments to treat conditions or diseases discussed above.\",\n \"For example, the described molecules can be used in combination with one or more known therapeutic agents to treat CNS injury, spinal cord injury, cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and/or other neurodegenerative disease states which respond to the modulation of NOGO and/or NOGO receptor expression.\",\n \"In another embodiment, the invention features nucleic acid-based inhibitors (e.g., enzymatic nucleic acid molecules (eg; ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of genes (e.g., NOGO and/or NOGO receptor) capable of progression and/or maintenance of CNS injury, spinal cord injury, cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and/or other neurodegenerative disease states which respond to the modulation of NOGO and/or NOGO receptor expression.\",\n \"The lack of axon regeneration capacity in the adult CNS manifests as a limiting factor in the treatment of CNS injury, cerebrovascular accident (CVA, stroke), chemotherapy-induced neuropathy, and possibly in neurodegenerative diseases such as Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, and/or muscular dystrophy.\",\n \"Neuron growth inhibition results from physical barriers imposed by glial scars, a lack of neurotrophic factors, and growth-inhibitory molecules associated with myelin.\",\n \"The abrogation of neurite growth inhibition creates the potential to treat conditions for which there is currently no definitive medical intervention.\",\n \"The inhibition of NOGO (Genbank Accession No AB020693) and NOGO-66 receptor (Genbank Accession No.\",\n \"AF283463) is demonstrated in the following examples.\",\n \"Identification of Potential Target Sites in Human NOGO RNA The sequence of human NOGO and NOGO receptor genes are screened for accessible sites using a computer-folding algorithm.\",\n \"Regions of the RNA that do not form secondary folding structures and contained potential enzymatic nucleic acid molecule and/or antisense binding/cleavage sites are identified.\",\n \"The sequences of these binding/cleavage sites are shown in Tables III-VII.\",\n \"Selection of Enzymatic Nucleic Acid Cleavage Sites in Human NOGO and NOGO Receptor RNA Enzymatic nucleic acid molecule target sites are chosen by analyzing sequences of Human NOGO (Genbank accession No: AB020693) and prioritizing the sites on the basis of folding.\",\n \"Enzymatic nucleic acid molecules are designed that can bind each target and are individually analyzed by computer folding (Christoffersen et al., 1994 J. Mol.\",\n \"Struc.\",\n \"Theochem, 311, 273; Jaeger et al., 1989, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 86, 7706) to assess whether the enzymatic nucleic acid molecule sequences fold into the appropriate secondary structure.\",\n \"Those enzymatic nucleic acid molecules with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration.\",\n \"As noted below, varying binding arm lengths can be chosen to optimize activity.\",\n \"Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.\",\n \"Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient Cleavage and/or Blocking of NOGO and NOGO Receptor RNA Enzymatic nucleic acid molecules and antisense constructs are designed to anneal to various sites in the RNA message.\",\n \"The binding arms of the enzymatic nucleic acid molecules are complementary to the target site sequences described above, while the antisense constructs are fully complimentary to the target site sequences described above.\",\n \"The enzymatic nucleic acid molecules and antisense constructs were chemically synthesized.\",\n \"The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.\",\n \"The average stepwise coupling yields were typically >98%.\",\n \"Enzymatic nucleic acid molecules and antisense constructs are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol.\",\n \"180, 51).\",\n \"Enzymatic nucleic acid molecules and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and are resuspended in water.\",\n \"The sequences of the chemically synthesized enzymatic nucleic acid molecules used in this study are shown below in Table III-VII.\",\n \"The sequences of the chemically synthesized antisense constructs used in this study are complimentary sequences to the Substrate sequences shown below as in Table III-VII.\",\n \"Enzymatic Nucleic Acid Molecule Cleavage of NOGO and NOGO Receptor RNA Target In Vitro Enzymatic nucleic acid molecules targeted to the human NOGO RNA are designed and synthesized as described above.\",\n \"These enzymatic nucleic acid molecules can be tested for cleavage activity in vitro, for example, using the following procedure.\",\n \"The target sequences and the nucleotide location within the NOGO receptor RNA are given in Tables III-VII.\",\n \"Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid molecule cleavage assay is prepared by in vitro transcription in the presence of [α-32P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification.\",\n \"Alternately, substrates are 5′-32P-end labeled using T4 polynucleotide kinase enzyme.\",\n \"Assays are performed by pre-warming a 2× concentration of purified enzymatic nucleic acid molecule in enzymatic nucleic acid molecule cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37° C., 10 mM MgCl2) and the cleavage reaction was initiated by adding the 2× enzymatic nucleic acid molecule mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer.\",\n \"As an initial screen, assays are carried out for 1 hour at 37° C. using a final concentration of either 40 nM or 1 mM enzymatic nucleic acid molecule, i.e., enzymatic nucleic acid molecule excess.\",\n \"The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95° C. for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel.\",\n \"Substrate RNA and the specific RNA cleavage products generated by enzymatic nucleic acid molecule cleavage are visualized on an autoradiograph of the gel.\",\n \"The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.\",\n \"Nucleic Acid Inhibition of NOGO and NOGO Receptor Target RNA In Vivo Nucleic acid molecules targeted to the human NOGO and NOGO receptor RNA are designed and synthesized as described above.\",\n \"These nucleic acid molecules can be tested for cleavage activity in vivo, for example using the procedures described below.\",\n \"The target sequences and the nucleotide location within the NOGO receptor RNA are given in Tables III-VII.\",\n \"Cell Culture Spillmann et al., 1998, J. Biol.\",\n \"Chem., 273, 19283-19293, describe the purification and biochemical characterization of a high molecular mass protein of bovine spinal cord myelin (bNI-220) which exerts potent inhibition of neurite outgrowth of NGF-primed PC12 cells and chick DRG cells.\",\n \"This protein can be used to inhibit spreading of 3T3 fibroblasts and to induce collapse of chick DRG growth cones.\",\n \"The monoclonal antibody, mAb IN-1, can be used to fully neutralize the inhibitory activity of bNI-220, which is a presumed NOGO gene product.\",\n \"As such, nucleic acid molecules of the instant invention directed at the inhibition of NOGO expression can be used in place of mAb IN-1 in studying the inhibition of bNI-220 in cell culture experiments described in detail by Spillmann et al., supra.\",\n \"Criteria used in these experiments include the evaluation of spreading behavior of 3T3 fibroblasts, the neurite outgrowth response of PC12 cells, and the growth cone motility of chick DRG growth cones.\",\n \"Similarly, nucleic acid molecules of the instant invention that target NOGO or NOGO receptors can be used to evaluate inhibition of NOGO mediated activity in these cell types using the criteria described above.\",\n \"Fournier et al., 2001, Nature, 409, 341 describe a mouse clone of the NOGO-66 receptor which is expressed in non-neuronal COS-7 cells.\",\n \"The transfected COS-7 cell line expresses NOGO-66 receptor protein on the cell surface.\",\n \"An antiserum developed to the NOGO-66 receptor can be used to specifically stain NOGO-66 receptor expressing cells by immunohistochemical staining.\",\n \"As such, an assay for screening nucleic acid-based inhibitors of NOGO-66 receptor expression is provided.\",\n \"Animal Models Bregman et al., 1995, Nature, 378, 498-501 and Z'Graggen et al., 1998, J. Neuroscience, 18, 4744, describe a rat based system for evaluating the role of myelin-associated neurite growth inhibitory proteins in vivo.\",\n \"Young adult Lewis rats receive a mid-thoracic microsurgical spinal cord lesion or a unilateral pyramidotomy.\",\n \"These animals are then treated with mAb IN-1 secreting hybridoma cell explants.\",\n \"A control population receive hybridoma explants which secrete horsreradish peroxidase (HRP) antibodies.\",\n \"Cyclosporin is used during the treatment period to allow hybridoma survival.\",\n \"Additional control rats receive either the spinal cord lesion without any further treatment or no lesion.\",\n \"After a 4-6 week recovery period, behavioral training is followed by the quantitative analysis of reflex and locomotor function.\",\n \"IN-1 treated animals demonstrate growth of corticospinal axons around the lesion site and into the spinal cord which persist past the longest time point of analysis (12 weeks).\",\n \"Furthermore, both reflex and locomotor function, including the functional recovery of fine motor control, is restored in IN-1 treated animals.\",\n \"As such, a robust animal model as described by Bregman et al.\",\n \"supra and Z'Graggen et al., supra, can be used to evaluate nucleic acid molecules of the instant invention when used in place of or in conjunction with mAb IN-1 toward use as modulators of neurite growth inhibitor function (eg.\",\n \"NOGO and NOGO receptor) in vivo.\",\n \"Indications The nucleic acids of the present invention can be used to treat a patient having a condition associated with the level of NOGO or NOGO receptor.\",\n \"One method of treatment comprises contacting cells of a patient with a nucleic acid molecule of the present invention, under conditions suitable for said treatment.\",\n \"Delivery methods and other methods of administration have been discussed herein and are commonly known in the art.\",\n \"Particular degenerative and disease states that can be associated with NOGO and NOGO receptor expression modulation include, but are not limited to, CNS injury, specifically spinal cord injury, cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and/or other neurodegenerative disease states which respond to the modulation of NOGO and NOGO receptor expression.\",\n \"The present body of knowledge in NOGO research indicates the need for methods to assay NOGO activity and for compounds that can regulate NOGO expression for research, diagnostic, and therapeutic use.\",\n \"Other treatment methods comprise contacting cells of a patient with a nucleic acid molecule of the present invention and further comprise the use of one or more drug therapies under conditions suitable for said treatment.\",\n \"The use of monoclonal antibody (eg; mAb IN-1) treatment, growth factors, antiinflammatory compounds, for example methylprednisolone, calcium blockers, apoptosis inhibiting compounds, for example GM-1 ganglioside, and physical therapies, for example treadmill therapy, are all non-limiting examples of methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g.\",\n \"ribozymes and antisense molecules) of the instant invention.\",\n \"Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g.\",\n \"ribozymes and antisense molecules) are hence within the scope of the instant invention.\",\n \"Diagnostic Uses The nucleic acid molecules of this invention (e.g., enzymatic nucleic acid molecules) can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of NOGO and/or NOGO receptor RNA in a cell.\",\n \"The close relationship between enzymatic nucleic acid molecule activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA.\",\n \"By using multiple enzymatic nucleic acid molecules described in this invention, one can map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues.\",\n \"Cleavage of target RNAs with enzymatic nucleic acid molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease.\",\n \"In this manner, other genetic targets can be defined as important mediators of the disease.\",\n \"These experiments can lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules and/or other chemical or biological molecules).\",\n \"Other in vitro uses of enzymatic nucleic acid molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with NOGO-related condition.\",\n \"Such RNA is detected by determining the presence of a cleavage product after treatment with a enzymatic nucleic acid molecule using standard methodology.\",\n \"In a specific example, enzymatic nucleic acid molecules which cleave only wild-type or mutant forms of the target RNA are used for the assay.\",\n \"The first enzymatic nucleic acid molecule is used to identify wild-type RNA present in the sample and the second enzymatic nucleic acid molecule is used to identify mutant RNA in the sample.\",\n \"As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both enzymatic nucleic acid molecules to demonstrate the relative enzymatic nucleic acid molecule efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species.\",\n \"The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population.\",\n \"Thus each analysis requires two enzymatic nucleic acid molecules, two substrates and one unknown sample which is combined into six reactions.\",\n \"The presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel.\",\n \"It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells.\",\n \"The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., NOGO) is adequate to establish risk.\",\n \"If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis.\",\n \"Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.\",\n \"The use of enzymatic nucleic acid molecules in diagnostic applications contemplated by the instant invention is more fully described in George et al., U.S. Pat.\",\n \"Nos.\",\n \"5,834,186 and 5,741,679, Shih et al., U.S. Pat.\",\n \"No.\",\n \"5,589,332, Nathan et al., U.S. Pat.\",\n \"No.\",\n \"5,871,914, Nathan and Ellington, International PCT publication No.\",\n \"WO 00/24931, and Sullenger et al., International PCT publication No.\",\n \"WO 99/29842.Example 3 Nucleic Acid Inhibition of IKK-Gamma and PKR Target RNA The invention features nucleic acid molecules, for example enzymatic nucleic acid molecules, antisense nucleic acid molecules, 2,5-A chimeras, decoys, siRNA, triplex oligonucleotides, siRNA and/or aptamers, and methods to modulate gene expression, for example, genes encoding a member of the IκB kinase IKK complex, such as IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKKγ) and/or a protein kinase PKR protein.\",\n \"In particular, the instant invention features nucleic-acid based molecules and methods to modulate the expression of IKK-gamma (IKKγ) and protein kinase PKR.\",\n \"The invention features one or more nucleic acid-based molecules and methods that independently or in combination modulate the expression of gene(s) encoding a member of the IκB kinase IKK complex or PKR.\",\n \"In particular embodiments, the invention features nucleic acid-based molecules and methods that modulate the expression of a member of the IκB kinase IKK complex, for example IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKKγ) and/or a protein kinase PKR protein, such as IKK-alpha (IKK1) gene (Genbank Accession No.\",\n \"NM—001278); IKK-beta (IKK2) gene, for example (Genbank Accession No.\",\n \"AF080158), IKK-gamma (IKKγ) gene, for example (Genbank Accession No.\",\n \"NM—003639), and protein kinase PKR gene, for example (Genbank Accession No.\",\n \"NM—002759).\",\n \"In one embodiment, an enzymatic nucleic acid molecule of the invention comprises a sequence selected from the group consisting of SEQ ID NOs.\",\n \"7056-7249.In another embodiment, an enzymatic nucleic acid molecule of the invention comprises at least one binding arm wherein one or more of said binding arms comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.\",\n \"1024-4414.In another embodiment, an antisense nucleic acid molecule or siRNA molecule of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.\",\n \"1024-4414.In another embodiment, an nucleic acid molecule of the invention is adapted to treat cancer.\",\n \"In yet another embodiment, an enzymatic nucleic acid molecule of the invention has an endonuclease activity to cleave RNA having IKK-gamma or PKR sequence.\",\n \"In one embodiment, an enzymatic nucleic acid molecule of the invention is in an Inozyme, Zinzyme, G-cleaver, Amberzyme, DNAzyme, or Hammerhead configuration.\",\n \"In another embodiment, an Inozyme of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.\",\n \"1218-1721 and 3051-3549.In another embodiment, an Inozyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs.\",\n \"7250-7753 and 9701-10199.In another embodiment, a Zinzyme of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.\",\n \"1722-1998 and 3550-3768.In another embodiment, a Zinzyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 7754-8030 and 10200-10418.In another embodiment, an Amberzyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 8441-9069 and 11001-11547.In another embodiment, a DNAzyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 8031-8440 and 10419-11000.In another embodiment, a Hammerhead of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs.\",\n \"1024-1217 and 2420-3050.In another embodiment, a Hammerhead of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 7056-7249 and 9070-9700.In one embodiment, a nucleic acid molecule of the invention comprises between 12 and 100 bases complementary to RNA having sequence of IKK-gamma or PKR.\",\n \"In another embodiment, a nucleic acid molecule of the invention comprises between 14 and 24 bases complementary to RNA having sequence of IKK-gamma or PKR.\",\n \"In yet another embodiment, a nucleic acid molecule of the invention is chemically synthesized.\",\n \"In another embodiment, a nucleic acid molecule or antisense nucleic acid molecule of the invention comprises at least one 2′-sugar modification, at least one nucleic acid base modification, or at least one phosphate backbone modification.\",\n \"In one embodiment, the invention features a mammalian cell, for example a human cell, including an enzymatic nucleic acid molecule of the invention.\",\n \"The present invention features method of down-regulating PKR activity in a cell, comprising contacting the cell with a nucleic acid molecule of the invention, under conditions suitable for down-regulating of PKR activity.\",\n \"The present invention also features method of treatment of a patient having a condition associated with the level of PKR, comprising contacting cells of the patient with a nucleic acid molecule of the invention under conditions suitable for the treatment.\",\n \"The present invention features method of down-regulating IKK-gamma activity in a cell, comprising contacting the cell with a nucleic acid molecule of the invention, under conditions suitable for down-regulating of IKK-gamma activity.\",\n \"The present invention also features method of treatment of a patient having a condition associated with the level of IKK-gamma, comprising contacting cells of the patient with the nucleic acid molecule of the invention, under conditions suitable for the treatment.\",\n \"In one embodiment, a method of treatment of the invention comprises the use of one or more drug therapies under conditions suitable for said treatment.\",\n \"The present invention features method of cleaving RNA comprising a sequence of PKR gene comprising contacting an enzymatic nucleic acid molecule of the invention with the RNA of a PKR gene under conditions suitable for the cleavage.\",\n \"The present invention also features method of cleaving RNA comprising a sequence of IKK-gamma gene comprising contacting an enzymatic nucleic acid molecule of the invention with the RNA of an IKK-gamma gene under conditions suitable for the cleavage.\",\n \"In one embodiment, a method of cleavage of the invention is carried out in the presence of a divalent cation, for example Mg2+.\",\n \"In another embodiment, a nucleic acid molecule of the invention comprises a cap structure, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic derivative.\",\n \"The present invention also features an expression vector comprising a nucleic acid sequence encoding at least one nucleic acid molecule the invention in a manner which allows expression of the nucleic acid molecule.\",\n \"In one embodiment, the invention features a mammalian cell, for example a human cell, including an expression vector contemplated by the invention.\",\n \"In another embodiment, an expression vector of the invention further comprises a sequence for a nucleic acid molecule complementary to the RNA of a subunit of IKK-gamma or PKR.\",\n \"In yet another embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more nucleic acid molecules, which can be the same or different.\",\n \"The present invention also features a method for treatment of cancer, for example breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer, comprising administering to a patient a nucleic acid molecule of the invention under conditions suitable for said treatment.\",\n \"In one embodiment, a nucleic acid molecule of the invention comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification such as a 3′-3′ inverted abasic moiety, and/or phosphorothioate linkages on at least three of the 5′ terminal nucleotides.\",\n \"In another embodiment, other drug therapies contemplated by the invention include monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.\",\n \"Specific chemotherapy contemplated by the invention include paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.\",\n \"The invention also features a method for treatment of an inflammatory disease, for example rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury, glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, or infection, comprising the step of administering to a patient a nucleic acid molecule of the invention under conditions suitable for the treatment.\",\n \"The present invention features pharmaceutical compositions comprising the nucleic acid molecules of the invention in a pharmaceutically acceptable carrier.\",\n \"The invention also features a method of administering to a cell, such as mammalian cell (e.g.\",\n \"human cell), where the cell can be in culture or in a mammal, such as a human, an enzymatic nucleic acid molecule or antisense molecule of the instant invention, comprising contacting the cell with the nucleic acid molecule under conditions suitable for such administration.\",\n \"The method of administration can be in the presence of a delivery reagent, for example a lipid, cationic lipid, phospholipid, or liposome.\",\n \"The nucleic acid molecules that target specific sites in IKK-gamma or PKR-specific RNAs represent a therapeutic approach to treat a variety of inflammatory-related diseases and conditions, including but not limited to rheumatoid arthritis, restenosis, asthma, Crohn's disease, incontinentia pigmenti, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, and any other inflammatory disease or condition which respond to the modulation of IKK-gamma or PKR function.\",\n \"The enzymatic nucleic acid molecules that cleave the specified sites in IKK-gamma or PKR-specific RNAs also represent a therapeutic approach to treat a variety of cancers, including but not limited to breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, and/or other cancers which respond to the modulation of IKK-gamma or PKR function.\",\n \"In one embodiment, a nucleic acid molecule that modulates, for example, down-regulates IKK-gamma or PKR expression comprises between 12 and 100 bases complementary to a RNA molecule of IKK-gamma or PKR.\",\n \"In another embodiment, a nucleic acid molecule that modulates, for example IKK-gamma or PKR expression comprises between 14 and 24 bases complementary to a RNA molecule of IKK-gamma or PKR.\",\n \"Nucleic acid-based inhibitors of IKK-gamma or PKR function are useful for the prevention and/or treatment of cancers and cancerous conditions such as breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, and any other diseases or conditions that are related to or will respond to the levels of IKK-gamma or PKR in a cell or tissue, alone or in combination with other therapies.\",\n \"Nucleic acid-based inhibitors of IKK-gamma or PKR function are also useful for the prevention and/or treatment of inflammatory related diseases and conditions, including but not limited to rheumatoid arthritis, restenosis, asthma, Crohn's disease, incontinentia pigmenti, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, and any other inflammatory disease or condition which respond to the modulation of IKK-gamma or PKR function.\",\n \"In a further embodiment, the described nucleic acid molecules, such as antisense, siRNA or enzymatic nucleic acid molecules, can be used in combination with other known treatments to treat conditions or diseases discussed above.\",\n \"For example, the described molecules can be used in combination with one or more known therapeutic agents to treat breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, incontinentia pigmenti, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, and any other cancerous disease or inflammatory disease or condition which respond to the modulation of IKK-gamma or PKR expression.\",\n \"Identification of Potential Target Sites in Human IKK-Gamma and PKR RNA The sequence of human IKK-gamma or PKR genes are screened for accessible sites using a computer-folding algorithm.\",\n \"Regions of the RNA that do not form secondary folding structures and contained potential enzymatic nucleic acid molecule and/or antisense binding/cleavage sites are identified.\",\n \"The sequences of these binding/cleavage sites are shown in Tables VIII-XVIII.\",\n \"Selection of Enzymatic Nucleic Acid Cleavage Sites in Human IKK-Gamma and PKR RNA Enzymatic nucleic acid molecule target sites are chosen by analyzing sequences of Human IKK-gamma (Genbank accession No: NM—003639) and PKR (Genbank accession No: NM—002759) and prioritizing the sites on the basis of folding.\",\n \"Enzymatic nucleic acid molecules are designed that can bind each target and are individually analyzed by computer folding (Christoffersen et al., 1994 J. Mol.\",\n \"Struc.\",\n \"Theochem, 311, 273; Jaeger et al., 1989, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 86, 7706) to assess whether the enzymatic nucleic acid molecule sequences fold into the appropriate secondary structure.\",\n \"Those enzymatic nucleic acid molecules with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration.\",\n \"As noted below, varying binding arm lengths can be chosen to optimize activity.\",\n \"Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.\",\n \"Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient Cleavage and/or Blocking of IKK-Gamma and PKR RNA Enzymatic nucleic acid molecules and antisense constructs are designed to anneal to various sites in the RNA message.\",\n \"The binding arms of the enzymatic nucleic acid molecules are complementary to the target site sequences described above, while the antisense constructs are fully complementary to the target site sequences described above.\",\n \"The enzymatic nucleic acid molecules and antisense constructs were chemically synthesized.\",\n \"The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.\",\n \"The average stepwise coupling yields were typically >98%.\",\n \"Enzymatic nucleic acid molecules and antisense constructs are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol.\",\n \"180, 51).\",\n \"Enzymatic nucleic acid molecules and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and are resuspended in water.\",\n \"The sequences of the chemically synthesized enzymatic nucleic acid molecules used in this study are shown below in Table XVIII.\",\n \"The sequences of the chemically synthesized antisense constructs used in this study are complementary sequences to the Substrate sequences shown below as in Tables VIII-XVIII.\",\n \"Enzymatic Nucleic Acid Molecule Cleavage of IKK-Gamma and PKR RNA Target In Vitro Enzymatic nucleic acid molecules targeted to the human IKK-gamma or PKR RNA are designed and synthesized as described above.\",\n \"These enzymatic nucleic acid molecules can be tested for cleavage activity in vitro, for example, using the following procedure.\",\n \"The target sequences and the nucleotide location within the IKK-gamma or PKR RNA are given in Tables VIII-XVIII.\",\n \"Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid molecule cleavage assay is prepared by in vitro transcription in the presence of [a-32P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification.\",\n \"Alternately, substrates are 5′-32P-end labeled using T4 polynucleotide kinase enzyme.\",\n \"Assays are performed by pre-warming a 2× concentration of purified enzymatic nucleic acid molecule in enzymatic nucleic acid molecule cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37° C., 10 mM MgCl2) and the cleavage reaction was initiated by adding the 2× enzymatic nucleic acid molecule mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer.\",\n \"As an initial screen, assays are carried out for 1 hour at 37° C. using a final concentration of either 40 nM or 1 mM enzymatic nucleic acid molecule, i.e., enzymatic nucleic acid molecule excess.\",\n \"The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95° C. for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel.\",\n \"Substrate RNA and the specific RNA cleavage products generated by enzymatic nucleic acid molecule cleavage are visualized on an autoradiograph of the gel.\",\n \"The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.\",\n \"Nucleic Acid Down-Regulation of IKK-Gamma and PKR Target RNA In Vivo Nucleic acid molecules targeted to the human IKK-gamma or PKR RNA are designed and synthesized as described above.\",\n \"These nucleic acid molecules can be tested for cleavage activity in vivo, for example using the procedures described below.\",\n \"The target sequences and the nucleotide location within the IKK-gamma or PKR RNA are given in Tables VIII-XVIII.\",\n \"In Vivo Models Used to Evaluate the Down-Regulation of IKK-Gamma or PKR Gene Expression A variety of endpoints have been used in cell culture models to evaluate IKK-gamma or PKR-mediated effects after treatment with anti-IKK-gamma or PKR agents.\",\n \"Phenotypic endpoints include inhibition of cell proliferation, apoptosis assays and reduction of IKK-gamma or PKR protein expression, or a decrease in NFKB expression.\",\n \"Since IKK-gamma and PKR are both involved in the induction of NFKB, NFKB can be used as a surrogate marker in cell culture, animal, and clinical studies.\",\n \"Because overexpression of NFKB is directly associated with increased proliferation of tumor cells, a proliferation endpoint for cell culture assays is preferably used as a primary screen.\",\n \"There are several methods by which this endpoint can be measured.\",\n \"Following treatment of cells with nucleic acid molecules, cells are allowed to grow (typically 5 days) after which either the cell viability, the incorporation of [3H]thymidine into cellular DNA and/or the cell density can be measured.\",\n \"The assay of cell density is very straightforward and can be performed in a 96-well format using commercially available fluorescent nucleic acid stains (such as Syto® 13 or CyQuant®).\",\n \"The assay using CyQuant® is described herein As a secondary, confirmatory endpoint a nucleic acid-mediated decrease in the level of IKK-gamma or PKR RNA and/or IKK-gamma or PKR protein expression can be evaluated.\",\n \"Alternately, a decrease in the level of NFKB RNA can be evaluated.\",\n \"Cell Culture Cell types that express/over-express NFKB include HeLa, macrophages, peripheral blood lymphocytes, hepatocytes, fibroblasts, endothelial cells and epithelial cells.\",\n \"In culture, these cells can be stimulated to express/over-express NFKB by addition of TNF-alpha PMA or IL-1-beta to the culture medium.\",\n \"Some of these cell types also can respond with a similar activation of NFKB following LPS treatment.\",\n \"Activation of NFKB in cultured cells can be evaluated by electrophoretic mobility shift assay (EMSA).\",\n \"Delineation of alterations in the subunits can be determined by Western blot.\",\n \"Primary Screen A useful cell culture system in evaluating NFKB modulation is human colonic epithelial cells.\",\n \"One suitable cell line is SW620 colon carcinoma cells (CCL227).\",\n \"These cells respond to stimulation with TNF-alpha, LPS and/or IL-1-beta with an increase in NFKB activation.\",\n \"SW620 cells are grown in MEM supplemented with 10% heat-inactivated FBS and glutamine (2 mmol/L).\",\n \"TNF-alpha dose-response curves in these cells are determined by incubating cells with various concentrations of recombinant human TNF-alpha (Sigma Chemical Co.).\",\n \"Maximal DNA binding activity induction can occur with 150 U/ml TNF-alpha in the culture medium.\",\n \"Induction is typically evident within 10 minutes of treatment with TNF-alpha reaches a peak at one hour post-treatment and persists for up to 4 hours post-treatment.\",\n \"The primary readout can be NFKB DNA activity in nuclear extracts of SW620 cells as determined by electrophoretic mobility shift assays (EMSA).\",\n \"Once the appropriate TNF-alpha dose/response profile has been determined, inhibition of IKK-gamma, PKR, or NFKB activation is evaluated using specific and non-specific inhibitors of activation, sulfasalazine and steroids, respectively.\",\n \"Cells are incubated with inhibitors or control media for 30 minutes prior to stimulation with TNF-alpha Nuclear extracts are prepared and evaluated for DNA binding activity by EMSA.\",\n \"Once the activity of positive controls has been established, enzymatic nucleic acids targeting the IKK-gamma or PKR are evaluated in this system.\",\n \"Supershift assays using polyclonal antibodies against the NFKB or PKR protein subunits can be performed to confirm down-regulation of NFKB.\",\n \"Secondary Screens SW620 cells can be transfected with the 3xIg-kappa-B-Luc reporter construct 18 hours before challenge with TNF-alpha, LPS or PMA.\",\n \"The readout for this assay is luciferase activity.\",\n \"Test compounds are applied 17.5 hours after transfection (30 minutes before challenge).\",\n \"Cells are harvested 24 hours after challenge and relative changes in luciferase activity is used as the endpoint.\",\n \"Lastly, the activation of NFKB can be visualized fluorescently.\",\n \"Inactive NFKB heterodimers are held in the cytoplasm by inhibitory proteins.\",\n \"Once activated, the free heterodimers translocate to the nucleus.\",\n \"Thus, the relative change in cytoplasmic versus nuclear fluorescence can indicate the degree of NFKB activation.\",\n \"Cells can be grown on chamber slides, treated with TNF-alpha with and without test compounds), and the location of the NFKB subunit can be determined by immunofluorescence using a FITC-labeled antibody to NFKB.\",\n \"Animal Models Evaluating the efficacy of anti-IKK-gamma or PKR agents in animal models is an important prerequisite to human clinical trials.\",\n \"Studies have shown that human breast carcinoma cell lines express high levels of NFKB (Sovak et al., 1997, J. Clin.\",\n \"Invest., 100, 2952-2960).\",\n \"High levels of NFKB have also been observed in carcinogen-induced primary rat mammary tumors and in human breast cancer specimins.\",\n \"Additionally, HER2/neu overexpression has been shown to activate NFKB (Pianetti et al., 2001, Oncogene, 20, 1287-1299).\",\n \"As such, xenografts of cell lines that over-express NFKB can be used in animal models of tumorigenesis and/or inflammation to study the inhibition of NFKB.\",\n \"Oncology Animal Model Development Tumor cell lines are characterized to establish their growth curves in mice.\",\n \"These cell lines are implanted into both nude and SCID mice and primary tumor volumes are measured 3 times per week.\",\n \"Growth characteristics of these tumor lines using a Matrigel implantation format can also be established.\",\n \"The use of other cell lines that have been engineered to express high levels of NFKB can also be used in the described studies.\",\n \"The tumor cell line(s) and implantation method that supports the most consistent and reliable tumor growth is used in animal studies testing the lead IKK-gamma or PKR nucleic acid(s).\",\n \"Nucleic acids are administered by daily subcutaneous injection or by continuous subcutaneous infusion from Alzet mini osmotic pumps beginning 3 days after tumor implantation and continuing for the duration of the study.\",\n \"Group sizes of at least 10 animals are employed.\",\n \"Efficacy is determined by statistical comparison of tumor volume of nucleic acid-treated animals to a control group of animals treated with saline alone.\",\n \"Because the growth of these tumors is generally slow (45-60 days), an initial endpoint is the time in days it takes to establish an easily measurable primary tumor (i.e.\",\n \"50-100 mm3) in the presence or absence of nucleic acid treatment.\",\n \"Inflammation Animal Model Development Chronic, sublethal administration of indomethacin to outbred rats produces an enteropathy characterized by thickening of the small intestine and mesentery, ulcerations, granulomatous inflammation, crypt abcesses and adhesions.\",\n \"These lesions are similar to those that are characteristic findings in human patients with Crohn's disease (CD).\",\n \"Thus, any beneficial therapeutic effects revealed using this model can be extrapolated to potential benefit for patients with CD.\",\n \"Male Sprague-Dawley rats (200-275 g) are utilized for these studies.\",\n \"Chronic intestinal inflammation is induced by two subcutaneous injections of indomethacin (7.5 mg/kg in 5% NaHCO3) administered on subsequent days (Day-0 and Day-1).\",\n \"Animals are followed for four days following the first indomethacin injection.\",\n \"The mortality rate associated with this model is typically less than 10%.\",\n \"On the last day of the study, animals are euthanized by CO2 asphyxiation, small intestines excised and gross pathologic findings ranked according to the following criteria: 0, normal; 1, minimal abnormalities, slight thickening of the small intestine, no adhesions; 2, obvious thickening of small intestine with 1 adhesion; 3, obvious thickening of small intestine with 2 or 3 adhesions; 4, massive adhesions to the extent that the intestine cannot be separated, contents primarily fluid; 5, severe peritonitis resulting in death.\",\n \"A 10-cm portion of the most affected region of the small intestine is weighed, placed in 10% neutral buffered formalin and submitted for histopathologic evaluation.\",\n \"The 10 cm portion of gut from each animal is cut into five equal sections.\",\n \"Transverse and longitudinal sections of each portion are cut and stained with hematoxylin and eosin.\",\n \"All slides are read in a blinded fashion and each section is scored for necrosis (% area of involvement) and inflammatory response according to the following scale: Necrosis—1, 10%; 2, 10-25%; 3, 25-50%; 4, 50-75%; 5, 75-100%; Inflammation— 1=minimal in mesentery and muscle or lesion 2=mild in mesentery and muscle or lesion 3=moderate in mesentery and muscle or lesion 4=marked in lesion 5=severe in lesion The scores for each of the five sections are averaged for necrosis and for inflammation.\",\n \"NFKB Levels for Patient Screening and as a Potential Endpoint Because elevated NFKB levels can be detected in cancers, cancer patients can be pre-screened for elevated NFKB prior to admission to initial clinical trials testing an anti-IKK-gamma or PKR nucleic acid.\",\n \"Initial NFKB levels can be determined (by ELISA) from tumor biopsies or resected tumor samples.\",\n \"During clinical trials, it can be possible to monitor circulating NFKB protein by ELISA.\",\n \"Evaluation of serial blood/serum samples over the course of the anti-IKK-gamma or PKR nucleic acid treatment period could be useful in determining early indications of efficacy.\",\n \"Activity of Nucleic Acid Molecules Used to Down-Regulate IKK-Gamma and PKR Gene Expression Applicant has designed and synthesized several nucleic acid molecules targeted against IKK-gamma or PKR RNA.\",\n \"These nucleic acid molecules can be tested in cell proliferation and RNA reduction assays described herein.\",\n \"Proliferation Assay A model proliferation assay can be done using a cell-plating density of 2,000 cells/well in 96-well plates and at least 2 cell doublings over a 5-day treatment period.\",\n \"Cells used in proliferation studies can be, for example, were either lung or ovarian cancer cells (A549 and SKOV-3 cells respectively).\",\n \"To calculate cell density for proliferation assays, the FIPS (fluoro-imaging processing system) method known in the art can be used.\",\n \"This method allows for cell density measurements after nucleic acids are stained with CyQuant® dye, and has the advantage of accurately measuring cell densities over a very wide range 1,000-100,000 cells/well in 96-well format.\",\n \"Enzymatic nucleic acid molecules (50-200 nM) are delivered in the presence of cationic lipid at 2.5-5.0 μg/mL and inhibition of proliferation can be determined on day 5 post-treatment.\",\n \"RNA Assay RNA is harvested 24 hours post-treatment using the Qiagen RNeasy® 96 procedure.\",\n \"Real time RT-PCR (TaqMan® assay) is performed on purified RNA samples using separate primer/probe sets specific for target IKK-gamma or PKR RNA.\",\n \"Indications Particular degenerative and disease states that can be associated with IKK-gamma or PKR expression modulation include but are not limited to cancerous and/or inflammatory diseases and conditions such as breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, incontinentia pigmenti and any other diseases or conditions that are related to or respond to the levels of IKK-gamma or PKR in a cell or tissue.\",\n \"The present body of knowledge in IKK-gamma and PKR research indicates the need for methods to assay IKK-gamma and PKR activity and for compounds that can regulate IKK-gamma and PKR expression for research, diagnostic, and therapeutic use.\",\n \"The use of monoclonal antibodies, chemotherapy, radiation therapy, analgesics, and/or anti-inflammatory compounds, are all non-limiting examples of a methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g.\",\n \"ribozymes and antisense molecules) of the instant invention.\",\n \"Common chemotherapies that can be combined with nucleic acid molecules of the instant invention include various combinations of cytotoxic drugs to kill cancer cells.\",\n \"These drugs include but are not limited to paclitaxel (Taxol), docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, vinorelbine etc.\",\n \"Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g.\",\n \"ribozymes and antisense molecules) are hence within the scope of the instant invention.\",\n \"Diagnostic Uses The nucleic acid molecules of this invention (e.g., enzymatic nucleic acid molecules) can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of IKK-gamma or PKR RNA in a cell.\",\n \"The close relationship between enzymatic nucleic acid molecule activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA.\",\n \"By using multiple enzymatic nucleic acid molecules described in this invention, one can map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues.\",\n \"Cleavage of target RNAs with enzymatic nucleic acid molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease.\",\n \"In this manner, other genetic targets can be defined as important mediators of the disease.\",\n \"These experiments can lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules and/or other chemical or biological molecules).\",\n \"Other in vitro uses of enzymatic nucleic acid molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with IKK-gamma or PKR-related condition.\",\n \"Such RNA is detected by determining the presence of a cleavage product after treatment with an enzymatic nucleic acid molecule using standard methodology.\",\n \"In a specific example, enzymatic nucleic acid molecules which cleave only wild-type or mutant forms of the target RNA are used for the assay.\",\n \"The first enzymatic nucleic acid molecule is used to identify wild-type RNA present in the sample and the second enzymatic nucleic acid molecule is used to identify mutant RNA in the sample.\",\n \"As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both enzymatic nucleic acid molecules to demonstrate the relative enzymatic nucleic acid molecule efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species.\",\n \"The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population.\",\n \"Thus each analysis requires two enzymatic nucleic acid molecules, two substrates and one unknown sample which is combined into six reactions.\",\n \"The presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel.\",\n \"It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells.\",\n \"The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., IKK-gamma or PKR) is adequate to establish risk.\",\n \"If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis.\",\n \"Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.\",\n \"The use of enzymatic nucleic acid molecules in diagnostic applications contemplated by the instant invention is more fully described in George et al., U.S. Pat.\",\n \"Nos.\",\n \"5,834,186 and 5,741,679, Shih et al., U.S. Pat.\",\n \"No.\",\n \"5,589,332, Nathan et al., U.S. Pat.\",\n \"No.\",\n \"5,871,914, Nathan and Ellington, International PCT publication No.\",\n \"WO 00/24931, Breaker et al., International PCT Publication Nos.\",\n \"WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No.\",\n \"WO 99/29842.Additional Uses Potential uses of sequence-specific enzymatic nucleic acid molecules of the instant invention can have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA (Nathans et al., 1975 Ann.\",\n \"Rev.\",\n \"Biochem.\",\n \"44:273).\",\n \"For example, the pattern of restriction fragments can be used to establish sequence relationships between two related RNAs, and large RNAs can be specifically cleaved to fragments of a size more useful for study.\",\n \"The ability to engineer sequence specificity of the enzymatic nucleic acid molecule is ideal for cleavage of RNAs of unknown sequence.\",\n \"Applicant has described the use of nucleic acid molecules to down-regulate gene expression of target genes in bacterial, microbial, fungal, viral, and eukaryotic systems including plant, or mammalian cells.\",\n \"All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains.\",\n \"All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.\",\n \"One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein.\",\n \"The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention.\",\n \"Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.\",\n \"It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.\",\n \"Thus, such additional embodiments are within the scope of the present invention and the following claims.\",\n \"The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that is not specifically disclosed herein.\",\n \"Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” can be replaced with either of the other two terms.\",\n \"The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.\",\n \"Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.\",\n \"In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.\",\n \"Other embodiments are within the following claims.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00001 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00002 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00003 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00004 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00005 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00006 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00007 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00008 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00009 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00010 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00011 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00012 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00013 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00014 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00015 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00016 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00017 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00018 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00019 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00020 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00021 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00022 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070026394A1-20070201-T00023 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE The patent application contains a lengthy table section.\",\n \"A copy of the table is available in electronic form from the USPTO web site () An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471271"}}},{"rowIdx":584,"cells":{"text":{"kind":"list like","value":[["Glazing laminates","There is provided a glazing laminate comprising a polyvinyl butyral core layer having polyvinyl butyral/IR dye layers on each side.","The dye containing layers are solvent cast from methanol onto the core layer.","The dyes in each layer are different comprising BN 18 type PVB containing either 2 mg Epoline III-125 or 2 mg of Epolite III-57 dyes per grams of resin and dissolved in 10 ml of analytical grade of methanol for casting.","The polymer is sandwiched between two glass layers under pressure and heat treatment at 135-140° C. An overall concentration of the dyes of ˜0.1 g/m2 results in an energy absorption over 65%.","The heating effect of absorption is reduced by conduction through the polymer to the laminate edges, internal reflection, spherical reradiation and the relative insulative effect of the glass layers."],["1.A laminated glazing element including an optically clear laminated sheet including at least two polymer layers having dispersed therein respective dyes, said layers having between them at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, said polymer layers being sandwiched between a UV absorbent outer support layer and an insulative inner support layer, said glazing element having at least one edge portion associated with a heat sink selected to disperse heat from said laminated sheet.","2.The laminated glazing element according to claim 1, wherein said polymer layers are selected to have higher thermal conductivity than said inner support layer.","3.The laminated glazing element according to claim 1, wherein said heat sink is configured to disperse heat to either the interior or exterior of a glazed structure from the at least one edge portion.","4.A The laminated glazing element according to claim 1, wherein said heat sink is configured to selectively disperse the heat to the interior or exterior of the glazed structure.","5.The laminated glazing element according to claim 1, wherein said heat sink is a thermal mass configured to radiate and/or convect heat.","6.The laminated glazing element according to claim 1, wherein the IR dyes are selected from aminium, bisammonium and bisimmonium salt dyes.","7.The laminated glazing sheet according to claim 1, wherein said polymer layers are selected from polyvinyl butyral (PVB) polymers.","8.The laminated glazing element according to claim 1, wherein said polymer layers are selected from polyvinyl butryal (PVB) polymers.","9.The laminated glazing element according to claim 1, wherein the inner support layer has a range of colouring dyes.","10.The laminated glazing element according to claim 6, wherein one or more of the IR dyes have graduated wavelengths from 900 to 1700 nanometers.","11.The laminated glazing sheet according to any of the preceding claims, excluding claim 7, wherein said polymers layers are selected from methyl acrylate polymers.","12.The laminated glazing sheet according to any of the preceding claims 1 to 10, excluding claim 7, wherein said polymer layers are selected from polycarbonate polymers.","13.The laminated glazing sheet according to claim 7, wherein a plolyvinyl butral layer is complimented by a liquid polycarbonate polymer containing selected dyes.","14.A laminated glazing sheet including a polymer layer comprising a polyvinyl butyral having a hydroxyl content of at least 18 wt % and at least one aminium salt IR absorbing dye, said at least one dye being selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, and at least one UV absorbent outer support layer, wherein said polymer layer is formed by forming a mixture of methanolic solution of said polyvinyl butyral having a hydroxyl content of at least 18 wt % and a saturated methanolic solution of said at least one dye, and drying said mixture.","15.A method of forming a glazing laminate including the steps of: (a) solvent casting a polyvinyl butyral film; (b) solvent casting a polyvinyl butyral/dye film to each surface of said polyvinyl butyral film, said polyvinyl butyral/dye films collectively including at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation; and (c) laminating the multilayer film of step (b) between glass sheets under heat and pressure.","16.The laminated glazing element according to claim 7, wherein one or more of the IR dyes have graduated wavelengths from 900 to 1700 nanometers.","17.The laminated glazing element according to claim 8, wherein one or more of the IR dyes have graduated wavelengths from 900 to 1700 nanometers.","18.The laminated glazing element according to claim 9, wherein one or more of the IR dyes have graduated wavelengths from 900 to 1700 nanometers."],["<SOH> BACKGROUND OF THE INVENTION <EOH>This invention relates to glazing laminates.","This invention has particular but not exclusive application to glazing laminates for glazing in construction, and for illustrative purposes reference will be made to such application.","However, it is to be understood that this invention could be used in other applications, such as automotive glass and the like.","A significant contributor to the cost of running commercial buildings or maintenance of comfort in residences is the energy required to provide control of the internal climate of the building.","In buildings in hot climates having windows that are exposed to a relatively high flux of solar radiation, or buildings with large areas exposed to solar radiation, a major cost is air cooling by airconditioning.","The conventional means of controlling heat passage into a building is to reduce the flux of solar radiation through the glazing by tinting to reduce a broad spectrum of radiation passing through the glass, with or without the provision of an outer partially reflective layer.","By these means, a reduction of the total flux effects a partial reduction of the heat transmitted to the interior of the building.","Clear window glass has a natural absorption maximum in the UV spectrum.","Whilst the UV part of the solar spectrum at the Earth's surface is of high energy, the proportion of the total EM flux in the UV at the earth's surface is quite small due to upper atmospheric absorption.","The visible spectrum is not substantially absorbed by clear window glass, but is quite strongly absorbed by tinted glass.","The absorbed energy is converted to heat, which is in part spherically radiated in the infrared, and otherwise dispersed by convective transfer and conduction.","The infrared component of the incident light is generally transmitted directly (with refraction) to the interior of the building in addition to the internally directed IR component arising from absorbed visible/UV radiation.","In terms of energy density, the IR/visible bands contribute the greatest part if the incident energy density transmitted to the interior of a building through the windows or glass walls.","In fact, energy density reaches a maximum at a wavelength of about 600 nm, with about 90% of the energy of solar radiation incident at the earth's surface being of wavelengths between 500 nm and 1750 nm, with several distinct maxima.","Where tinting and/or partially reflective films is used, this generally reduces the visible flux and requires stronger lighting, which carries its own heat burden.","In the case of tinting alone, the heat loading transmitted is only reduced by the proportion of IR reradiation back out of the building.","International patent publication WO97/44690 discloses an optical element comprising a transparent layers comprising one or more passive layers and one or more active layers wherein said passive layers facilitate the transmission of electromagnetic radiation in a substantially unaltered form and the at least one active layers include an active material dispersed through the active layer and having the capacity to intercept electromagnetic radiation of a wavelength or range of wavelengths and redirect some of the energy of the intercepted radiation into the interior of the optical element, the layers being in face to face relationship and being optically coupled to each other.","The described embodiments use a luminophore as the active material to absorb IR in the active layer and to spherically reemit IR by luminescent decay of the luminophore from the excited state.","The basic concept of causing dissipation of IR by absorbance dye in a polymer film was shown to work, but no practical polymer film was produced."],["<SOH> SUMMARY OF THE INVENTION <EOH>This invention in one aspect resides broadly in a laminated glazing sheet including at least one polymer layer having dispersed therein at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, and at least one UV absorbent outer support layer.","The at least two dyes may be dispersed in one, both or more of the polymer layers depending on the number of layers, choice of polymers and compatibility of the dyes in polyemer dispersion in practice.","The polymer layer may include a tint such as by means of a dye absorbing in the visible band.","A practical difficulty experienced with such constructs is that suitable dyes tend to be barely compatible with the polymer matrices in which they are dispersed, and may be reactive with the IR dyes.","The problems range from lack of homogeneity in monolayer films, films changed colour and destroyed the IR properties, and when laminated in two layers to keep the dyes separate, the panels were blotchy, patchy, uneven in colour and torn.","Accordingly, in a further aspect this invention resides in a laminated glazing sheet including at least two polymer layers having dispersed therein respective dyes, said layers having between them at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, at least one other of said layers including a dye absorbing in the visible spectrum, a polymer interlayer between adjacent dye bearing ones of said polymer layers, and at least one UV absorbent outer support layer.","The absorbed IR radiation will be for the most part dispersed in the laminate as heat.","In order to minimize the heat transmitted to the interior of the building, the heat is preferably dispersed from the laminate to reduce heat build-up and the black body effect to the interior of a glazed structure.","Accordingly, in a yet further aspect the present invention resides broadly in a laminated glazing element including an optically clear laminated sheet including at least two polymer layers having dispersed therein respective dyes, said layers having between them at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, said polymer layers being sandwiched between a UV absorbent outer support layer and an insulative inner support layer, said glazing element having at least one edge portion associated with a heat sink selected to disperse heat from said laminated sheet.","The inner support layer is of an insulative material to reduce heat transfer to the interior of a glazed structure by convection.","The at least one polymer layer may be selected from polymers that have a higher then usual thermal conductivity, and that the heat sink be configured to disperse heat to the outside of the glazed structure from the at least one edge thereof.","For example, the heat sink may be a thermal mass being configured to either or both of radiate and convect heat to the exterior.","Alternatively, the heat sink may be configured to selectively disperse the heat to the interior or exterior of the glazed structure.","By this means, in winter the filtered IR may be used to heat the building, whilst in summer the trapped heat is disposed to the exterior.","In one embodiment the heat sink is the atmosphere per se, rendered useful by installing the laminate having the at least one edge exposed.","The relative refractive indices of the inner and outer support layers and the at least one polymer layer may be selected to maximize the amount of black body radiation which is internally reflected to the edges of the laminate."],["BACKGROUND OF THE INVENTION This invention relates to glazing laminates.","This invention has particular but not exclusive application to glazing laminates for glazing in construction, and for illustrative purposes reference will be made to such application.","However, it is to be understood that this invention could be used in other applications, such as automotive glass and the like.","A significant contributor to the cost of running commercial buildings or maintenance of comfort in residences is the energy required to provide control of the internal climate of the building.","In buildings in hot climates having windows that are exposed to a relatively high flux of solar radiation, or buildings with large areas exposed to solar radiation, a major cost is air cooling by airconditioning.","The conventional means of controlling heat passage into a building is to reduce the flux of solar radiation through the glazing by tinting to reduce a broad spectrum of radiation passing through the glass, with or without the provision of an outer partially reflective layer.","By these means, a reduction of the total flux effects a partial reduction of the heat transmitted to the interior of the building.","Clear window glass has a natural absorption maximum in the UV spectrum.","Whilst the UV part of the solar spectrum at the Earth's surface is of high energy, the proportion of the total EM flux in the UV at the earth's surface is quite small due to upper atmospheric absorption.","The visible spectrum is not substantially absorbed by clear window glass, but is quite strongly absorbed by tinted glass.","The absorbed energy is converted to heat, which is in part spherically radiated in the infrared, and otherwise dispersed by convective transfer and conduction.","The infrared component of the incident light is generally transmitted directly (with refraction) to the interior of the building in addition to the internally directed IR component arising from absorbed visible/UV radiation.","In terms of energy density, the IR/visible bands contribute the greatest part if the incident energy density transmitted to the interior of a building through the windows or glass walls.","In fact, energy density reaches a maximum at a wavelength of about 600 nm, with about 90% of the energy of solar radiation incident at the earth's surface being of wavelengths between 500 nm and 1750 nm, with several distinct maxima.","Where tinting and/or partially reflective films is used, this generally reduces the visible flux and requires stronger lighting, which carries its own heat burden.","In the case of tinting alone, the heat loading transmitted is only reduced by the proportion of IR reradiation back out of the building.","International patent publication WO97/44690 discloses an optical element comprising a transparent layers comprising one or more passive layers and one or more active layers wherein said passive layers facilitate the transmission of electromagnetic radiation in a substantially unaltered form and the at least one active layers include an active material dispersed through the active layer and having the capacity to intercept electromagnetic radiation of a wavelength or range of wavelengths and redirect some of the energy of the intercepted radiation into the interior of the optical element, the layers being in face to face relationship and being optically coupled to each other.","The described embodiments use a luminophore as the active material to absorb IR in the active layer and to spherically reemit IR by luminescent decay of the luminophore from the excited state.","The basic concept of causing dissipation of IR by absorbance dye in a polymer film was shown to work, but no practical polymer film was produced.","SUMMARY OF THE INVENTION This invention in one aspect resides broadly in a laminated glazing sheet including at least one polymer layer having dispersed therein at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, and at least one UV absorbent outer support layer.","The at least two dyes may be dispersed in one, both or more of the polymer layers depending on the number of layers, choice of polymers and compatibility of the dyes in polyemer dispersion in practice.","The polymer layer may include a tint such as by means of a dye absorbing in the visible band.","A practical difficulty experienced with such constructs is that suitable dyes tend to be barely compatible with the polymer matrices in which they are dispersed, and may be reactive with the IR dyes.","The problems range from lack of homogeneity in monolayer films, films changed colour and destroyed the IR properties, and when laminated in two layers to keep the dyes separate, the panels were blotchy, patchy, uneven in colour and torn.","Accordingly, in a further aspect this invention resides in a laminated glazing sheet including at least two polymer layers having dispersed therein respective dyes, said layers having between them at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, at least one other of said layers including a dye absorbing in the visible spectrum, a polymer interlayer between adjacent dye bearing ones of said polymer layers, and at least one UV absorbent outer support layer.","The absorbed IR radiation will be for the most part dispersed in the laminate as heat.","In order to minimize the heat transmitted to the interior of the building, the heat is preferably dispersed from the laminate to reduce heat build-up and the black body effect to the interior of a glazed structure.","Accordingly, in a yet further aspect the present invention resides broadly in a laminated glazing element including an optically clear laminated sheet including at least two polymer layers having dispersed therein respective dyes, said layers having between them at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, said polymer layers being sandwiched between a UV absorbent outer support layer and an insulative inner support layer, said glazing element having at least one edge portion associated with a heat sink selected to disperse heat from said laminated sheet.","The inner support layer is of an insulative material to reduce heat transfer to the interior of a glazed structure by convection.","The at least one polymer layer may be selected from polymers that have a higher then usual thermal conductivity, and that the heat sink be configured to disperse heat to the outside of the glazed structure from the at least one edge thereof.","For example, the heat sink may be a thermal mass being configured to either or both of radiate and convect heat to the exterior.","Alternatively, the heat sink may be configured to selectively disperse the heat to the interior or exterior of the glazed structure.","By this means, in winter the filtered IR may be used to heat the building, whilst in summer the trapped heat is disposed to the exterior.","In one embodiment the heat sink is the atmosphere per se, rendered useful by installing the laminate having the at least one edge exposed.","The relative refractive indices of the inner and outer support layers and the at least one polymer layer may be selected to maximize the amount of black body radiation which is internally reflected to the edges of the laminate.","DETAILED DECRIPTION OF THE INVENTION The polymer material comprising respective ones of the at least two polymer layers may be the same or different from each other, and may respectively be the same or different from the polymer interlayer.","In order to provide a constant refractive milieu, the polymer of the at least two polymer layers are the same, and preferably all polymers layers are of the same material.","The polymers may be of any known optically clear film forming or castable thermoplastic or thermoset resin, selected on the basis of compatibility with the respective dyestuffs.","The respective polymers may be formed into films and assembled into the layered structure in an appropriate forming process such as pressing with or without the application of heat, or the like.","Where the polymers are extrudable thermoplastics, the layers of polymer may be concurrently formed such as by coextrusion.","For example, where the polymers permit, the polymer layers may be formed by a melt blowing process such as the double bubble process for the production of laminated films.","In order to permit intimate lamination of the polymers with disparate materials such as glass by thermal pressing or the like, the polymer may be a thermoplastic or a crosslinked polymer that is thermoplastic or thermoset with a thermoplastic uncrosslinked form.","The polymer may be selected as to refractive index relative to the UV absorbent outer support layer so as to provide for a degree of total internal reflection of spherically dispersed long-wave IR emanating from the polymer layers in use.","By this means a degree of selective transmission of energy absorbed by said dyes is to the periphery of the sheet by conduction and a degree of total internal reflection.","The respective dyes may be selected having regard to compatibility with the polymer of choice.","The IR dye may for example be selected from organic dyes, aminium, bisammonium and bisimmonium salt dyes, or metal salts of organic acids.","It has surprisingly been found that polyvinyl butyral (PVB) polymers are compatible with aminium, bisammonium and bisimmonium salt dyes, a specific class of dyes which are generally N-organic salts in which the counter anion is derived from the salt of a strong acid such as ClO4−, PF6−, AsF6−, SbF6− and the like.","For example the dye may be divalent immonium salts which are near infrared dyes of the formula (I).","(R2NAr)2—N+═Art═N+R2+2X− (I) wherein R═C1 to C6 alkyl; Ar=divalent phenyl which may or may not be ring substituted with one or more alkyl, alkoxy, halogen, nitro, cyano or carboalkoxy groups; Art=quinoidal phenyl which may or may not be ring substituted with one or more alkyl, alkoxy, halogen, nitro, cyano or carboalkoxy groups; and X=an anion of a strong acid, such as an SbF6− anion.","Experimental work such as that of Resch-Genger U. and Wolfbeis, O., “Near Infrared Dyes for High Technology Applications”, (Kluwer Academic Publishers) has resulted in the production of a series of such dyes including those of the Epolin series.","Typical structures of amimium and bisammonium salt dyes are given in formulae II and III respectively.","It is envisaged that many other polymer/dye combinations may be appropriate.","The visible spectrum dyes may be selected from dyes known for the purpose.","The dyes may be dispersed in the polymer layers by any suitable means.","The dyes may for example be directly blended into the polymer material prior to casting or forming the polymer layer.","Alternatively, the dye may be dissolved in a solvent which is preferably a cosolvent for the polymer, whereby the polymer layer may be formed as a cast film by solvent evaporation.","The UV absorbent outer support layer is preferably glass, but may also be a further polymer layer doped with a suitable UV absorbent substance.","The UV absorbent outer support layer is preferably selected to have refractive qualities relative to those of the polymer layers whereby the boundary between the polymer layer and the UV absorbent outer support layer forms a surface for internal reflection of longwave IR emitted by the warmed polymer layers.","According to a further preferred feature of the invention at least a portion of the edge of the optically clear laminated sheet is configured to facilitate emission of the emitted radiation from the optical element.","In one form at least some of the edges may be chamfered.","Alternatively the sheet edges may be in contact with an absorber comprising a conductive thermal mass such as metal.","The thermal mass may include means to disperse heat to the outside of the building, or to convective chimneys in the walls.","The heat may be recovered by exchange means such as water to be harvested for heating or heat pump applications.","At least one face of the optically clear laminated sheet may be formed with a discontinuity to facilitate the emission of emitted radiation from the optical element.","The form of discontinuity may comprise at least one groove formed in the at least one face.","Another form of discontinuity may comprise one or more depressions or dimples provided in the at least one face.","Another form of discontinuity may comprise at least one rib or protrusion on the at least one face.","Another form of discontinuity may comprise an etched surface on a portion of the at least one face.","It has surprisingly been determined that the suitability of a particular dye/polymer combination in a film does not in general predict the performance or stability of the polymer layers in a laminate.","Without wishing to be bound by theory, it is believed that the dyes in adjacent layers of a laminate exhibit a cross reactivity that can affect the IR performance, the stability of the dye systems, and even the physical properties of the polymers in the laminate.","It has been found that the provision of a polymer interlayer reduces the interactions between the respective dye/polymer systems, and that as a preference the same polymer may be used in the dye-containing layers and interlayers to provide a substantially consistent refractive qualities in the visible band.","It has been found that the provision of a UV absorbent outer layer tends to improve the stability of the polymers and polymer/dye systems.","In one aspect the invention resides in a method of forming a glazing laminate including the steps of: (a) solvent casting a polyvinyl butyral film; (b) solvent casting a polyvinyl butyral/dye film to each surface of said polyvinyl butyral film, said polyvinyl butyral/dye films collectively including at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation; and (c) laminating the multilayer film of step (b) between glass sheets under heat and pressure.","It has further been determined that certain compositions for film forming by solvent evaporation and comprising relatively reactive dyes and polymers having reactive groups may be stabilized by selection of the solvent.","For example, polyvinyl butyrals are polymers having significant hydroxyl functionality.","Such polymers are usually worked up and are stable in technical grade ethanol.","Aminium salt dyes such as the EPOLIN dyes are also generally worked up in polar solvents such as acetone, and are reasonably soluble in ethanol.","However, in the use of polyvinyl butyral in forming the dye containing polymer layers of the present invention, the PVB/ethanol//dye/ethanol and PVB/ethanol//dye/acetone systems results in films of poor physical characteristics and markedly reduced IR performance.","The films so produced were also intractable in being formed up under heat and pressure to glass substrates, with excessive bubble formation.","However, despite limited solubility of aminium salt dyes such as aminium salts in methanol, it has been surprisingly found that solvent cast films based on a PVB/methanol//dye/methanol system exhibit superior film forming and dye-stability performance.","The polymer compositions formed by this system have also shown an ability to be subsequently melt-processed such as by extrusion into films with suitable physical and IR dye performance.","In a further aspect the present invention relates to a film forming polymer composition comprising a mixture of methanolic solution of a polyvinyl butyral having a hydroxyl content of at least 18 wt % and a saturated methanolic solution of an aminium salt IR absorbing dye.","In a yet further aspect of the present invention, there is provided a method of forming an IR absorbing polymer film comprising the steps of forming a mixture of methanolic solution of a polyvinyl butyral having a hydroxyl content of at least 18 wt % and a saturated methanolic solution of an aminium salt IR absorbing dye, forming said mixture into a liquid layer, and drying said layer to a film by solvent evaporation.","In a yet further aspect of the present invention, there is provided a method of forming an IR absorbing polymer film comprising the steps of forming a mixture of methanolic solution of a polyvinyl butyral having a hydroxyl content of at least 18 wt % and a saturated methanolic solution of an aminium salt IR absorbing dye, drying said mixture, and melt extruding said dried mixture to form a film.","It has further been determined that such cast and extruded films may include more than one dye and may further be laminated to form an optically clear laminated sheet.","Accordingly, in another aspect there may be provided an optically clear laminated sheet including a polymer layer comprising a polyvinyl butyral having a hydroxyl content of at least 18 wt % and at least one aminium salt IR absorbing dye, said dyes being selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, and at least one UV absorbent outer support layer, wherein said polymer is formed by forming a mixture of methanolic solution of said polyvinyl butyral having a hydroxyl content of at least 18 wt % and a saturated methanolic solution of said dyes, and drying said mixture.","In order that this invention may be more readily understood and put into practical effect, reference will now be made to the following examples which illustrates preferred embodiments and comparative formulations in respect of the invention, and wherein: FIG.","1 is a FT-NIR absorption of dye combinations in polymers in accordance with the present invention; FIG.","2 is a FT-NIR absorption of Epolite 125 and Epoline III-57 combinations in polymers in accordance with the present invention; FIG.","3 is a FT-NIR absorption of Epolite dye combinations in polymers in accordance with the present invention; FIG.","4 is the energy spectrum of the Sun; FIG.","5 is the energy absorption of laminates of glass and PVB; FIG.","6 is the energy absorption of some Epolin combinations; FIG.","7 is the energy absorption spectra of H.W.","Sands dye systems; FIG.","8 is the energy absorption spectrum of a sample; FIG.","9 is the energy absorption spectrum of a sample, and FIG.","10 is the energy absorption spectrum of a sample.","EXAMPLE 1 Methanol was tested for the dissolution of the dyes and of the PVB.","BN18 polymer (Wacker-Chemie, PIOLOFORM) was used.","Initally 2 mg dye/1 g of PVB mixture was produced and dissolved in ethanol forming a ˜40% solution with respect to the resin.","This solution was diluted by methanol to have an overall concentration of 20% of resin.","The glass plates were cast in consecutive steps by this solution.","During the drying process the layer turned to be turbulent then it cleared.","The next consecutive layer was cast in this stage.","A dye density of 0.010-0.026 g/m2 covered the individual slides.","Additional layers of PVB were cast to have an expected layer thickness of 0.05 mm after the heat treatment.","Following by an overnight drying the slides were heat treated at 135° C. for 5-10 min.","Two slides were pressed together to have a homogeneous and transparent layer.","Four dyes were tested.","Epolite 125-(AN), Epolite 178 (AM), Epoline III-57 (AL) and Spectra IR 920 (AO).","The combinations and their properties are shown in Table I.","TABLE 1 Thickness, Combination Density of dyes, g/m2 IR930 absorption, % μm AN-AN 0.052 57 515 AN-AM 0.050 50 495 AN-AL 0.047 53 473 AN-AO 0.038 60 517 AM-AM 0.049 33 412 AM-AL 0.060 67 450 AM-AO 0.046 50 530 AL-AL 0.065 87 525 AL-AO 0.059 67 525 AO-AO 0.017 17 522 PVB 0 3 552 The near infrared (NIR) spectra of the samples were determined by a Perkin-Elmer FT spectrometer from 13000 to 4000 cm−1 energy range what corresponds to the wave length of 670 to 2500 nm.","FIG.","1 shows the absorption spectra of the different combinations.","Epoline III-57 has an extraordinary high absorption at the wavelength near to 1000 nm but it has practically no absorption at longer wavelengths.","Epolite 125 has good absorption at the lower wavelengths but it is relatively ineffective at the higher energy range.","The IR absorption of the combinations of these two dyes are shown in FIG.","2.Next a solution of Epolite 125 and Epolite 178 was prepared in pure methanol in 8 mg dye/1 g of resin.","Epolite 178 did not dissolve completely in the solution.","One layer of this solution was cast on glass and combined by itself and by double layers of Epoline III-57 of 1 mg dye in 1 g of resin of the methanol solution.","The density of the dye and the IR930 absorption with the layer thickness are given in Table II.","TABLE II Thickness, Combination Density of dyes, g/m2 IR930 absorption, % μm 57/25.78 0.06/0.95 92 300 25.78/57 0.95/0.06 86 320 25.78/25.78 2.36 95 135 57/57 0.11 90 230 Transparent layers of yellow/green colour were formed.","The FT-NIR absorption of the layers are shown in FIG.","3.The layers have extremely high NIR absorption.","There might be some scaling error in the equipment showing absorption values higher than 1.The IR930 absorption values correspond to the FT-NIR data.","Epolite 125 assures high absorption values at longer wavelength, Epoline III-57 assures the good absorption at the range of 1000 nm, what corresponds to the middle of the IR energy peak of the incident solar radiation spectrum (see in FIG.","4).","The FT-NIR absorption of the PVB with a layer thickness of 0.5 mm enclosed in between two glass sheets of 1 mm thickness is shown in FIG.","5.The Figure also shows the absorption of a single and a double sheets of glass plates of 3 mm thickness.","This kind of glass plates is supposed to be used for the final sample preparation.","PVB has homogeneously low absorption up to a wavelength of 2000 nm, than there are two absorbing bands.","These bands are also visible in FIGS.","1-3.The thick glass plates show higher and homogeneous absorption level for the whole range with a broad peak at the 900-1400 nm range.","Conclusion Epolite 125 and Epoline III-57 dyes are suitable for the preparation of the laminates.","They can be employed in a concentration of 0.1-0.3 g/m2.This amount of dyes can be cast as couple of layers of 2 mg dye in 1 g of BN18 resin from a methanol solution on a glass surface.","The necessary layer thickness is achieved by casting of additional layers of PVB from methanol solution.","Two coated glass surfaces are heated and pressed together after having been properly dried at temperature of 135° C. to form the laminated glass of the present invention.","EXAMPLE 2 Two new dyes were obtained from Epolin Inc. for the purpose of producing blue and grey laminates.","The dyes were Sole Blue 33 and Violet B. Epolin give the ratio of colour dye with respect to IR dye of 0.1-0.15 w/w.","Types of PVB (BN18) and glass used were as previously reported.","Methods for preparation were used as previously developed.","Results Sole Blue 33 dissolved well in methanol (5 mg/10 ml) but Violet B was soluble only up to 1 mg/10 ml.","Initial experiments on a small scale were carried out to match the colour to blue or gray from that of the IR dye (i.e.","green-yellow).","Levels of the new dye of 0.1, 0.3 and 1 w/w dye per IR dye were tested.","4 mg/g IR dye with respect to PVB was used dissolved in 10 ml MeOH/g of PVB.","Sole Blue 33 dissolved properly at each concentration.","However, it did not produce a good colour match—it formed various green colours.","Violet B did not dissolve perfectly at the highest concentration—it also produced only a yellow-brown colour.","Some particles remained not dissolved at each concentration indicating some inhomogeneities in the dye.","During heat treatment at 135° C. the undissolved part of Violet B dissolved and produced very heterogeneous samples i.e.","the colour varied throughout the sample.","It was theorized that new solvents were needed to overcome these problems.","Acetone/butanol-1 (4:1) solvent was selected for the next set of experiments, 2 mg/g IR dye was used (1.25 mg/g Epolight III-57 and 0.75 mg/g Epolight III-25 with respect to the PVB).","Violet B was added at 0.3 w/w with respect to the IR dye.","10 ml acetone mixed with 2.5 ml butanol-1 was used for 1 g of PVB.","A clear grey colour formed.","2 or 3 layers of mixture were cast to glass, dried and heat treated at 135° in the oven, as required to form the flat adhered laminate structure needed for window glass.","Unfortunately, the colour changed.","The grey became violet.","FTNIR spectra showed that the IR dye function had been destroyed, its initial good IR absorption decreased to less than one third of its initial value.","IR transmission measured by laser showed the same result.","These results show that the Violet B dye destroyed the IR dye after a short heat treatment at 135° for 3-5 min.","To overcome this problem a sample was prepared with two separate layers, one layer containing Violet B and one the IR dye.","A 400×400 ml panel was prepared using two independent layers, one with IR dye cast to one glass plate, the other with Violet B to the other glass plate.","The IR dye containing layer was prepared using 128 mg of IR dye (80 mg Epolight III-57 and 48 mg of Epolight III-125) mixed to 32 g of PVB (4 mg/g), what corresponds to 0.8 g dye/m2.The other layer contained 53 mg of Violet B dispersed in 32 g PVB (0.4 w/w Violet blue with respect or IR dye).","Both glasses were cast in two steps using acetone/butanol-1 solvent and the solvent was evaporated overnight.","Heat treatment in the oven at 135° was performed for 5 min and then the plates were pressed together.","During the heat treatment a 5 kg weight was on the laminate, as previously developed.","During this treatment the layer of Violet B unexpectedly coagulated, lost its lamellar shape and formed totally inhomogeneous laminate.","The IR dye containing layer remained intact as expected—FTNIR spectra of the system showed that absorption was homogeneous across the layer, indicating that the IR dye remained intact.","There was a small reduction in absorption power in some spots where there was a high concentration of Violet B.","Hence the problems with the large panel were related to the unexpected effects of the Violet B dye on the laminate.","The visual aspect of the panel is blotchy, patchy, with some tears in the layer structure.","The colour is very varied from yellow to blue.","Conclusion Violet B is chemically not neutral with respect to IR dyes.","It destroys the activity of the IR dyes at higher temperatures within a short time.","The dyes should not be mixed in one layer OR a different violet dye is needed.","The surface tension of the PVB makes the two layered techniques vulnerable.","The smallest inhomogeneity in the layer thickness is apparent as a colour variation.","The greenish-yellow colour of the IR dye was matched to gray (before heating) but much higher levels of Violet B were needed than expected, hence the transparency of the laminate was decreased significantly.","ATAU 0.100 96 66 AUAU 0.038 96 71 ASAU 0.108 97 94 ASAV 0.069 93 92 AUAU 0.091 97 71 AUAV 0.098 80 76 AVAV 0.085 97 75 The FTNIR data and the calculation supported the IR930 absorption data The overall energy absorption is less than IR930 in the case of Epoline III-57 due to its lower absorption at longer wave length.","Some of the energy transmittance curves are shown in FIGS.","5 to 7.In a comparison of H.W.","Sands SDA 2072 to Epoline dyes, this corresponds to Epoline III-125.They FTNIR absorption curves are identical.","The glass with 5 mm of thickness has an energy absorption value of 24%, two glass plates have 44%, 0.5 mm thick PVB between two microscope slides has 24%.","The Final Samples The first sample was prepared using Epoline III-125 and Epolite III-57.First 50-50 and 100-100 mm sample sizes were tested and coated.","The necessary layer thickness was achieved using multiple layers from PVB.","The layers cracked after a complete drying and partly pealed from the glass surface.","The resulted laminates were inhomogeneous, the cracks could not have been eliminated neither by pressing together or at higher temperatures.","The 50-50 mm samples were FT-NIR tested and their energy absorption was found to be 73-86%.","A second sample was prepared using the Du Pont PVB film (0.375 m).","The glass surface was covered by one layer of BN 18 type of PVB containing either 2 mg Epoline III-125 or 2 mg of Epolite III-57 dyes per grams of resin and dissolved in 10 ml of analytical grade of methanol.","The layers were dried overnight than two glass plates containing different dyes were pressed together with an inner layer of Du Pont PVB film.","The glass laminates were put into an oven preheated to 135° C. and kept there for 15-20 min.","The laminates formed transparent layers.","The energy absorption of the laminates was tested on 50-50 mm samples.","The samples were prepared for the weathering test.","FIG.","8 shows the energy absorption spectrum of sample 122/126.The integrated absorption values are: Sample 122/126: 86%, sample 128/124: 73%, sample aq1/as1: 65% and as2/as2: 81%.","The homogeneity of the laminate was unsatisfactory.","The dyed PVB layer showed a very strong surface tension and had the tendency to collect into lines during the evaporation of the methanol.","The surface tension was big enough to remove the layer from great parts of the total surface.","Disregarding the inhomogeneities the demonstration laminates showed excellent heat filtering when tested by an IR lamp.","The hot component of the radiation of the lamp was missing.","The reference laminate without dye showed a burning heat radiation.","A third laminate was prepared using glass plates of 5 mm.","The dye containing PVB layer was not cast to the surface of the Du Pont PVB film.","First Epoline III-125 was cast to the one side of the film, then Epolite III-57 on the other side.","There was an overnight drying period between the two casts.","The film was dried again for 20 hours and then used to form laminate.","The 50-50 mm parallel sample showed good homogeneity and its energy absorption spectrum is shown in FIG.","9.The total absorption is 64% The density of the dyes is 0.14 g/m2.CONCLUSION IR dyes can be used to form laminates with heat resistant properties.","An overall concentration of the dyes of ˜0.1 g/m2 results in an energy absorption over 65%.","The laminates formed after a heat treatment under pressure at 135-140° C. for 15-20 min are pale lemon.","The laminates fill the requirements of the energy retardation.","The heating energy is not absorbed by the system which would result in the increase of the glass temperature.","The energy is conducted out of the laminate.","This kind of laminate may be used as security glazing for windows to reduce of the air conditioning costs of buildings in territories with very high solar heat radiation.","It will of course be realised that while the above has been given by way of illustrative example of this invention, all such and other modifications and variations thereto as would be apparent to persons skilled in the art are deemed to fall within the broad scope and ambit of this invention as defined in the claims appended hereto."]],"string":"[\n [\n \"Glazing laminates\",\n \"There is provided a glazing laminate comprising a polyvinyl butyral core layer having polyvinyl butyral/IR dye layers on each side.\",\n \"The dye containing layers are solvent cast from methanol onto the core layer.\",\n \"The dyes in each layer are different comprising BN 18 type PVB containing either 2 mg Epoline III-125 or 2 mg of Epolite III-57 dyes per grams of resin and dissolved in 10 ml of analytical grade of methanol for casting.\",\n \"The polymer is sandwiched between two glass layers under pressure and heat treatment at 135-140° C. An overall concentration of the dyes of ˜0.1 g/m2 results in an energy absorption over 65%.\",\n \"The heating effect of absorption is reduced by conduction through the polymer to the laminate edges, internal reflection, spherical reradiation and the relative insulative effect of the glass layers.\"\n ],\n [\n \"1.A laminated glazing element including an optically clear laminated sheet including at least two polymer layers having dispersed therein respective dyes, said layers having between them at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, said polymer layers being sandwiched between a UV absorbent outer support layer and an insulative inner support layer, said glazing element having at least one edge portion associated with a heat sink selected to disperse heat from said laminated sheet.\",\n \"2.The laminated glazing element according to claim 1, wherein said polymer layers are selected to have higher thermal conductivity than said inner support layer.\",\n \"3.The laminated glazing element according to claim 1, wherein said heat sink is configured to disperse heat to either the interior or exterior of a glazed structure from the at least one edge portion.\",\n \"4.A The laminated glazing element according to claim 1, wherein said heat sink is configured to selectively disperse the heat to the interior or exterior of the glazed structure.\",\n \"5.The laminated glazing element according to claim 1, wherein said heat sink is a thermal mass configured to radiate and/or convect heat.\",\n \"6.The laminated glazing element according to claim 1, wherein the IR dyes are selected from aminium, bisammonium and bisimmonium salt dyes.\",\n \"7.The laminated glazing sheet according to claim 1, wherein said polymer layers are selected from polyvinyl butyral (PVB) polymers.\",\n \"8.The laminated glazing element according to claim 1, wherein said polymer layers are selected from polyvinyl butryal (PVB) polymers.\",\n \"9.The laminated glazing element according to claim 1, wherein the inner support layer has a range of colouring dyes.\",\n \"10.The laminated glazing element according to claim 6, wherein one or more of the IR dyes have graduated wavelengths from 900 to 1700 nanometers.\",\n \"11.The laminated glazing sheet according to any of the preceding claims, excluding claim 7, wherein said polymers layers are selected from methyl acrylate polymers.\",\n \"12.The laminated glazing sheet according to any of the preceding claims 1 to 10, excluding claim 7, wherein said polymer layers are selected from polycarbonate polymers.\",\n \"13.The laminated glazing sheet according to claim 7, wherein a plolyvinyl butral layer is complimented by a liquid polycarbonate polymer containing selected dyes.\",\n \"14.A laminated glazing sheet including a polymer layer comprising a polyvinyl butyral having a hydroxyl content of at least 18 wt % and at least one aminium salt IR absorbing dye, said at least one dye being selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, and at least one UV absorbent outer support layer, wherein said polymer layer is formed by forming a mixture of methanolic solution of said polyvinyl butyral having a hydroxyl content of at least 18 wt % and a saturated methanolic solution of said at least one dye, and drying said mixture.\",\n \"15.A method of forming a glazing laminate including the steps of: (a) solvent casting a polyvinyl butyral film; (b) solvent casting a polyvinyl butyral/dye film to each surface of said polyvinyl butyral film, said polyvinyl butyral/dye films collectively including at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation; and (c) laminating the multilayer film of step (b) between glass sheets under heat and pressure.\",\n \"16.The laminated glazing element according to claim 7, wherein one or more of the IR dyes have graduated wavelengths from 900 to 1700 nanometers.\",\n \"17.The laminated glazing element according to claim 8, wherein one or more of the IR dyes have graduated wavelengths from 900 to 1700 nanometers.\",\n \"18.The laminated glazing element according to claim 9, wherein one or more of the IR dyes have graduated wavelengths from 900 to 1700 nanometers.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>This invention relates to glazing laminates.\",\n \"This invention has particular but not exclusive application to glazing laminates for glazing in construction, and for illustrative purposes reference will be made to such application.\",\n \"However, it is to be understood that this invention could be used in other applications, such as automotive glass and the like.\",\n \"A significant contributor to the cost of running commercial buildings or maintenance of comfort in residences is the energy required to provide control of the internal climate of the building.\",\n \"In buildings in hot climates having windows that are exposed to a relatively high flux of solar radiation, or buildings with large areas exposed to solar radiation, a major cost is air cooling by airconditioning.\",\n \"The conventional means of controlling heat passage into a building is to reduce the flux of solar radiation through the glazing by tinting to reduce a broad spectrum of radiation passing through the glass, with or without the provision of an outer partially reflective layer.\",\n \"By these means, a reduction of the total flux effects a partial reduction of the heat transmitted to the interior of the building.\",\n \"Clear window glass has a natural absorption maximum in the UV spectrum.\",\n \"Whilst the UV part of the solar spectrum at the Earth's surface is of high energy, the proportion of the total EM flux in the UV at the earth's surface is quite small due to upper atmospheric absorption.\",\n \"The visible spectrum is not substantially absorbed by clear window glass, but is quite strongly absorbed by tinted glass.\",\n \"The absorbed energy is converted to heat, which is in part spherically radiated in the infrared, and otherwise dispersed by convective transfer and conduction.\",\n \"The infrared component of the incident light is generally transmitted directly (with refraction) to the interior of the building in addition to the internally directed IR component arising from absorbed visible/UV radiation.\",\n \"In terms of energy density, the IR/visible bands contribute the greatest part if the incident energy density transmitted to the interior of a building through the windows or glass walls.\",\n \"In fact, energy density reaches a maximum at a wavelength of about 600 nm, with about 90% of the energy of solar radiation incident at the earth's surface being of wavelengths between 500 nm and 1750 nm, with several distinct maxima.\",\n \"Where tinting and/or partially reflective films is used, this generally reduces the visible flux and requires stronger lighting, which carries its own heat burden.\",\n \"In the case of tinting alone, the heat loading transmitted is only reduced by the proportion of IR reradiation back out of the building.\",\n \"International patent publication WO97/44690 discloses an optical element comprising a transparent layers comprising one or more passive layers and one or more active layers wherein said passive layers facilitate the transmission of electromagnetic radiation in a substantially unaltered form and the at least one active layers include an active material dispersed through the active layer and having the capacity to intercept electromagnetic radiation of a wavelength or range of wavelengths and redirect some of the energy of the intercepted radiation into the interior of the optical element, the layers being in face to face relationship and being optically coupled to each other.\",\n \"The described embodiments use a luminophore as the active material to absorb IR in the active layer and to spherically reemit IR by luminescent decay of the luminophore from the excited state.\",\n \"The basic concept of causing dissipation of IR by absorbance dye in a polymer film was shown to work, but no practical polymer film was produced.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>This invention in one aspect resides broadly in a laminated glazing sheet including at least one polymer layer having dispersed therein at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, and at least one UV absorbent outer support layer.\",\n \"The at least two dyes may be dispersed in one, both or more of the polymer layers depending on the number of layers, choice of polymers and compatibility of the dyes in polyemer dispersion in practice.\",\n \"The polymer layer may include a tint such as by means of a dye absorbing in the visible band.\",\n \"A practical difficulty experienced with such constructs is that suitable dyes tend to be barely compatible with the polymer matrices in which they are dispersed, and may be reactive with the IR dyes.\",\n \"The problems range from lack of homogeneity in monolayer films, films changed colour and destroyed the IR properties, and when laminated in two layers to keep the dyes separate, the panels were blotchy, patchy, uneven in colour and torn.\",\n \"Accordingly, in a further aspect this invention resides in a laminated glazing sheet including at least two polymer layers having dispersed therein respective dyes, said layers having between them at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, at least one other of said layers including a dye absorbing in the visible spectrum, a polymer interlayer between adjacent dye bearing ones of said polymer layers, and at least one UV absorbent outer support layer.\",\n \"The absorbed IR radiation will be for the most part dispersed in the laminate as heat.\",\n \"In order to minimize the heat transmitted to the interior of the building, the heat is preferably dispersed from the laminate to reduce heat build-up and the black body effect to the interior of a glazed structure.\",\n \"Accordingly, in a yet further aspect the present invention resides broadly in a laminated glazing element including an optically clear laminated sheet including at least two polymer layers having dispersed therein respective dyes, said layers having between them at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, said polymer layers being sandwiched between a UV absorbent outer support layer and an insulative inner support layer, said glazing element having at least one edge portion associated with a heat sink selected to disperse heat from said laminated sheet.\",\n \"The inner support layer is of an insulative material to reduce heat transfer to the interior of a glazed structure by convection.\",\n \"The at least one polymer layer may be selected from polymers that have a higher then usual thermal conductivity, and that the heat sink be configured to disperse heat to the outside of the glazed structure from the at least one edge thereof.\",\n \"For example, the heat sink may be a thermal mass being configured to either or both of radiate and convect heat to the exterior.\",\n \"Alternatively, the heat sink may be configured to selectively disperse the heat to the interior or exterior of the glazed structure.\",\n \"By this means, in winter the filtered IR may be used to heat the building, whilst in summer the trapped heat is disposed to the exterior.\",\n \"In one embodiment the heat sink is the atmosphere per se, rendered useful by installing the laminate having the at least one edge exposed.\",\n \"The relative refractive indices of the inner and outer support layers and the at least one polymer layer may be selected to maximize the amount of black body radiation which is internally reflected to the edges of the laminate.\"\n ],\n [\n \"BACKGROUND OF THE INVENTION This invention relates to glazing laminates.\",\n \"This invention has particular but not exclusive application to glazing laminates for glazing in construction, and for illustrative purposes reference will be made to such application.\",\n \"However, it is to be understood that this invention could be used in other applications, such as automotive glass and the like.\",\n \"A significant contributor to the cost of running commercial buildings or maintenance of comfort in residences is the energy required to provide control of the internal climate of the building.\",\n \"In buildings in hot climates having windows that are exposed to a relatively high flux of solar radiation, or buildings with large areas exposed to solar radiation, a major cost is air cooling by airconditioning.\",\n \"The conventional means of controlling heat passage into a building is to reduce the flux of solar radiation through the glazing by tinting to reduce a broad spectrum of radiation passing through the glass, with or without the provision of an outer partially reflective layer.\",\n \"By these means, a reduction of the total flux effects a partial reduction of the heat transmitted to the interior of the building.\",\n \"Clear window glass has a natural absorption maximum in the UV spectrum.\",\n \"Whilst the UV part of the solar spectrum at the Earth's surface is of high energy, the proportion of the total EM flux in the UV at the earth's surface is quite small due to upper atmospheric absorption.\",\n \"The visible spectrum is not substantially absorbed by clear window glass, but is quite strongly absorbed by tinted glass.\",\n \"The absorbed energy is converted to heat, which is in part spherically radiated in the infrared, and otherwise dispersed by convective transfer and conduction.\",\n \"The infrared component of the incident light is generally transmitted directly (with refraction) to the interior of the building in addition to the internally directed IR component arising from absorbed visible/UV radiation.\",\n \"In terms of energy density, the IR/visible bands contribute the greatest part if the incident energy density transmitted to the interior of a building through the windows or glass walls.\",\n \"In fact, energy density reaches a maximum at a wavelength of about 600 nm, with about 90% of the energy of solar radiation incident at the earth's surface being of wavelengths between 500 nm and 1750 nm, with several distinct maxima.\",\n \"Where tinting and/or partially reflective films is used, this generally reduces the visible flux and requires stronger lighting, which carries its own heat burden.\",\n \"In the case of tinting alone, the heat loading transmitted is only reduced by the proportion of IR reradiation back out of the building.\",\n \"International patent publication WO97/44690 discloses an optical element comprising a transparent layers comprising one or more passive layers and one or more active layers wherein said passive layers facilitate the transmission of electromagnetic radiation in a substantially unaltered form and the at least one active layers include an active material dispersed through the active layer and having the capacity to intercept electromagnetic radiation of a wavelength or range of wavelengths and redirect some of the energy of the intercepted radiation into the interior of the optical element, the layers being in face to face relationship and being optically coupled to each other.\",\n \"The described embodiments use a luminophore as the active material to absorb IR in the active layer and to spherically reemit IR by luminescent decay of the luminophore from the excited state.\",\n \"The basic concept of causing dissipation of IR by absorbance dye in a polymer film was shown to work, but no practical polymer film was produced.\",\n \"SUMMARY OF THE INVENTION This invention in one aspect resides broadly in a laminated glazing sheet including at least one polymer layer having dispersed therein at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, and at least one UV absorbent outer support layer.\",\n \"The at least two dyes may be dispersed in one, both or more of the polymer layers depending on the number of layers, choice of polymers and compatibility of the dyes in polyemer dispersion in practice.\",\n \"The polymer layer may include a tint such as by means of a dye absorbing in the visible band.\",\n \"A practical difficulty experienced with such constructs is that suitable dyes tend to be barely compatible with the polymer matrices in which they are dispersed, and may be reactive with the IR dyes.\",\n \"The problems range from lack of homogeneity in monolayer films, films changed colour and destroyed the IR properties, and when laminated in two layers to keep the dyes separate, the panels were blotchy, patchy, uneven in colour and torn.\",\n \"Accordingly, in a further aspect this invention resides in a laminated glazing sheet including at least two polymer layers having dispersed therein respective dyes, said layers having between them at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, at least one other of said layers including a dye absorbing in the visible spectrum, a polymer interlayer between adjacent dye bearing ones of said polymer layers, and at least one UV absorbent outer support layer.\",\n \"The absorbed IR radiation will be for the most part dispersed in the laminate as heat.\",\n \"In order to minimize the heat transmitted to the interior of the building, the heat is preferably dispersed from the laminate to reduce heat build-up and the black body effect to the interior of a glazed structure.\",\n \"Accordingly, in a yet further aspect the present invention resides broadly in a laminated glazing element including an optically clear laminated sheet including at least two polymer layers having dispersed therein respective dyes, said layers having between them at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, said polymer layers being sandwiched between a UV absorbent outer support layer and an insulative inner support layer, said glazing element having at least one edge portion associated with a heat sink selected to disperse heat from said laminated sheet.\",\n \"The inner support layer is of an insulative material to reduce heat transfer to the interior of a glazed structure by convection.\",\n \"The at least one polymer layer may be selected from polymers that have a higher then usual thermal conductivity, and that the heat sink be configured to disperse heat to the outside of the glazed structure from the at least one edge thereof.\",\n \"For example, the heat sink may be a thermal mass being configured to either or both of radiate and convect heat to the exterior.\",\n \"Alternatively, the heat sink may be configured to selectively disperse the heat to the interior or exterior of the glazed structure.\",\n \"By this means, in winter the filtered IR may be used to heat the building, whilst in summer the trapped heat is disposed to the exterior.\",\n \"In one embodiment the heat sink is the atmosphere per se, rendered useful by installing the laminate having the at least one edge exposed.\",\n \"The relative refractive indices of the inner and outer support layers and the at least one polymer layer may be selected to maximize the amount of black body radiation which is internally reflected to the edges of the laminate.\",\n \"DETAILED DECRIPTION OF THE INVENTION The polymer material comprising respective ones of the at least two polymer layers may be the same or different from each other, and may respectively be the same or different from the polymer interlayer.\",\n \"In order to provide a constant refractive milieu, the polymer of the at least two polymer layers are the same, and preferably all polymers layers are of the same material.\",\n \"The polymers may be of any known optically clear film forming or castable thermoplastic or thermoset resin, selected on the basis of compatibility with the respective dyestuffs.\",\n \"The respective polymers may be formed into films and assembled into the layered structure in an appropriate forming process such as pressing with or without the application of heat, or the like.\",\n \"Where the polymers are extrudable thermoplastics, the layers of polymer may be concurrently formed such as by coextrusion.\",\n \"For example, where the polymers permit, the polymer layers may be formed by a melt blowing process such as the double bubble process for the production of laminated films.\",\n \"In order to permit intimate lamination of the polymers with disparate materials such as glass by thermal pressing or the like, the polymer may be a thermoplastic or a crosslinked polymer that is thermoplastic or thermoset with a thermoplastic uncrosslinked form.\",\n \"The polymer may be selected as to refractive index relative to the UV absorbent outer support layer so as to provide for a degree of total internal reflection of spherically dispersed long-wave IR emanating from the polymer layers in use.\",\n \"By this means a degree of selective transmission of energy absorbed by said dyes is to the periphery of the sheet by conduction and a degree of total internal reflection.\",\n \"The respective dyes may be selected having regard to compatibility with the polymer of choice.\",\n \"The IR dye may for example be selected from organic dyes, aminium, bisammonium and bisimmonium salt dyes, or metal salts of organic acids.\",\n \"It has surprisingly been found that polyvinyl butyral (PVB) polymers are compatible with aminium, bisammonium and bisimmonium salt dyes, a specific class of dyes which are generally N-organic salts in which the counter anion is derived from the salt of a strong acid such as ClO4−, PF6−, AsF6−, SbF6− and the like.\",\n \"For example the dye may be divalent immonium salts which are near infrared dyes of the formula (I).\",\n \"(R2NAr)2—N+═Art═N+R2+2X− (I) wherein R═C1 to C6 alkyl; Ar=divalent phenyl which may or may not be ring substituted with one or more alkyl, alkoxy, halogen, nitro, cyano or carboalkoxy groups; Art=quinoidal phenyl which may or may not be ring substituted with one or more alkyl, alkoxy, halogen, nitro, cyano or carboalkoxy groups; and X=an anion of a strong acid, such as an SbF6− anion.\",\n \"Experimental work such as that of Resch-Genger U. and Wolfbeis, O., “Near Infrared Dyes for High Technology Applications”, (Kluwer Academic Publishers) has resulted in the production of a series of such dyes including those of the Epolin series.\",\n \"Typical structures of amimium and bisammonium salt dyes are given in formulae II and III respectively.\",\n \"It is envisaged that many other polymer/dye combinations may be appropriate.\",\n \"The visible spectrum dyes may be selected from dyes known for the purpose.\",\n \"The dyes may be dispersed in the polymer layers by any suitable means.\",\n \"The dyes may for example be directly blended into the polymer material prior to casting or forming the polymer layer.\",\n \"Alternatively, the dye may be dissolved in a solvent which is preferably a cosolvent for the polymer, whereby the polymer layer may be formed as a cast film by solvent evaporation.\",\n \"The UV absorbent outer support layer is preferably glass, but may also be a further polymer layer doped with a suitable UV absorbent substance.\",\n \"The UV absorbent outer support layer is preferably selected to have refractive qualities relative to those of the polymer layers whereby the boundary between the polymer layer and the UV absorbent outer support layer forms a surface for internal reflection of longwave IR emitted by the warmed polymer layers.\",\n \"According to a further preferred feature of the invention at least a portion of the edge of the optically clear laminated sheet is configured to facilitate emission of the emitted radiation from the optical element.\",\n \"In one form at least some of the edges may be chamfered.\",\n \"Alternatively the sheet edges may be in contact with an absorber comprising a conductive thermal mass such as metal.\",\n \"The thermal mass may include means to disperse heat to the outside of the building, or to convective chimneys in the walls.\",\n \"The heat may be recovered by exchange means such as water to be harvested for heating or heat pump applications.\",\n \"At least one face of the optically clear laminated sheet may be formed with a discontinuity to facilitate the emission of emitted radiation from the optical element.\",\n \"The form of discontinuity may comprise at least one groove formed in the at least one face.\",\n \"Another form of discontinuity may comprise one or more depressions or dimples provided in the at least one face.\",\n \"Another form of discontinuity may comprise at least one rib or protrusion on the at least one face.\",\n \"Another form of discontinuity may comprise an etched surface on a portion of the at least one face.\",\n \"It has surprisingly been determined that the suitability of a particular dye/polymer combination in a film does not in general predict the performance or stability of the polymer layers in a laminate.\",\n \"Without wishing to be bound by theory, it is believed that the dyes in adjacent layers of a laminate exhibit a cross reactivity that can affect the IR performance, the stability of the dye systems, and even the physical properties of the polymers in the laminate.\",\n \"It has been found that the provision of a polymer interlayer reduces the interactions between the respective dye/polymer systems, and that as a preference the same polymer may be used in the dye-containing layers and interlayers to provide a substantially consistent refractive qualities in the visible band.\",\n \"It has been found that the provision of a UV absorbent outer layer tends to improve the stability of the polymers and polymer/dye systems.\",\n \"In one aspect the invention resides in a method of forming a glazing laminate including the steps of: (a) solvent casting a polyvinyl butyral film; (b) solvent casting a polyvinyl butyral/dye film to each surface of said polyvinyl butyral film, said polyvinyl butyral/dye films collectively including at least two dyes selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation; and (c) laminating the multilayer film of step (b) between glass sheets under heat and pressure.\",\n \"It has further been determined that certain compositions for film forming by solvent evaporation and comprising relatively reactive dyes and polymers having reactive groups may be stabilized by selection of the solvent.\",\n \"For example, polyvinyl butyrals are polymers having significant hydroxyl functionality.\",\n \"Such polymers are usually worked up and are stable in technical grade ethanol.\",\n \"Aminium salt dyes such as the EPOLIN dyes are also generally worked up in polar solvents such as acetone, and are reasonably soluble in ethanol.\",\n \"However, in the use of polyvinyl butyral in forming the dye containing polymer layers of the present invention, the PVB/ethanol//dye/ethanol and PVB/ethanol//dye/acetone systems results in films of poor physical characteristics and markedly reduced IR performance.\",\n \"The films so produced were also intractable in being formed up under heat and pressure to glass substrates, with excessive bubble formation.\",\n \"However, despite limited solubility of aminium salt dyes such as aminium salts in methanol, it has been surprisingly found that solvent cast films based on a PVB/methanol//dye/methanol system exhibit superior film forming and dye-stability performance.\",\n \"The polymer compositions formed by this system have also shown an ability to be subsequently melt-processed such as by extrusion into films with suitable physical and IR dye performance.\",\n \"In a further aspect the present invention relates to a film forming polymer composition comprising a mixture of methanolic solution of a polyvinyl butyral having a hydroxyl content of at least 18 wt % and a saturated methanolic solution of an aminium salt IR absorbing dye.\",\n \"In a yet further aspect of the present invention, there is provided a method of forming an IR absorbing polymer film comprising the steps of forming a mixture of methanolic solution of a polyvinyl butyral having a hydroxyl content of at least 18 wt % and a saturated methanolic solution of an aminium salt IR absorbing dye, forming said mixture into a liquid layer, and drying said layer to a film by solvent evaporation.\",\n \"In a yet further aspect of the present invention, there is provided a method of forming an IR absorbing polymer film comprising the steps of forming a mixture of methanolic solution of a polyvinyl butyral having a hydroxyl content of at least 18 wt % and a saturated methanolic solution of an aminium salt IR absorbing dye, drying said mixture, and melt extruding said dried mixture to form a film.\",\n \"It has further been determined that such cast and extruded films may include more than one dye and may further be laminated to form an optically clear laminated sheet.\",\n \"Accordingly, in another aspect there may be provided an optically clear laminated sheet including a polymer layer comprising a polyvinyl butyral having a hydroxyl content of at least 18 wt % and at least one aminium salt IR absorbing dye, said dyes being selected to absorb IR radiation of different or overlapping ranges of frequencies, said ranges including frequencies corresponding to IR intensity peaks in incident solar radiation, and at least one UV absorbent outer support layer, wherein said polymer is formed by forming a mixture of methanolic solution of said polyvinyl butyral having a hydroxyl content of at least 18 wt % and a saturated methanolic solution of said dyes, and drying said mixture.\",\n \"In order that this invention may be more readily understood and put into practical effect, reference will now be made to the following examples which illustrates preferred embodiments and comparative formulations in respect of the invention, and wherein: FIG.\",\n \"1 is a FT-NIR absorption of dye combinations in polymers in accordance with the present invention; FIG.\",\n \"2 is a FT-NIR absorption of Epolite 125 and Epoline III-57 combinations in polymers in accordance with the present invention; FIG.\",\n \"3 is a FT-NIR absorption of Epolite dye combinations in polymers in accordance with the present invention; FIG.\",\n \"4 is the energy spectrum of the Sun; FIG.\",\n \"5 is the energy absorption of laminates of glass and PVB; FIG.\",\n \"6 is the energy absorption of some Epolin combinations; FIG.\",\n \"7 is the energy absorption spectra of H.W.\",\n \"Sands dye systems; FIG.\",\n \"8 is the energy absorption spectrum of a sample; FIG.\",\n \"9 is the energy absorption spectrum of a sample, and FIG.\",\n \"10 is the energy absorption spectrum of a sample.\",\n \"EXAMPLE 1 Methanol was tested for the dissolution of the dyes and of the PVB.\",\n \"BN18 polymer (Wacker-Chemie, PIOLOFORM) was used.\",\n \"Initally 2 mg dye/1 g of PVB mixture was produced and dissolved in ethanol forming a ˜40% solution with respect to the resin.\",\n \"This solution was diluted by methanol to have an overall concentration of 20% of resin.\",\n \"The glass plates were cast in consecutive steps by this solution.\",\n \"During the drying process the layer turned to be turbulent then it cleared.\",\n \"The next consecutive layer was cast in this stage.\",\n \"A dye density of 0.010-0.026 g/m2 covered the individual slides.\",\n \"Additional layers of PVB were cast to have an expected layer thickness of 0.05 mm after the heat treatment.\",\n \"Following by an overnight drying the slides were heat treated at 135° C. for 5-10 min.\",\n \"Two slides were pressed together to have a homogeneous and transparent layer.\",\n \"Four dyes were tested.\",\n \"Epolite 125-(AN), Epolite 178 (AM), Epoline III-57 (AL) and Spectra IR 920 (AO).\",\n \"The combinations and their properties are shown in Table I.\",\n \"TABLE 1 Thickness, Combination Density of dyes, g/m2 IR930 absorption, % μm AN-AN 0.052 57 515 AN-AM 0.050 50 495 AN-AL 0.047 53 473 AN-AO 0.038 60 517 AM-AM 0.049 33 412 AM-AL 0.060 67 450 AM-AO 0.046 50 530 AL-AL 0.065 87 525 AL-AO 0.059 67 525 AO-AO 0.017 17 522 PVB 0 3 552 The near infrared (NIR) spectra of the samples were determined by a Perkin-Elmer FT spectrometer from 13000 to 4000 cm−1 energy range what corresponds to the wave length of 670 to 2500 nm.\",\n \"FIG.\",\n \"1 shows the absorption spectra of the different combinations.\",\n \"Epoline III-57 has an extraordinary high absorption at the wavelength near to 1000 nm but it has practically no absorption at longer wavelengths.\",\n \"Epolite 125 has good absorption at the lower wavelengths but it is relatively ineffective at the higher energy range.\",\n \"The IR absorption of the combinations of these two dyes are shown in FIG.\",\n \"2.Next a solution of Epolite 125 and Epolite 178 was prepared in pure methanol in 8 mg dye/1 g of resin.\",\n \"Epolite 178 did not dissolve completely in the solution.\",\n \"One layer of this solution was cast on glass and combined by itself and by double layers of Epoline III-57 of 1 mg dye in 1 g of resin of the methanol solution.\",\n \"The density of the dye and the IR930 absorption with the layer thickness are given in Table II.\",\n \"TABLE II Thickness, Combination Density of dyes, g/m2 IR930 absorption, % μm 57/25.78 0.06/0.95 92 300 25.78/57 0.95/0.06 86 320 25.78/25.78 2.36 95 135 57/57 0.11 90 230 Transparent layers of yellow/green colour were formed.\",\n \"The FT-NIR absorption of the layers are shown in FIG.\",\n \"3.The layers have extremely high NIR absorption.\",\n \"There might be some scaling error in the equipment showing absorption values higher than 1.The IR930 absorption values correspond to the FT-NIR data.\",\n \"Epolite 125 assures high absorption values at longer wavelength, Epoline III-57 assures the good absorption at the range of 1000 nm, what corresponds to the middle of the IR energy peak of the incident solar radiation spectrum (see in FIG.\",\n \"4).\",\n \"The FT-NIR absorption of the PVB with a layer thickness of 0.5 mm enclosed in between two glass sheets of 1 mm thickness is shown in FIG.\",\n \"5.The Figure also shows the absorption of a single and a double sheets of glass plates of 3 mm thickness.\",\n \"This kind of glass plates is supposed to be used for the final sample preparation.\",\n \"PVB has homogeneously low absorption up to a wavelength of 2000 nm, than there are two absorbing bands.\",\n \"These bands are also visible in FIGS.\",\n \"1-3.The thick glass plates show higher and homogeneous absorption level for the whole range with a broad peak at the 900-1400 nm range.\",\n \"Conclusion Epolite 125 and Epoline III-57 dyes are suitable for the preparation of the laminates.\",\n \"They can be employed in a concentration of 0.1-0.3 g/m2.This amount of dyes can be cast as couple of layers of 2 mg dye in 1 g of BN18 resin from a methanol solution on a glass surface.\",\n \"The necessary layer thickness is achieved by casting of additional layers of PVB from methanol solution.\",\n \"Two coated glass surfaces are heated and pressed together after having been properly dried at temperature of 135° C. to form the laminated glass of the present invention.\",\n \"EXAMPLE 2 Two new dyes were obtained from Epolin Inc. for the purpose of producing blue and grey laminates.\",\n \"The dyes were Sole Blue 33 and Violet B. Epolin give the ratio of colour dye with respect to IR dye of 0.1-0.15 w/w.\",\n \"Types of PVB (BN18) and glass used were as previously reported.\",\n \"Methods for preparation were used as previously developed.\",\n \"Results Sole Blue 33 dissolved well in methanol (5 mg/10 ml) but Violet B was soluble only up to 1 mg/10 ml.\",\n \"Initial experiments on a small scale were carried out to match the colour to blue or gray from that of the IR dye (i.e.\",\n \"green-yellow).\",\n \"Levels of the new dye of 0.1, 0.3 and 1 w/w dye per IR dye were tested.\",\n \"4 mg/g IR dye with respect to PVB was used dissolved in 10 ml MeOH/g of PVB.\",\n \"Sole Blue 33 dissolved properly at each concentration.\",\n \"However, it did not produce a good colour match—it formed various green colours.\",\n \"Violet B did not dissolve perfectly at the highest concentration—it also produced only a yellow-brown colour.\",\n \"Some particles remained not dissolved at each concentration indicating some inhomogeneities in the dye.\",\n \"During heat treatment at 135° C. the undissolved part of Violet B dissolved and produced very heterogeneous samples i.e.\",\n \"the colour varied throughout the sample.\",\n \"It was theorized that new solvents were needed to overcome these problems.\",\n \"Acetone/butanol-1 (4:1) solvent was selected for the next set of experiments, 2 mg/g IR dye was used (1.25 mg/g Epolight III-57 and 0.75 mg/g Epolight III-25 with respect to the PVB).\",\n \"Violet B was added at 0.3 w/w with respect to the IR dye.\",\n \"10 ml acetone mixed with 2.5 ml butanol-1 was used for 1 g of PVB.\",\n \"A clear grey colour formed.\",\n \"2 or 3 layers of mixture were cast to glass, dried and heat treated at 135° in the oven, as required to form the flat adhered laminate structure needed for window glass.\",\n \"Unfortunately, the colour changed.\",\n \"The grey became violet.\",\n \"FTNIR spectra showed that the IR dye function had been destroyed, its initial good IR absorption decreased to less than one third of its initial value.\",\n \"IR transmission measured by laser showed the same result.\",\n \"These results show that the Violet B dye destroyed the IR dye after a short heat treatment at 135° for 3-5 min.\",\n \"To overcome this problem a sample was prepared with two separate layers, one layer containing Violet B and one the IR dye.\",\n \"A 400×400 ml panel was prepared using two independent layers, one with IR dye cast to one glass plate, the other with Violet B to the other glass plate.\",\n \"The IR dye containing layer was prepared using 128 mg of IR dye (80 mg Epolight III-57 and 48 mg of Epolight III-125) mixed to 32 g of PVB (4 mg/g), what corresponds to 0.8 g dye/m2.The other layer contained 53 mg of Violet B dispersed in 32 g PVB (0.4 w/w Violet blue with respect or IR dye).\",\n \"Both glasses were cast in two steps using acetone/butanol-1 solvent and the solvent was evaporated overnight.\",\n \"Heat treatment in the oven at 135° was performed for 5 min and then the plates were pressed together.\",\n \"During the heat treatment a 5 kg weight was on the laminate, as previously developed.\",\n \"During this treatment the layer of Violet B unexpectedly coagulated, lost its lamellar shape and formed totally inhomogeneous laminate.\",\n \"The IR dye containing layer remained intact as expected—FTNIR spectra of the system showed that absorption was homogeneous across the layer, indicating that the IR dye remained intact.\",\n \"There was a small reduction in absorption power in some spots where there was a high concentration of Violet B.\",\n \"Hence the problems with the large panel were related to the unexpected effects of the Violet B dye on the laminate.\",\n \"The visual aspect of the panel is blotchy, patchy, with some tears in the layer structure.\",\n \"The colour is very varied from yellow to blue.\",\n \"Conclusion Violet B is chemically not neutral with respect to IR dyes.\",\n \"It destroys the activity of the IR dyes at higher temperatures within a short time.\",\n \"The dyes should not be mixed in one layer OR a different violet dye is needed.\",\n \"The surface tension of the PVB makes the two layered techniques vulnerable.\",\n \"The smallest inhomogeneity in the layer thickness is apparent as a colour variation.\",\n \"The greenish-yellow colour of the IR dye was matched to gray (before heating) but much higher levels of Violet B were needed than expected, hence the transparency of the laminate was decreased significantly.\",\n \"ATAU 0.100 96 66 AUAU 0.038 96 71 ASAU 0.108 97 94 ASAV 0.069 93 92 AUAU 0.091 97 71 AUAV 0.098 80 76 AVAV 0.085 97 75 The FTNIR data and the calculation supported the IR930 absorption data The overall energy absorption is less than IR930 in the case of Epoline III-57 due to its lower absorption at longer wave length.\",\n \"Some of the energy transmittance curves are shown in FIGS.\",\n \"5 to 7.In a comparison of H.W.\",\n \"Sands SDA 2072 to Epoline dyes, this corresponds to Epoline III-125.They FTNIR absorption curves are identical.\",\n \"The glass with 5 mm of thickness has an energy absorption value of 24%, two glass plates have 44%, 0.5 mm thick PVB between two microscope slides has 24%.\",\n \"The Final Samples The first sample was prepared using Epoline III-125 and Epolite III-57.First 50-50 and 100-100 mm sample sizes were tested and coated.\",\n \"The necessary layer thickness was achieved using multiple layers from PVB.\",\n \"The layers cracked after a complete drying and partly pealed from the glass surface.\",\n \"The resulted laminates were inhomogeneous, the cracks could not have been eliminated neither by pressing together or at higher temperatures.\",\n \"The 50-50 mm samples were FT-NIR tested and their energy absorption was found to be 73-86%.\",\n \"A second sample was prepared using the Du Pont PVB film (0.375 m).\",\n \"The glass surface was covered by one layer of BN 18 type of PVB containing either 2 mg Epoline III-125 or 2 mg of Epolite III-57 dyes per grams of resin and dissolved in 10 ml of analytical grade of methanol.\",\n \"The layers were dried overnight than two glass plates containing different dyes were pressed together with an inner layer of Du Pont PVB film.\",\n \"The glass laminates were put into an oven preheated to 135° C. and kept there for 15-20 min.\",\n \"The laminates formed transparent layers.\",\n \"The energy absorption of the laminates was tested on 50-50 mm samples.\",\n \"The samples were prepared for the weathering test.\",\n \"FIG.\",\n \"8 shows the energy absorption spectrum of sample 122/126.The integrated absorption values are: Sample 122/126: 86%, sample 128/124: 73%, sample aq1/as1: 65% and as2/as2: 81%.\",\n \"The homogeneity of the laminate was unsatisfactory.\",\n \"The dyed PVB layer showed a very strong surface tension and had the tendency to collect into lines during the evaporation of the methanol.\",\n \"The surface tension was big enough to remove the layer from great parts of the total surface.\",\n \"Disregarding the inhomogeneities the demonstration laminates showed excellent heat filtering when tested by an IR lamp.\",\n \"The hot component of the radiation of the lamp was missing.\",\n \"The reference laminate without dye showed a burning heat radiation.\",\n \"A third laminate was prepared using glass plates of 5 mm.\",\n \"The dye containing PVB layer was not cast to the surface of the Du Pont PVB film.\",\n \"First Epoline III-125 was cast to the one side of the film, then Epolite III-57 on the other side.\",\n \"There was an overnight drying period between the two casts.\",\n \"The film was dried again for 20 hours and then used to form laminate.\",\n \"The 50-50 mm parallel sample showed good homogeneity and its energy absorption spectrum is shown in FIG.\",\n \"9.The total absorption is 64% The density of the dyes is 0.14 g/m2.CONCLUSION IR dyes can be used to form laminates with heat resistant properties.\",\n \"An overall concentration of the dyes of ˜0.1 g/m2 results in an energy absorption over 65%.\",\n \"The laminates formed after a heat treatment under pressure at 135-140° C. for 15-20 min are pale lemon.\",\n \"The laminates fill the requirements of the energy retardation.\",\n \"The heating energy is not absorbed by the system which would result in the increase of the glass temperature.\",\n \"The energy is conducted out of the laminate.\",\n \"This kind of laminate may be used as security glazing for windows to reduce of the air conditioning costs of buildings in territories with very high solar heat radiation.\",\n \"It will of course be realised that while the above has been given by way of illustrative example of this invention, all such and other modifications and variations thereto as would be apparent to persons skilled in the art are deemed to fall within the broad scope and ambit of this invention as defined in the claims appended hereto.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471272"}}},{"rowIdx":585,"cells":{"text":{"kind":"list like","value":[["Micromechanical capacitive acceleration sensor","A micromechanical capacitive acceleration sensor is described for picking up the acceleration of an object in at least one direction.","The sensor includes a frame structure (110), a sensor inertia mass (101) made of a wafer and movably mounted relative to the frame structure (110) about a rotation axis, and a capacitive pick-up unit (120) for producing at least one capacitive output signal representing the position of the sensor mass (101) relative to the frame structure (110).","The sensor inertia mass (101) has a center of gravity which offset relative to the rotation axis in a direction perpendicularly to a wafer plane for measuring accelerations laterally to the wafer plane.","The sensor mass (101) and the frame structure (110) are made monolithically of one single crystal silicon wafer.","A cover section (112) forms a common connector plane (150) for the connection of capacitor electrodes (125,126).","Torqueable elements (105) form an electrically conducting bearing device for the sensor mass (101)."],["1.A micromechanical capacitive acceleration sensor for picking up the acceleration of an object in at least one direction, said sensor comprising a frame structure, a sensor inertia mass made of a wafer movably supported about a rotation axis (Ax, Ay) relative to said frame structure, and a capacitive pick-up unit for producing at least one capacitive output signal representing the position of said sensor inertia mass relative to said frame structure, wherein said sensor inertia mass comprises a center of gravity (Mx, My) which is offset relative to said rotation axis (Ax, Ay) in a direction extending perpendicularly to a plane of said wafer.","2.The acceleration sensor of claim 1, wherein said sensor inertia mass and/or said frame structure are made monolithically of a single crystal silicon wafer.","3-48.","(canceled) 49.The acceleration sensor of claim 1, further comprising a bearing device including torqueable elements made of silicon for supporting said sensor inertia mass.","50.The acceleration sensor of claim 1, further comprising an electrical contacting on said frame structure and a bearing device for supporting said sensor inertia mass, said bearing device forming an electrically conducting connection between said sensor inertia mass and said electrical contacting on said frame structure.","51.The acceleration sensor of claim 1, further comprising a bearing device arranged on said sensor inertia mass close to a surface of said wafer.","52.The acceleration sensor of claim 1, wherein said sensor inertia mass has a shape selected from any one of the following shapes: a cubic shape, a parallelepiped, a frustum and a truncated pyramid.","53.The acceleration sensor of claim 1, comprising a plurality of sensor inertia masses movably mounted relative to a respective rotation axis (AX, AY, AZ) for measuring accelerations in a plurality of directions (x, y, z) extending perpendicularly to one another, wherein one or more sensor inertia masses respectively have a center of gravity (MZ, MY) which is offset relative to its respective rotation axis (AX, AY) in a direction extending perpendicularly to a plane of said wafer.","54.The acceleration sensor of claim 1, wherein said center of gravity of said sensor inertia mass is arranged relative to the respective rotation axis (AX, AY) and laterally displaced in a direction of said wafer plane.","55.The acceleration sensor of claim 1, wherein said capacitive pick-up unit comprises first capacitor electrodes provided on said sensor inertia mass and second capacitor electrodes provided on said frame structure opposite said first capacitor electrodes, said pick-up unit further comprising first connector elements for electrically connecting said first capacitor electrodes and/or second connector elements electrically connecting said second capacitor electrodes, said first and second connector elements being arranged in a common connector plane.","56.The acceleration sensor of claim 55, wherein said first connector elements and/or said second connector elements are contacted on a single side.","57.The acceleration sensor of claim 1, wherein said capacitive pick-up unit is contacted electrically on a single side.","58.The acceleration sensor of claim 55, wherein said second capacitor electrodes on said frame structure and said first and second connector elements are arranged on or in said common connector plane.","59.The acceleration sensor of claim 1, further comprising a coupling arrangement for coupling of first capacitor electrodes provided on said sensor inertia mass to first connector elements provided on said frame structure, said coupling arrangement comprising a galvanic or capacitive connection for bridging an elevational difference between said first capacitor electrodes and a common connector plane.","60.The acceleration sensor of claim 1, wherein said frame structure comprises a central frame section laterally enclosing said sensor inertia mass and a first cover section elevationally displaced relative to said sensor inertia mass and arranged on said central frame section and second capacitor electrodes provided on a facing side of said first cover section which faces said sensor inertia mass.","61.The acceleration sensor of claim 60, wherein said facing side of said first cover section facing said sensor inertia mass forms a common connector plane for an electrical connection of said capacitive pick-up unit.","62.The acceleration sensor of claim 55, wherein said second capacitor electrodes are formed either by a metallizing layer on said sensor inertia mass or by said sensor inertia mass that is made of an electrically conducting material.","63.A micromechanical capacitive acceleration sensor, for picking up acceleration of an object in at least one direction (x, y, z), comprising a frame structure which is stationary relative to said object, a sensor inertia mass which is movable relative to said frame structure, a bearing device elastically supporting said sensor inertia mass relative to said frame structure for movement about a starting position, said capacitive acceleration sensor further comprising a capacitive pick-up unit for producing at least one capacitive output signal representing a position of said sensor inertia mass relative to said frame structure, said capacitive pick-up unit comprising first capacitor electrodes provided on said sensor inertia mass and second capacitor electrodes provided on said frame structure opposite said first capacitor electrodes, and a coupling arrangement including first connector elements coupled to said first capacitor electrodes, said first connector elements being provided on said frame structure, and second connector elements coupled to said second capacitor electrodes, and wherein said first connector elements and/or said second connector elements are arranged in a common connector plane which is elevationally displaced relative to said first capacitor electrodes provided on said sensor mass and/or relative to said second capacitor electrodes provided on said frame structure.","64.The acceleration sensor of claim 63, wherein said coupling arrangement comprises means for bridging an elevational difference between said first capacitor electrodes and/or said second capacitor electrodes and said common connector plane.","65.The acceleration sensor of claim 63, wherein said second capacitor electrodes provided on said frame structure and said first and second connector elements are arranged in or on said common connector plane, and wherein said coupling arrangement comprises means for bridging an elevational difference to said common connector plane.","66.The acceleration sensor of claim 63, wherein said coupling arrangement forms a galvanic or capacitive connection which bridges said elevational difference to said common connector plane.","67.The acceleration sensor of claim 63, wherein said frame structure comprises a central section laterally surrounding said sensor inertia mass and a first cover section elevationally displaced relative to said sensor inertia mass and arranged on said central section, wherein said second capacitor electrodes are provided on that side of said first cover section which faces said sensor inertia mass, and wherein said first capacitor electrodes are provided on said sensor inertia mass opposite said second capacitor electrodes.","68.The acceleration sensor of claim 67, wherein said common connector plane in which said first connector elements and said second connector elements are arranged, is formed by that side of said first cover section which faces said sensor inertia mass and wherein said second capacitor electrodes are also provided in or on said side of said first cover section facing said sensor inertia mass.","69.The acceleration sensor of claim 68, wherein said first connector elements, said second connector elements and said second capacitor electrodes which are connected to the second connector elements, are made of a single conductive layer formed on the side of the first cover section which faces said sensor inertia mass.","70.The acceleration sensor of claim 69, further comprising a second coupling electrode formed on that side of said first cover section which faces said sensor inertia mass, wherein a first coupling electrode is provided on said sensor inertia mass opposite said second coupling electrode, whereby said first and second coupling electrodes form a capacitive connection of the coupling arrangement for coupling the first capacitor electrodes provided on the sensor inertia mass with a first connector element provided on said frame structure.","71.The acceleration sensor of claim 70, wherein said second coupling electrode and said first connector element are provided in said common connector plane which is formed by that side of said first cover section which faces the sensor inertia mass.","72.The acceleration sensor of claim 71, wherein said second coupling electrode and said first connector element connected to said first coupling electrode, said second capacitor electrodes and said second connector elements connected with the second capacitor electrodes are made of a single conducting layer formed on that side of said first cover section which faces said sensor inertia mass.","73.The acceleration sensor of claim 67, further comprising a second cover section provided elevationally displaced relative to said sensor inertia mass, said second cover section being positioned at said central section opposite said first cover section, said second cover section forming together with said central section and with said first cover section a hermetically sealed housing enclosing said sensor inertia mass, said first capacitor electrodes and said second capacitor electrodes.","74.The acceleration sensor of claim 63, wherein said bearing device comprises torsion elements to which said sensor inertia mass is mounted to the frame structure in a torsion elastic manner for motion relative to and about said starting position.","75.The acceleration sensor of claim 74, wherein said two torsion elements forming said bearing device are positioned relative to a common rotation axis, said two torsion elements being effective on opposite sides of said sensor inertia mass, whereby said sensor inertia mass is movable only relative to a single direction (x, y, z) relative to the frame structure.","76.The acceleration sensor of claim 74, wherein said torsion elements are rigidly secured, on the one hand, to said sensor inertia mass and, on the other hand, to said central section of said frame structure.","77.The acceleration sensor of claim 74, wherein said coupling arrangement is formed by a galvanic connection which includes said torsion elements.","78.The acceleration sensor of claim 63, wherein said bearing device comprises one or more bending elements on which said sensor inertia mass is supported in a bending elastic manner around said starting position and relative to said frame structure.","79.The acceleration sensor of claim 78, wherein two bending elements are provided in a common plane spaced from each other and rigidly connected to said sensor inertia mass, whereby said sensor inertia mass is movable only in a single direction (x) relative to said frame structure.","80.The acceleration sensor of claim 78, wherein two bending elements are rigidly connected, on the one hand, to the sensor inertia mass and, on the other hand, to said first cover section of said frame structure.","81.The acceleration sensor of claim 78, wherein said coupling arrangement is formed by a galvanic connection which includes said bending elements.","82.The acceleration sensor of claim 78, comprising a single bending element which is centrally and rigidly connected to said sensor inertia mass, whereby said sensor inertia mass is movable with respect to two different directions (x, y) relative to said frame structure, and wherein said pick-up unit is adapted for producing two capacitive output signals which represent the position of said sensor inertia mass relative to said frame structure.","83.The acceleration sensor of claim 82, wherein said pick-up unit comprises four first capacitor electrodes secured to said sensor inertia mass in pairs positioned opposite each other and on opposite sides of said bending element, and four second capacitor electrodes secured to said first cover section of said frame structure, said second capacitor electrodes being arranged in pairs positioned opposite each other on opposite sides of said bending element and opposite said first capacitor electrodes.","84.The acceleration sensor of claim 82, wherein said coupling arrangement comprises a first coupling electrode provided on said sensor inertia mass and surrounding said bending element, and a second coupling electrode provided on said first cover section of said frame structure, and wherein said second coupling electrode is positioned opposite said first coupling electrode and surrounding said bending element.","85.The acceleration sensor of claim 82, wherein said coupling arrangement comprises a galvanic connection including said bending element.","86.The acceleration sensor of claim 1, comprising a plurality of sensor inertia masses and wherein each sensor inertia mass is equipped with a respective capacitive pick-up unit for picking up the acceleration of an object in a plurality of directions (x, y, z), a plurality of bearing devices, each of said plurality of sensor inertia masses being supported by a respective bearing device in said frame structure independently of one another, each sensor inertia mass comprising a respective capacitive pick-up unit for producing an output signal representing the position of the respective sensor inertia mass relative to the frame structure.","87.The acceleration sensor of claim 86, wherein said sensor inertia masses are mounted in a common plane in said frame structure, and wherein first and second connector elements of all sensor inertia masses are provided in a common connector plane.","88.The acceleration sensor of claim 63, wherein said sensor inertia mass is adapted for picking-up an acceleration of said object in a horizontal direction (x, y) and wherein said bearing device of said sensor inertia mass is elevationally displaced upwardly relative to the mass center of gravity (MZ, MY), whereby the sensor inertia mass performs, in response to an acceleration of said object, a rotational motion about a rotation axis (AX, AY) defined by said bearing device.","89.The acceleration sensor of claim 63, wherein said sensor inertia mass is adapted for picking-up an acceleration of said object in a vertical direction (z), and wherein said bearing device of said sensor inertia mass is laterally offset relative to the mass center of gravity (MZ) whereby the sensor mass performs, in response to an acceleration of the object, a rotational motion about a rotation axis (AZ) defined by the bearing device.","90.The acceleration sensor of claim 63, wherein said first capacitor electrodes are provided on said sensor inertia mass in pairs and respectively on opposite sides of a rotation axis (AX, AY, AZ) defined by said bearing device wherein said second capacitor electrodes are provided on said frame structure also in pairs and respectively positioned opposite said first capacitor electrodes, and wherein a differential capacity of said capacitor electrodes forming pairs is used for producing a capacitive output signal of said pick-up unit.","91.The acceleration sensor of claim 63, wherein said first capacitor electrodes provided on the sensor inertia mass are formed of a conducting material of which the sensor mass is made.","92.The acceleration sensor of claim 67, wherein said first cover section of said frame structure is made of a glass containing an alkali metal.","93.The acceleration sensor of claim 73, wherein said second cover section of said frame structure is made of a glass containing an alkali metal.","94.A method for producing an acceleration sensor as defined in claim 86, said method comprising the following steps: a) first producing each of said plurality of sensor inertia masses as a single sensor with its own frame structure and b) assembling said single sensors to a complete sensor."],["The invention relates to a micromechanical capacitive acceleration sensor in accordance with the preamble of patent claim 1 and the preamble of patent claim 17.Micromechanical capacitive acceleration sensors made of a wafer are known for picking up the acceleration of an object in one or more directions.","The sensor has a mounting which is for example a frame structure which is fixed relative to the object and a sensor inertia mass which is movable relative to the mounting or frame structure of the acceleration sensor, with a bearing device by means of which the sensor mass is elastically mounted relative to the mounting about a starting position.","The sensor further includes a capacitive pick-up unit for producing at least one capacitive output signal that represents the position of the sensor mass relative to the mounting or frame structure.","The capacitive pick-up unit comprises for example first capacitor electrodes provided on the sensor mass and second capacitor electrodes provided on the mounting opposite the first capacitor electrodes.","For example, a coupling arrangement is provided for coupling the first capacitor electrodes provided on the sensor mass which first connector elements provided on the frame structure.","The second capacitor electrodes are coupled with second connector elements.","Micromechanical capacitive acceleration sensors of this type or similar type are known from the following German patent publications: DE 38 37 883 A1; DE 196 37 265 A1; DE 41 26 100 A1, DE 197 09 520 A1 and DE 197 09 731 A1.It is the object of the invention to provide a micromechanical capacitive acceleration sensor which is as simple as possible and thus cost effective to produce, which has a small size and a sensitivity as small as possible relative to interfering influences.","This object has been achieved by the micromechanical capacitive acceleration sensors defined in patent claims 1 and 17.Advantageous further embodiments of the invention are characterized in the dependent claims.","According to a first aspect of the invention a micromechanical capacitive acceleration sensor is provided for picking up the acceleration of an object in at least one direction.","The sensor includes a frame structure, a sensor inertia mass made of a wafer and movably supported relative to the frame structure.","The sensor further includes a capacitive pick-up unit for producing at least one capacitive output signal representing the position of the sensor mass relative to the frame structure whereby the sensor inertia mass has a center of gravity which is offset relative to the rotation axis in a direction extending perpendicularly to a plane of the wafer.","It is an important advantage of this acceleration sensor according to the invention that it can be manufactured in an especially simple way and that it has a large sensitivity and a compact construction.","The acceleration sensor has a small sensitivity relative to interference influences.","The acceleration sensor is capable of measuring accelerations parallel to the wafer surface.","The present sensor is further adapted to produce two or three axes acceleration sensors or sensor systems which, for example, may be produced monolithically.","For example, either the sensor mass and/or the frame structure may be constructed monolithically out of one single crystal silicon wafer.","This feature provides an especially cost effective production while providing a most compact structure and a high measuring precision.","The acceleration sensor has, for example, a bearing device for supporting the sensor inertia mass.","The bearing device comprises particularly torsionable elements, for example, made of silicon.","This feature also enhances the simple and cost effective production of a compact structure.","The bearing device forms preferably an electrically conducting connection between the sensor mass and an electrical contacting provided on the frame structure.","This feature achieves that the contacting of the movable sensor mass takes place on the frame structure whereby the electrical connection to the sensor mass is produced in an especially simple and reliable manner.","Preferably the bearing device is arranged on the sensor mass close to the wafer surface.","This features provides, for example, an especially large measuring sensitivity.","The sensor mass can, for example, be formed to have a cubic shape, or a parallelepiped shape, or a frustum shape, or a truncated pyramid shape.","These geometries provide even additionally large measuring sensitivities while requiring little space and assuring a cost effective production.","A further embodiment preferably provides several sensor inertia masses which are respectively supported for movement about a rotation axis in order to measure accelerations in several directions extending perpendicularly to one another.","One or more sensor masses have a respective center of gravity which is offset relative to the respective rotation axis in a direction perpendicularly to the wafer plane.","Thus, a sensor system is provided which in spite of a small structural size and a cost effective production permits measurements in two or three axial directions or precise acceleration measurements in several directions of space.","Preferably the center of gravity of the sensor mass is additionally offset laterally in a direction of the wafer plane relative to the respective rotation axis.","As a result of the additional lateral offset of the center of gravity a measuring sensitivity is achieved in a further direction of space.","The acceleration sensor has preferably first capacitor electrodes provided on the sensor mass and second capacitor electrodes provided on the frame structure in positions opposite the first capacitor electrodes.","In this embodiment for example first connector elements for the electrical connection of the first capacitor electrodes and/or a second connector elements for the electrical connection of the second capacitor electrodes are arranged preferably in a common connector plane.","The contacting of the first connector elements and/or the second connector elements can take place from a single side.","The electrical contacting of the capacitive pick-up unit can also take place from a single side.","The second capacitor electrodes and the first and second connector elements are provided in a common connector plane preferably on the frame structure.","Each of these features provides an especially advantageous possibility for contacting which simplifies the production and additionally reduces costs.","The acceleration sensor preferably has a coupling arrangement for coupling the first capacitor electrodes provided on the sensor mass with first connector elements provided on the frame structure whereby the coupling arrangement comprises for example a galvanic or capacitive connection for bridging an elevational displacement between the first capacitor electrodes and a common connector plane.","Thus, for example all connector elements can be positioned in a single plane.","Preferably the frame structure comprises a central section which surrounds the sensor mass laterally and a first cover section elevationally displaced relative to the sensor mass and arranged on the central section whereby particularly second capacitor electrodes are provided on the first cover section on its side facing the sensor mass.","The side of the first cover section facing the sensor mass forms for example a common connector plane for the electrical connection of the capacitive pick-up unit.","Preferably the second capacitor electrodes are formed by a metallizing of the sensor mass or by the sensor mass itself which is at an electrical potential.","According a second aspect of the invention the acceleration sensor comprises a frame structure which is stationary relative to the object, a sensor inertia mass which is movable relative to the frame structure of the acceleration sensor, a bearing device by means of which the sensor mass is elastically supported relative to the frame structure and about a starting position.","The second embodiment further includes a capacitive pick-up unit for producing at least one capacitive output signal representing the position of the sensor mass relative to the frame structure.","The pick-up unit comprises first capacitor electrodes provided on the sensor mass and second capacitor electrodes provided on the frame structure in positions opposite to the first capacitor electrodes.","A coupling arrangement is provided for coupling the first capacitor electrodes provided on the sensor mass with first connector elements provided on the frame structure and second connector elements to which the second capacitor electrodes are coupled.","In this embodiment is it particularly provided that the first connector elements and/or the second connector elements are arranged in a common connector plane which is, for example, elevationally displaced upwardly relative to the first capacitor electrodes provided on the sensor mass and/or relative to the second capacitor electrodes provided on the frame structure.","Further, the coupling arrangement comprises particularly means for bridging the elevational displacement between the first capacitor electrodes and/or the second capacitor electrodes and the common connector plane.","The essential advantage of this aspect of the micromechanical capacitive acceleration sensor according to the invention is seen in that it is particularly simple to produce.","This advantage is particularly due to the fact that all electrical connections in the form of the connector elements for the first and second capacitor electrodes are positioned in a single plane.","According to a preferred embodiment of the acceleration sensor according to the invention it is provided that the second capacitor electrodes provided at the frame structure and the first and second connector elements are arranged in the common connector plane and that the coupling arrangement comprises means for bridging the elevational displacement to the common connector plane.","This embodiment is especially advantageous because the arrangement of the first and second connector elements together with the second capacitor electrodes in a common plane makes the production especially simple.","According to a further aspect of the invention it is provided that the coupling arrangement forms a galvanic connection that bridges the elevational displacement to the common connector plane.","According to a still other aspect of the invention it is provided that the coupling arrangement forms a capacitive connection that bridges the elevational displacement to the common connector plane.","According to an especially preferred embodiment of the invention it is provided that the frame structure comprises a central section laterally surrounding the sensor mass and a first cover section elevationally displaced relative to the sensor mass and arranged on said central section, whereby the second capacitor electrodes are provided on the side of the first cover section that faces the sensor mass and wherein the first capacitor electrodes are provided on the sensor mass opposite said second capacitor electrodes.","The advantage of this arrangement is seen in the simple contacting and in small stray capacities.","An advantageous further development of the just mentioned embodiment provides that the common connector plane is formed by the side of the first cover section which faces the sensor mass and on which side said first connector elements and said second connector elements are arranged in said common connector-plane and in which side the second capacitor electrodes are also provided.","A preferred embodiment hereof provides that the first connector elements, the second connector elements, and the second capacitor electrodes which are connected with the second connector elements, are produced of a single conducting layer which is formed on the side of the first cover section facing the sensor mass.","In connection with embodiments in which the coupling arrangements comprises a capacitive connection it is advantageously provided that a second coupling electrode is formed on the side of the first cover section facing the sensor mass and that a first coupling electrode is provided on the sensor mass opposite the second coupling electrode.","In this embodiment the first and the second coupling electrodes form a capacitive connection of the coupling arrangement for coupling the first capacitor electrodes provided on the sensor mass with a first connector element provided on the frame structure.","This embodiment is advantageously further developed in that the second coupling electrode together with the first connector element are provided in the common connector plane which is formed by that side of the first cover section which faces the sensor mass.","Furthermore, it is preferably provided that the second coupling electrode and the first connector element connected therewith are produced together with the second capacitor electrodes and the second connector element connected with the second capacitor electrodes, of a single conducting layer formed on that side of the first cover section which faces the sensor mass.","In those embodiments in which the frame structure comprises a central section laterally surrounding the sensor mass and a first cover section elevationally displaced relative to the central section and arranged on the central section, it is advantageously provided that a second cover section is provided on the central section opposite the first cover section and elevationally displaced relative to the sensor mass.","The first cover section, the central section and the second cover section form a housing that hermetically encloses the sensor mass, the first capacitor electrodes and to the second capacitor electrodes.","The housing that hermetically seals the acceleration sensor provides on the one hand reliable protection relative to environmental influences and on the other hand makes it possible to adjust inside the housing a small gas pressure for reducing the damping.","According to a type of embodiment of the acceleration sensor according to the invention it is provided that the bearing device is formed by torsion elements to which the sensor mass is mounted in a torsion elastic manner relative to the frame structure and about said starting position.","A preferred embodiment of this version provides that two torsion elements forming the bearing device are positioned on a common rotation axis and wherein the two torsion elements are effective on two opposite sides of the sensor mass whereby the sensor mass is movable only in a single direction relative to the frame structure.","A preferred embodiment of the acceleration sensor according the invention provides that the torsion elements are secured on the one hand to the sensor mass and on the other hand to the central section of the frame structure.","According to an aspect of the invention it is provided that the coupling arrangement is formed by a galvanic connection which comprises the torsion elements.","Another type of the embodiment of the acceleration sensor according to the invention provides that the bearing device is formed by one or more bending elements on which the sensor mass is supported in a bending elastic manner about said starting position and relative to the frame structure.","A preferred embodiment hereof provides that two bending elements are positioned in a common plane, spaced from each other and connected to the sensor mass whereby the sensor mass is movable only in a single direction relative to the frame structure.","According to an embodiment it is provided that the bending elements are connected on the one hand to the sensor mass and on the other hand to the first cover section of the frame structure.","Another aspect of the invention provides that the coupling arrangement is formed by a galvanic connection that includes the bending elements.","Another type of embodiment of the acceleration sensor according to the invention provides that a single bending element is centrally secured to the sensor mass whereby the sensor mass is movable relative to two different directions and relative to the frame structure.","This embodiment further includes the pick-up unit for producing two capacitive output signals which are representing the position of the sensor mass relative to the frame structure.","The advantage of this type of embodiment is seen in that with a single sensor mass it is possible to pick up accelerations in two different directions for example in the two directions of the horizontal plane.","According to a preferred embodiment hereof it is provided that the pick-up unit comprises four first capacitor electrodes which are secured to the sensor mass in pairs positioned opposite each other and on opposite sides of the bending element.","Four second capacitor electrodes are secured to the first cover section of the frame structure whereby the second capacitor electrodes are arranged in pairs positioned opposite each other on opposite sides of the bending element and opposite the first capacitor electrodes.","In case a capacitive connection is to be formed in the last mentioned embodiment by the coupling arrangement, it is particularly advantageous to provide that the coupling arrangement comprises a first coupling electrode secured to the sensor mass and surrounding the bending element and a second coupling electrode secured to the first cover section of the frame structure said second coupling electrode being positioned opposite the first coupling electrode and surrounding the bending element.","In connection with the embodiments in which the coupling arrangement forms a galvanic connection it is advantageous to provide that the coupling arrangement is formed by a galvanic connection which comprises the bending element.","According to a further type of embodiment of the acceleration sensor according to the invention it is provided that for picking up the acceleration of an object in several directions, several sensor masses are provided each equipped with a capacitive pick-up unit.","These several sensor masses are mounted independently of one another each by means of a corresponding bearing device in a frame structure.","Each sensor mass is provided with a capacitive pick-up unit for producing an output signal representing the position of the corresponding sensor mass relative to the frame structure.","Hereby, it is advantageously provided that the sensor masses are mounted in a common plane in the frame structure and that the first and second connector elements of all sensor masses are provided in a common connector plane.","According to an embodiment of the acceleration sensor according to the invention it is provided that a sensor mass is provided for picking up the acceleration of the object in a horizontal direction, wherein the sensor mass comprises a bearing device which is elevationally displaced upwardly relative to the mass center of gravity whereby the sensor mass performs, in response to an acceleration of the object, a rotational motion about a rotation axis defined by the bearing device.","According to another embodiment of the acceleration sensor according to the invention it is provided that a sensor mass for picking up the acceleration of the object in a vertical direction is provided which comprises a bearing device laterally offset relative to the mass center of gravity whereby the sensor mass performs, in response to an acceleration of the object, a rotational motion about a rotation axis defined by the bearing device.","In this connection it is particularly advantageous to provide that the first capacitor electrodes are respectively provided in pairs on the sensor mass on opposite sides of the rotational axis defined by the bearing device and in that the second capacitor electrodes are provided on the frame structure also in pairs and respectively positioned opposite the first capacitor electrodes, and that the differential capacity of the capacitor electrodes forming pairs is used for producing the capacitive output signal of the pick-up unit.","Picking up the difference capacity for producing the output signal is particularly advantageous for minimizing interfering influences.","Advantageously it is provided that the first capacitor electrodes provided on the sensor mass are formed by a conducting material of which the sensor mass is made.","In the embodiments in which the frame structure comprises a center section laterally surrounding the sensor mass and a first cover section secured thereto, it is advantageous if the first cover section of the frame structure is made of a glass containing an alkali metal.","It is also advantageous to provide that the second cover section of the frame structure is made of a glass containing an alkali metal.","Finally there is provided a method for producing an acceleration sensor in accordance with one of the preceding embodiments whereby each of the sensor masses is first produced as a single sensor with its own and wherein the single sensors are then assembled to a complete sensor.","In the following example embodiments of the invention are described with reference to the drawing wherein: FIG.","1a shows a perspective partially sectional view of a micromechanical capacitive acceleration sensor for picking up the acceleration of an object in one direction according to a first example embodiment of the invention; FIG.","1b shows an electrical equivalent circuit diagram of the capacitive acceleration sensor according to FIG.","1a; FIG.","2 is a cross sectional view of a micromechanical capacitive acceleration sensor according to the first example embodiment shown in FIG.","1a; FIG.","3 shows the perspective views of three sensor masses of a micromechanical capacitive acceleration sensor for picking up the acceleration of an object in three directions (x, y, z) as an expansion of the first example embodiment of the invention shown in FIG.","1.FIG.","4a is a perspective view partially in section of a micromechanical capacitive acceleration sensor for picking up the acceleration of an object in one direction (x) according to a second example embodiment of the invention; FIG.","4b shows an electrical equivalent circuit diagram of the capacitive acceleration sensor of FIG.","4 according to a variant with a capacitive coupling; FIG.","4c is an electrical equivalent circuit diagram of the capacitive acceleration sensor of FIG.","4a according to a variant with a galvanic coupling; FIG.","5 shows a cross sectional view of the micromechanical capacitive acceleration sensor according to the example embodiment of FIG.","4a; FIG.","6 shows a sectional view of a micromechanical capacitive acceleration sensor according to a third example embodiment of the invention; FIG.","7 is a perspective view of a sensor mass of a micromechanical capacitive acceleration sensor for picking up the acceleration of an object in two directions (x, y) according to a fourth and according to a fifth example embodiment of the invention; and FIGS.","8a and 8b respectively show plan views of arrangements of capacitor and connector elements of a micromechanical capacitive acceleration sensor for picking up the acceleration of an object in two directions (x, y) according to the fourth and fifth example embodiment of the invention.","In FIGS.","1a and 2 a first example embodiment of the micromechanical capacitive acceleration sensor for picking up the acceleration of an object in one direction namely the direction x is altogether designated with the reference character 100.The acceleration sensor 100 comprises a frame structure 110 including a central section 111, a first cover section 112 and a second cover section 113 whereby the latter is shown only in FIG.","2.The frame structure 110 is arranged in a fixed position relative to the object the acceleration of which is to be picked up.","The acceleration sensor 100 comprises a sensor inertia mass 101 which is movable relative to the frame structure 110 of the acceleration sensor 100 and which is elastically mounted about a starting position by a bearing device 105 relative to the frame structure 110.The sensor inertia mass 101 has, in the preferred example embodiment shown here, a center of gravity which is arranged offset relative to the rotation axis of the sensor mass 101 in a direction extending perpendicularly to the wafer plane.","That is, in this example embodiment the center of gravity of the sensor mass 101 is supported outside of the rotation axis, whereby the rotation axis is positioned near to the surface of the wafer or of the sensor mass 101, and the center of gravity is positioned below the rotation axis when the wafer surface is horizontally aligned.","Thus, an acceleration F oriented in parallel to the wafer surface can be measured.","The acceleration F causes a deflection of the sensor inertia mass 101 about the rotation axis.","The central section 111 of the frame structure 110 laterally surrounds the sensor mass 101 and the first cover section 112 is elevationally offset relative to the sensor mass 101 and arranged on the central section 111.The bearing device 105 in the example embodiment as shown in FIGS.","1a and 2 is formed by torsion elements to which the sensor mass 101 is mounted in a torque elastic manner relative to the frame structure 110 i.e.","relative to the central portion 111 for movement about said starting position.","The torsion elements that form the bearing device 105 are provided on sides of the sensor mass 110 positioned opposite each other on a common rotation axis.","Thus, the sensor mass 101 is only movable relative to a single direction, namely the coordinate direction designated by x in FIG.","1a relative to the frame structure 110.The sensor mass 101 moves in the direction of the x-axis about its rotation axis in response to the occurrence of an acceleration force Fx whereby the deflection at the edge of the sensor mass 101 is directed upwardly and downwardly.","The torsion elements 105 are connected on the one hand to the central section 111 of the frame structure 110 and on the other hand to the sensor mass 101 in a rotationally stable manner.","Furthermore the acceleration sensor 100 comprises a capacitive pick-up unit 120 for producing a capacitive output signal which represents the position of the sensor mass 101 relative to the frame structure 110.The pick-up unit comprises first capacitor electrodes 121, 122 provided on the sensor mass 101 and second capacitor electrodes 125, 126 provided at the frame structure 110 opposite the first capacitor electrodes 121, 122.The first capacitor electrodes 121, 122 provided on the sensor mass 101 are connected to a first connector element 142 provided on the first cover section 112.The connection is a galvanic connection formed by a coupling arrangement which is formed by the torsion elements 105.The galvanic connection also passes through a metallization 141 on the central section 111 of the frame structure 110.Second connector elements 145, 146 are coupled with the second capacitor electrodes.","The second connector elements are also provided on the first cover section 112 of the frame structure 110.The first capacitor electrodes 121, 122 provided on the sensor mass 101 are formed by a conducting material of which is the sensor mass 101 is made.","In this manner one obtains an equivalent electrical circuit diagram for the first and second capacitor electrodes 121, 122 and 125, 126 as shown in FIG.","1b.","Two capacities E1 and E3 are connected in series between two reference voltages +Vref (connector element 145) and −Vref (connector element 146).","The center junction of the capacitor series connection is connected to a terminal K1 (connector element 142) at which the output signal Vout can be tapped off.","The first connector element 142 and the second connector elements 145, 146 are arranged in a common connector plane 150.In this first example embodiment this common connector plane 150 is offset relative to the first capacitor electrodes 121, 122 provided on the sensor mass 101.This elevational offset is bridged by the coupling arrangement formed by the torsion elements 105, the metallization 141 and the first connector element 142.The second capacitor electrodes 125,126 are also arranged in said common connector plane 150 together with the first and second connector elements 142 and 145, 146.The connector plane 150 is formed by one side of the first cover section 112.The first connector element 142, the second connector elements 145, 146 and the second capacitor electrodes 125, 126, which are connected with the latter, are made of a single conductive layer formed on that side of the first cover section 112 which faces the sensor mass 101.As seen in FIG.","2, the central section 111, the first cover section 112 and the second cover section 113 form together a hermetically sealed housing in which the sensor mass 101 is arranged together with the first capacitor electrodes 121, 122 and the second capacitor electrodes 125, 126.The first cover section 112 and the second cover section 113 of the frame structure 110 are made of a glass containing an alkaline metal.","FIG.","3 shows the sensor masses 101, 102, 103 which are provided for picking up the acceleration of an object in several directions namely the directions x, y, z of the coordinate system shown in FIG.","3.The sensor mass 101 (partial Fig.","A) corresponds to the sensor mass 101 of the acceleration sensor shown in FIGS.","1a and 2.The sensor mass 101 is mounted in a torsion elastic manner by means of torsion elements 105 whereby it is movable about a starting position.","The torsion element 105 defines a rotation axis which is offset relative to the mass center of gravity Mx of the sensor mass 101 so that an acceleration force Fx effective in the x-direction causes a deflection Sx.","The sensor mass 102 (partial Fig.","B) corresponds in its configuration substantially to the sensor mass 101 and also has torsion elements 106 which form a bearing device for supporting the sensor mass in a torsion or rotational elastic manner.","However, the rotation axis defined by the torsion elements 106 is angularly displaced by 90 degrees in the horizontal plane relative to the rotation axis of the sensor mass 101.The rotation axis formed by the torsion elements 106 is again displaced relative to the mass center of gravity My of the sensor mass 102 in elevation so that an acceleration force Fy effective in the y-direction causes a deflection Sy of the sensor mass about its starting position.","The sensor masses 101 and 102 are provided, corresponding to the sensor mass 101 of FIGS.","1a and 2 with first capacitor electrodes 121 and 122 of a capacitive pick-up unit not particularly shown in FIG.","3.The capacitive pick-up unit produces a capacitive output signal representing the position of the sensor mass 102 relative to a frame structure also not shown in particular in FIG.","3.A third sensor mass 103 (partial Fig.","C of FIG.","3) is mounted by means of a bearing device formed by torsion elements 107 for rotation about a rotation axis.","The mass center of gravity Mz is laterally offset relative to the rotation axis formed by the torsion elements 107.A vertical acceleration Fz caused by an acceleration in the vertical z-direction causes a deflection Sz of the end of the sensor mass 103 as shown in partial Fig.","C. Each of the sensor masses 101, 102, 103 is provided with a capacitive pick-up unit for producing a respective output signal representing the corresponding position of the sensor mass relative to a frame structure not shown.","This capacitive pick-up unit comprises respectively first capacitor electrodes (not shown in FIG.","3).","These first capacitive electrodes are provided in pairs respectively on opposite sides of the rotation axis Ax, Ay, Az.","These rotation axis are defined by the torsion elements 105, 106 and 107 respectively.","Second capacitor electrodes of the capacitive pick-up unit are provided on the frame structure, not shown, of the acceleration sensor.","The second capacitor electrodes are positioned opposite the first capacitor electrodes.","For the sensor masses 101 and 102 shown in the partial Figs.","A & B, this means an arrangement similar to that shown in FIGS.","1a and 2.In connection with the sensor mass 103 shown in partial Fig.","C of FIG.","3, the first capacitor electrode is arranged to the left of the rotation axis defined by the torsion elements 107 on the short piece of the sensor mass 103.The other capacitor electrode is arranged symmetrically to the first capacitor electrode and to the right hand side of the rotation axis defined by the torsion elements 107.Here again the difference capacity of the capacitor electrodes arranged in pairs is used for producing the capacitive output signal of the capacitive pick-up units.","This utilization of the difference capacity is similar to that explained with reference to the example embodiment of FIGS.","1 & 2.The first capacitor electrodes provided on the sensor mass 101, 102 or 103 are formed in the just described example embodiment by a conducting material of which the sensor mass 101, 102, 103 is made.","Each of the sensor masses 101, 102 and 103 shown in FIG.","3 is initially and preferably produced as an individual sensor with its own frame structure.","These individual sensors are then assembled to form a complete sensor capable of picking up accelerations in all three directions of space x, y, z.","The acceleration sensor shown in FIGS.","4a, 5 and 6 according to a second and a third example embodiment of the invention is designated altogether with the reference characters 200; 300.The acceleration sensor 200; 300 serves for picking up the acceleration of an object in the x-direction shown in the coordinate system illustrated in FIG.","4a.","The sensor comprises a frame structure 210, 310 which is mounted in a fixed position relative to the object the acceleration of which is to be picked up.","The acceleration sensor further comprises a sensor inertia mass 201, 301 which is movable relative to the frame structure 210, 310 of the acceleration sensor 200, 300.The sensor mass 201, 301 is elastically mounted about a starting position by means of a bearing device 205, 305 relative to the frame structure 210, 310.The frame structure 210, 310 comprises a center section 211, 311 laterally surrounding the sensor mass 201, 301 and a first cover section 212, 312 arranged on the center section 211, 311 elevationally displaced relative to the sensor mass 201, 301.The bearing device of the second and third example embodiments shown in FIGS.","4 to 6 is formed by bending elements 205, 305.The sensor mass 201, 301 is mounted to the bending elements in a bending elastic manner about the starting position and relative to the frame structure 210, 310.As shown in FIG.","4a, two of these bending elements which are provided in a common plane and spaced from one another and connected to the sensor mass 201, 301.These bending elements 205, 305 define a rotation axis about which the sensor mass 201, 301 moves in response to the occurrence of an acceleration force Fx in the direction of the x-axis whereby the deflection Sx at an edge of the sensor mass 201, 301 is directed upwardly and downwardly.","The bending elements 205, 305 are connected on the one hand to the sensor mass 201, 301 and on the other hand to the first cover section 212, 312 of the frame structure 210, 310.The acceleration sensor 200, 300 comprises a capacitive pick-up unit altogether designated by the reference character 220, 320.This pick-up unit 220, 320 serves for producing an output signal representing the position of the sensor mass 201, 301 relative to the frame structure 210, 310 and thus the size of the acceleration force Fx that deflects the sensor mass 201, 301 out of its starting position.","This output signal is a measure for the acceleration of the object to which the acceleration sensor is attached.","The capacitive pick-up unit 220, 320 comprises first capacitor electrodes 221, 222, 321, 322 which are provided on the sensor mass 201, 301.The pick-up unit further comprises second capacitor electrodes 225, 226; 325, 326 which are secured to the frame structure 210, 310 more specifically on the first cover section 212, 312 in positions opposite the first capacitor electrodes 221, 222; 321, 322.Generally, a coupling arrangement is provided for coupling the first capacitor electrodes 221, 222 or 321, 322 provided on the sensor mass 201, 301 with first connector elements.","Of these connector elements, the first connector element 242 of the acceleration sensor 200 is seen in FIG.","4a according to the second example embodiment.","This first connector element 242 is arranged on the frame structure 210, more specifically on the first cover section 212 thereof.","The acceleration sensor 300 shown in FIG.","6 according to the third example embodiment, is arranged correspondingly, however, the connector element corresponding to the first connector element 242 of FIG.","4a is not visible in FIG.","6.The second capacitor electrodes 225, 226 or 325, 326 provided on the first cover section 212, 312 are coupled with the second connector elements.","Here again the second connector elements 245, 246 which are connected with the second capacitor electrodes 225, 226 of the acceleration sensor 200, are seen in FIG.","4a.","The acceleration sensors 300 of the third example embodiment shown in FIG.","6 has a corresponding arrangement.","The first connector elements, compare the connector element 242 in FIG.","4a, and the second connector elements, compare the connector elements 245, 246 in FIG.","4a, are arranged in a common connector plane 250, 350.This common connector plane is formed by that side of the first cover section 212, 312 which faces the sensor mass 201, 301.Further, this common connector plane is elevationally offset relative to the first capacitor electrodes 221, 222; 321, 322 provided on the sensor mass 201, 301.The coupling arrangement which connects the first capacitor electrodes 221, 222 or 321, 322 provided on the sensor mass 201 or 301, with the first connector elements, compare 242 in FIG.","4a, forms a bridging of the elevational offset between the sensor mass 201, 301 and said common connector plane 250, 350.The coupling arrangement in the acceleration sensor 200 of the second example embodiment shown in FIGS.","4a and 5 is formed by a capacitive connection.","This capacitive connection comprises first and second coupling electrodes 231 and 232 positioned opposite each other.","The first coupling electrode 231 is provided on the sensor mass 201.The second coupling electrode is provided on the first cover section 212.The second coupling element 232 is connected with the above mentioned first connector element 242.The second coupling electrode is provided on that side of the first cover 212 which faces the sensor mass 201, compare FIG.","4a.","FIG.","4b shows an electrical equivalent circuit diagram of the acceleration sensor 200 according to the second example embodiment as shown in FIGS.","4a and 5.As can be seen the capacities E1 and E3 formed by the first capacitor electrodes 221, 222 and by the second capacitor electrodes 225, 226, are connected in series between a positive reference voltage +Vref (at the terminal 245) and a negative reference voltage −Vref (at the terminal 246).","The output signal Vout is provided at the center junction through a capacity E2 formed by the capacitive coupling.","Contrary to the above, in the third example embodiment of the acceleration sensor 300 shown in FIG.","6, the first capacitor electrodes 321, 322 are galvanically connected with the first connector element corresponding to the connector element 242 in FIG.","4a.","Specifically, the connection extends through the bending elements 305 which are made of a conductive material.","In this manner a galvanic connection is formed between the capacities E1 and E3 formed by the capacitor electrodes 321, 322 and 325, 326.The output voltage Vout can be tapped off at this galvanic connection, compare FIG.","4c.","As in the first example embodiment of the acceleration sensor shown in FIG.","1a and FIG.","2, the first cover section 212, 312 and the second cover section 213, 313 in the acceleration sensors 200 and 300 according to the second and third example embodiment of FIGS.","4a, 5 and 6 are also made of a glass containing an alkali metal.","The first cover section 212, 312, the central section 211, 311 and the second cover section 213, 313 form together a hermetically sealed housing for the sensor mass 201, 301, for the first capacitor electrodes 221, 222; 321, 322 and for the second capacitor electrodes 225, 226; 325, 326.The second cover section 213, 313 is connected to the center section 211, 311 of the frame structure 210, 310 and positioned opposite the first cover section 212, 312.The first connector elements, shown at 242 in FIG.","4a, the second connector elements shown at 245, 246 in FIG.","4a and the second capacitor electrodes 225, 226 or 325, 326 are formed of a single conducting layer in the example embodiments shown in FIGS.","4a, 5 and 6.This single conducting layer is formed on that side of the first cover section 212, 312 that faces the sensor mass 201, 301.FIG.","7 shows a sensor mass 401, 501 of fourth and fifth example embodiments of the acceleration sensor capable of picking up acceleration in two directions namely the x-direction and the y-direction in the horizontal plane.","A single bending element 405, 505 is secured centrally to the sensor mass 401, 501 so that the sensor mass is movable in two different directions in response to acceleration forces Fx or Fy relative to a frame structure not shown in FIG.","7.In order to obtain output signals representing the position of the sensor mass 401, 501, capacitive pick-up units are provided which will be described with reference to FIGS.","8a and b.","FIG.","8 shows a portion of capacitive pick-up units 420, 520 for picking up the position of the sensor mass 401, 501 in two directions.","The pick-up unit comprises four second capacitor electrodes 425, 426, 427, 428; 525, 526, 527 and 528 which are provided in pairs facing each other on opposite sides of the bending element 405, 505 on a first cover section 412, 512 of a frame structure similar to the example embodiments illustrated in FIG.","1a and FIG.","2 or in FIGS.","4a to 6.These second capacitor electrodes 425, 426, 427, 428; 525, 526, 527, 528 are electrically connected with second connector elements 445, 446, 447, 448; 545, 546, 547 and 548.Similar first capacitor electrodes are provided on the sensor mass 401, 501 and positioned opposite the second capacitor electrodes 425, 426, 427, 428; 525, 526, 527 and 528.These first capacitor electrodes are not particularly shown in FIG.","7.FIG.","8a illustrates a fourth example embodiment including a coupling arrangement which connects the first capacitor electrodes provided on the sensor mass 401 with a first connector element 442 on the first cover section 412.The fourth example embodiment further comprises a first coupling electrode surrounding the bending element 405.The first coupling electrode is not shown but provided on the sensor mass 401, and a second coupling electrode 432 surrounding the bending element 405 and positioned opposite the first coupling electrode.","The second coupling electrode 432 is secured to and provided on the first cover section 412 of the frame structure.","Thus, a capacitive connection is formed between the first capacitor electrodes provided on the sensor mass 401 and the first connector element 442.Contrary to the above, in the example embodiment shown in FIG.","8b, the coupling arrangement is formed by a galvanic connection comprising the bending element 505 so that the first capacitor electrodes provided on the sensor mass 501, please compare FIG.","7, are connected galvanically through the bending element 505 with the first connector element 542.As in the above example embodiments, the first capacitor electrodes provided on the sensor mass 401, 501 are formed by a conducting material of which the sensor mass 401, 501 is made.","The sensor mass used in particularly preferred embodiments of the invention is made of a single crystal silicon.","Thereby the capacitor electrodes of the sensor mass are formed directly by the sensor mass itself.","In these cases the sensor mass is electrically connected with the frame structure whereby the torsion beams or the suspensions of the sensor mass form conducting connections to the sensor mass.","Thus, the sensor mass is maintained at a certain electrical potential and forms with the oppositely positioned capacitor electrodes capacitive elements for the measurement of the deflection of the sensor mass.","The electrical contacting of the sensor mass takes place for example at a location of the frame which is readily accessible and which is connected in an electrically conducting manner with the sensor mass through the torsion beams.","The contacting of all electrical connections can be achieved on a single side for example on the back side of the sensor or from its top side with the wafer alignment shown in FIG.","1a.","It is thereby possible to provide holes in the wafer which contain conducting material or are filled with conducting material in order to electrically contact from the outside the connector electrodes 142, 145 and 146 positioned on the inside for example on the side 150 of the cover section 112 positioned opposite the sensor mass 101.Instead of the coupling arrangement 141, 142 shown in FIG.","1a, with one connecting electrode 142 of the sensor mass 101 at the cover section 112, it is possible to arrange the connector electrodes of the sensor mass 101 at the lower frame structure 110.Thus, the connector electrode may for example be positioned on the bottom side of the shoulder or projection of the frame structure 110, more specifically, below the coupling arrangement 141 shown in FIG.","1a."]],"string":"[\n [\n \"Micromechanical capacitive acceleration sensor\",\n \"A micromechanical capacitive acceleration sensor is described for picking up the acceleration of an object in at least one direction.\",\n \"The sensor includes a frame structure (110), a sensor inertia mass (101) made of a wafer and movably mounted relative to the frame structure (110) about a rotation axis, and a capacitive pick-up unit (120) for producing at least one capacitive output signal representing the position of the sensor mass (101) relative to the frame structure (110).\",\n \"The sensor inertia mass (101) has a center of gravity which offset relative to the rotation axis in a direction perpendicularly to a wafer plane for measuring accelerations laterally to the wafer plane.\",\n \"The sensor mass (101) and the frame structure (110) are made monolithically of one single crystal silicon wafer.\",\n \"A cover section (112) forms a common connector plane (150) for the connection of capacitor electrodes (125,126).\",\n \"Torqueable elements (105) form an electrically conducting bearing device for the sensor mass (101).\"\n ],\n [\n \"1.A micromechanical capacitive acceleration sensor for picking up the acceleration of an object in at least one direction, said sensor comprising a frame structure, a sensor inertia mass made of a wafer movably supported about a rotation axis (Ax, Ay) relative to said frame structure, and a capacitive pick-up unit for producing at least one capacitive output signal representing the position of said sensor inertia mass relative to said frame structure, wherein said sensor inertia mass comprises a center of gravity (Mx, My) which is offset relative to said rotation axis (Ax, Ay) in a direction extending perpendicularly to a plane of said wafer.\",\n \"2.The acceleration sensor of claim 1, wherein said sensor inertia mass and/or said frame structure are made monolithically of a single crystal silicon wafer.\",\n \"3-48.\",\n \"(canceled) 49.The acceleration sensor of claim 1, further comprising a bearing device including torqueable elements made of silicon for supporting said sensor inertia mass.\",\n \"50.The acceleration sensor of claim 1, further comprising an electrical contacting on said frame structure and a bearing device for supporting said sensor inertia mass, said bearing device forming an electrically conducting connection between said sensor inertia mass and said electrical contacting on said frame structure.\",\n \"51.The acceleration sensor of claim 1, further comprising a bearing device arranged on said sensor inertia mass close to a surface of said wafer.\",\n \"52.The acceleration sensor of claim 1, wherein said sensor inertia mass has a shape selected from any one of the following shapes: a cubic shape, a parallelepiped, a frustum and a truncated pyramid.\",\n \"53.The acceleration sensor of claim 1, comprising a plurality of sensor inertia masses movably mounted relative to a respective rotation axis (AX, AY, AZ) for measuring accelerations in a plurality of directions (x, y, z) extending perpendicularly to one another, wherein one or more sensor inertia masses respectively have a center of gravity (MZ, MY) which is offset relative to its respective rotation axis (AX, AY) in a direction extending perpendicularly to a plane of said wafer.\",\n \"54.The acceleration sensor of claim 1, wherein said center of gravity of said sensor inertia mass is arranged relative to the respective rotation axis (AX, AY) and laterally displaced in a direction of said wafer plane.\",\n \"55.The acceleration sensor of claim 1, wherein said capacitive pick-up unit comprises first capacitor electrodes provided on said sensor inertia mass and second capacitor electrodes provided on said frame structure opposite said first capacitor electrodes, said pick-up unit further comprising first connector elements for electrically connecting said first capacitor electrodes and/or second connector elements electrically connecting said second capacitor electrodes, said first and second connector elements being arranged in a common connector plane.\",\n \"56.The acceleration sensor of claim 55, wherein said first connector elements and/or said second connector elements are contacted on a single side.\",\n \"57.The acceleration sensor of claim 1, wherein said capacitive pick-up unit is contacted electrically on a single side.\",\n \"58.The acceleration sensor of claim 55, wherein said second capacitor electrodes on said frame structure and said first and second connector elements are arranged on or in said common connector plane.\",\n \"59.The acceleration sensor of claim 1, further comprising a coupling arrangement for coupling of first capacitor electrodes provided on said sensor inertia mass to first connector elements provided on said frame structure, said coupling arrangement comprising a galvanic or capacitive connection for bridging an elevational difference between said first capacitor electrodes and a common connector plane.\",\n \"60.The acceleration sensor of claim 1, wherein said frame structure comprises a central frame section laterally enclosing said sensor inertia mass and a first cover section elevationally displaced relative to said sensor inertia mass and arranged on said central frame section and second capacitor electrodes provided on a facing side of said first cover section which faces said sensor inertia mass.\",\n \"61.The acceleration sensor of claim 60, wherein said facing side of said first cover section facing said sensor inertia mass forms a common connector plane for an electrical connection of said capacitive pick-up unit.\",\n \"62.The acceleration sensor of claim 55, wherein said second capacitor electrodes are formed either by a metallizing layer on said sensor inertia mass or by said sensor inertia mass that is made of an electrically conducting material.\",\n \"63.A micromechanical capacitive acceleration sensor, for picking up acceleration of an object in at least one direction (x, y, z), comprising a frame structure which is stationary relative to said object, a sensor inertia mass which is movable relative to said frame structure, a bearing device elastically supporting said sensor inertia mass relative to said frame structure for movement about a starting position, said capacitive acceleration sensor further comprising a capacitive pick-up unit for producing at least one capacitive output signal representing a position of said sensor inertia mass relative to said frame structure, said capacitive pick-up unit comprising first capacitor electrodes provided on said sensor inertia mass and second capacitor electrodes provided on said frame structure opposite said first capacitor electrodes, and a coupling arrangement including first connector elements coupled to said first capacitor electrodes, said first connector elements being provided on said frame structure, and second connector elements coupled to said second capacitor electrodes, and wherein said first connector elements and/or said second connector elements are arranged in a common connector plane which is elevationally displaced relative to said first capacitor electrodes provided on said sensor mass and/or relative to said second capacitor electrodes provided on said frame structure.\",\n \"64.The acceleration sensor of claim 63, wherein said coupling arrangement comprises means for bridging an elevational difference between said first capacitor electrodes and/or said second capacitor electrodes and said common connector plane.\",\n \"65.The acceleration sensor of claim 63, wherein said second capacitor electrodes provided on said frame structure and said first and second connector elements are arranged in or on said common connector plane, and wherein said coupling arrangement comprises means for bridging an elevational difference to said common connector plane.\",\n \"66.The acceleration sensor of claim 63, wherein said coupling arrangement forms a galvanic or capacitive connection which bridges said elevational difference to said common connector plane.\",\n \"67.The acceleration sensor of claim 63, wherein said frame structure comprises a central section laterally surrounding said sensor inertia mass and a first cover section elevationally displaced relative to said sensor inertia mass and arranged on said central section, wherein said second capacitor electrodes are provided on that side of said first cover section which faces said sensor inertia mass, and wherein said first capacitor electrodes are provided on said sensor inertia mass opposite said second capacitor electrodes.\",\n \"68.The acceleration sensor of claim 67, wherein said common connector plane in which said first connector elements and said second connector elements are arranged, is formed by that side of said first cover section which faces said sensor inertia mass and wherein said second capacitor electrodes are also provided in or on said side of said first cover section facing said sensor inertia mass.\",\n \"69.The acceleration sensor of claim 68, wherein said first connector elements, said second connector elements and said second capacitor electrodes which are connected to the second connector elements, are made of a single conductive layer formed on the side of the first cover section which faces said sensor inertia mass.\",\n \"70.The acceleration sensor of claim 69, further comprising a second coupling electrode formed on that side of said first cover section which faces said sensor inertia mass, wherein a first coupling electrode is provided on said sensor inertia mass opposite said second coupling electrode, whereby said first and second coupling electrodes form a capacitive connection of the coupling arrangement for coupling the first capacitor electrodes provided on the sensor inertia mass with a first connector element provided on said frame structure.\",\n \"71.The acceleration sensor of claim 70, wherein said second coupling electrode and said first connector element are provided in said common connector plane which is formed by that side of said first cover section which faces the sensor inertia mass.\",\n \"72.The acceleration sensor of claim 71, wherein said second coupling electrode and said first connector element connected to said first coupling electrode, said second capacitor electrodes and said second connector elements connected with the second capacitor electrodes are made of a single conducting layer formed on that side of said first cover section which faces said sensor inertia mass.\",\n \"73.The acceleration sensor of claim 67, further comprising a second cover section provided elevationally displaced relative to said sensor inertia mass, said second cover section being positioned at said central section opposite said first cover section, said second cover section forming together with said central section and with said first cover section a hermetically sealed housing enclosing said sensor inertia mass, said first capacitor electrodes and said second capacitor electrodes.\",\n \"74.The acceleration sensor of claim 63, wherein said bearing device comprises torsion elements to which said sensor inertia mass is mounted to the frame structure in a torsion elastic manner for motion relative to and about said starting position.\",\n \"75.The acceleration sensor of claim 74, wherein said two torsion elements forming said bearing device are positioned relative to a common rotation axis, said two torsion elements being effective on opposite sides of said sensor inertia mass, whereby said sensor inertia mass is movable only relative to a single direction (x, y, z) relative to the frame structure.\",\n \"76.The acceleration sensor of claim 74, wherein said torsion elements are rigidly secured, on the one hand, to said sensor inertia mass and, on the other hand, to said central section of said frame structure.\",\n \"77.The acceleration sensor of claim 74, wherein said coupling arrangement is formed by a galvanic connection which includes said torsion elements.\",\n \"78.The acceleration sensor of claim 63, wherein said bearing device comprises one or more bending elements on which said sensor inertia mass is supported in a bending elastic manner around said starting position and relative to said frame structure.\",\n \"79.The acceleration sensor of claim 78, wherein two bending elements are provided in a common plane spaced from each other and rigidly connected to said sensor inertia mass, whereby said sensor inertia mass is movable only in a single direction (x) relative to said frame structure.\",\n \"80.The acceleration sensor of claim 78, wherein two bending elements are rigidly connected, on the one hand, to the sensor inertia mass and, on the other hand, to said first cover section of said frame structure.\",\n \"81.The acceleration sensor of claim 78, wherein said coupling arrangement is formed by a galvanic connection which includes said bending elements.\",\n \"82.The acceleration sensor of claim 78, comprising a single bending element which is centrally and rigidly connected to said sensor inertia mass, whereby said sensor inertia mass is movable with respect to two different directions (x, y) relative to said frame structure, and wherein said pick-up unit is adapted for producing two capacitive output signals which represent the position of said sensor inertia mass relative to said frame structure.\",\n \"83.The acceleration sensor of claim 82, wherein said pick-up unit comprises four first capacitor electrodes secured to said sensor inertia mass in pairs positioned opposite each other and on opposite sides of said bending element, and four second capacitor electrodes secured to said first cover section of said frame structure, said second capacitor electrodes being arranged in pairs positioned opposite each other on opposite sides of said bending element and opposite said first capacitor electrodes.\",\n \"84.The acceleration sensor of claim 82, wherein said coupling arrangement comprises a first coupling electrode provided on said sensor inertia mass and surrounding said bending element, and a second coupling electrode provided on said first cover section of said frame structure, and wherein said second coupling electrode is positioned opposite said first coupling electrode and surrounding said bending element.\",\n \"85.The acceleration sensor of claim 82, wherein said coupling arrangement comprises a galvanic connection including said bending element.\",\n \"86.The acceleration sensor of claim 1, comprising a plurality of sensor inertia masses and wherein each sensor inertia mass is equipped with a respective capacitive pick-up unit for picking up the acceleration of an object in a plurality of directions (x, y, z), a plurality of bearing devices, each of said plurality of sensor inertia masses being supported by a respective bearing device in said frame structure independently of one another, each sensor inertia mass comprising a respective capacitive pick-up unit for producing an output signal representing the position of the respective sensor inertia mass relative to the frame structure.\",\n \"87.The acceleration sensor of claim 86, wherein said sensor inertia masses are mounted in a common plane in said frame structure, and wherein first and second connector elements of all sensor inertia masses are provided in a common connector plane.\",\n \"88.The acceleration sensor of claim 63, wherein said sensor inertia mass is adapted for picking-up an acceleration of said object in a horizontal direction (x, y) and wherein said bearing device of said sensor inertia mass is elevationally displaced upwardly relative to the mass center of gravity (MZ, MY), whereby the sensor inertia mass performs, in response to an acceleration of said object, a rotational motion about a rotation axis (AX, AY) defined by said bearing device.\",\n \"89.The acceleration sensor of claim 63, wherein said sensor inertia mass is adapted for picking-up an acceleration of said object in a vertical direction (z), and wherein said bearing device of said sensor inertia mass is laterally offset relative to the mass center of gravity (MZ) whereby the sensor mass performs, in response to an acceleration of the object, a rotational motion about a rotation axis (AZ) defined by the bearing device.\",\n \"90.The acceleration sensor of claim 63, wherein said first capacitor electrodes are provided on said sensor inertia mass in pairs and respectively on opposite sides of a rotation axis (AX, AY, AZ) defined by said bearing device wherein said second capacitor electrodes are provided on said frame structure also in pairs and respectively positioned opposite said first capacitor electrodes, and wherein a differential capacity of said capacitor electrodes forming pairs is used for producing a capacitive output signal of said pick-up unit.\",\n \"91.The acceleration sensor of claim 63, wherein said first capacitor electrodes provided on the sensor inertia mass are formed of a conducting material of which the sensor mass is made.\",\n \"92.The acceleration sensor of claim 67, wherein said first cover section of said frame structure is made of a glass containing an alkali metal.\",\n \"93.The acceleration sensor of claim 73, wherein said second cover section of said frame structure is made of a glass containing an alkali metal.\",\n \"94.A method for producing an acceleration sensor as defined in claim 86, said method comprising the following steps: a) first producing each of said plurality of sensor inertia masses as a single sensor with its own frame structure and b) assembling said single sensors to a complete sensor.\"\n ],\n [\n \"The invention relates to a micromechanical capacitive acceleration sensor in accordance with the preamble of patent claim 1 and the preamble of patent claim 17.Micromechanical capacitive acceleration sensors made of a wafer are known for picking up the acceleration of an object in one or more directions.\",\n \"The sensor has a mounting which is for example a frame structure which is fixed relative to the object and a sensor inertia mass which is movable relative to the mounting or frame structure of the acceleration sensor, with a bearing device by means of which the sensor mass is elastically mounted relative to the mounting about a starting position.\",\n \"The sensor further includes a capacitive pick-up unit for producing at least one capacitive output signal that represents the position of the sensor mass relative to the mounting or frame structure.\",\n \"The capacitive pick-up unit comprises for example first capacitor electrodes provided on the sensor mass and second capacitor electrodes provided on the mounting opposite the first capacitor electrodes.\",\n \"For example, a coupling arrangement is provided for coupling the first capacitor electrodes provided on the sensor mass which first connector elements provided on the frame structure.\",\n \"The second capacitor electrodes are coupled with second connector elements.\",\n \"Micromechanical capacitive acceleration sensors of this type or similar type are known from the following German patent publications: DE 38 37 883 A1; DE 196 37 265 A1; DE 41 26 100 A1, DE 197 09 520 A1 and DE 197 09 731 A1.It is the object of the invention to provide a micromechanical capacitive acceleration sensor which is as simple as possible and thus cost effective to produce, which has a small size and a sensitivity as small as possible relative to interfering influences.\",\n \"This object has been achieved by the micromechanical capacitive acceleration sensors defined in patent claims 1 and 17.Advantageous further embodiments of the invention are characterized in the dependent claims.\",\n \"According to a first aspect of the invention a micromechanical capacitive acceleration sensor is provided for picking up the acceleration of an object in at least one direction.\",\n \"The sensor includes a frame structure, a sensor inertia mass made of a wafer and movably supported relative to the frame structure.\",\n \"The sensor further includes a capacitive pick-up unit for producing at least one capacitive output signal representing the position of the sensor mass relative to the frame structure whereby the sensor inertia mass has a center of gravity which is offset relative to the rotation axis in a direction extending perpendicularly to a plane of the wafer.\",\n \"It is an important advantage of this acceleration sensor according to the invention that it can be manufactured in an especially simple way and that it has a large sensitivity and a compact construction.\",\n \"The acceleration sensor has a small sensitivity relative to interference influences.\",\n \"The acceleration sensor is capable of measuring accelerations parallel to the wafer surface.\",\n \"The present sensor is further adapted to produce two or three axes acceleration sensors or sensor systems which, for example, may be produced monolithically.\",\n \"For example, either the sensor mass and/or the frame structure may be constructed monolithically out of one single crystal silicon wafer.\",\n \"This feature provides an especially cost effective production while providing a most compact structure and a high measuring precision.\",\n \"The acceleration sensor has, for example, a bearing device for supporting the sensor inertia mass.\",\n \"The bearing device comprises particularly torsionable elements, for example, made of silicon.\",\n \"This feature also enhances the simple and cost effective production of a compact structure.\",\n \"The bearing device forms preferably an electrically conducting connection between the sensor mass and an electrical contacting provided on the frame structure.\",\n \"This feature achieves that the contacting of the movable sensor mass takes place on the frame structure whereby the electrical connection to the sensor mass is produced in an especially simple and reliable manner.\",\n \"Preferably the bearing device is arranged on the sensor mass close to the wafer surface.\",\n \"This features provides, for example, an especially large measuring sensitivity.\",\n \"The sensor mass can, for example, be formed to have a cubic shape, or a parallelepiped shape, or a frustum shape, or a truncated pyramid shape.\",\n \"These geometries provide even additionally large measuring sensitivities while requiring little space and assuring a cost effective production.\",\n \"A further embodiment preferably provides several sensor inertia masses which are respectively supported for movement about a rotation axis in order to measure accelerations in several directions extending perpendicularly to one another.\",\n \"One or more sensor masses have a respective center of gravity which is offset relative to the respective rotation axis in a direction perpendicularly to the wafer plane.\",\n \"Thus, a sensor system is provided which in spite of a small structural size and a cost effective production permits measurements in two or three axial directions or precise acceleration measurements in several directions of space.\",\n \"Preferably the center of gravity of the sensor mass is additionally offset laterally in a direction of the wafer plane relative to the respective rotation axis.\",\n \"As a result of the additional lateral offset of the center of gravity a measuring sensitivity is achieved in a further direction of space.\",\n \"The acceleration sensor has preferably first capacitor electrodes provided on the sensor mass and second capacitor electrodes provided on the frame structure in positions opposite the first capacitor electrodes.\",\n \"In this embodiment for example first connector elements for the electrical connection of the first capacitor electrodes and/or a second connector elements for the electrical connection of the second capacitor electrodes are arranged preferably in a common connector plane.\",\n \"The contacting of the first connector elements and/or the second connector elements can take place from a single side.\",\n \"The electrical contacting of the capacitive pick-up unit can also take place from a single side.\",\n \"The second capacitor electrodes and the first and second connector elements are provided in a common connector plane preferably on the frame structure.\",\n \"Each of these features provides an especially advantageous possibility for contacting which simplifies the production and additionally reduces costs.\",\n \"The acceleration sensor preferably has a coupling arrangement for coupling the first capacitor electrodes provided on the sensor mass with first connector elements provided on the frame structure whereby the coupling arrangement comprises for example a galvanic or capacitive connection for bridging an elevational displacement between the first capacitor electrodes and a common connector plane.\",\n \"Thus, for example all connector elements can be positioned in a single plane.\",\n \"Preferably the frame structure comprises a central section which surrounds the sensor mass laterally and a first cover section elevationally displaced relative to the sensor mass and arranged on the central section whereby particularly second capacitor electrodes are provided on the first cover section on its side facing the sensor mass.\",\n \"The side of the first cover section facing the sensor mass forms for example a common connector plane for the electrical connection of the capacitive pick-up unit.\",\n \"Preferably the second capacitor electrodes are formed by a metallizing of the sensor mass or by the sensor mass itself which is at an electrical potential.\",\n \"According a second aspect of the invention the acceleration sensor comprises a frame structure which is stationary relative to the object, a sensor inertia mass which is movable relative to the frame structure of the acceleration sensor, a bearing device by means of which the sensor mass is elastically supported relative to the frame structure and about a starting position.\",\n \"The second embodiment further includes a capacitive pick-up unit for producing at least one capacitive output signal representing the position of the sensor mass relative to the frame structure.\",\n \"The pick-up unit comprises first capacitor electrodes provided on the sensor mass and second capacitor electrodes provided on the frame structure in positions opposite to the first capacitor electrodes.\",\n \"A coupling arrangement is provided for coupling the first capacitor electrodes provided on the sensor mass with first connector elements provided on the frame structure and second connector elements to which the second capacitor electrodes are coupled.\",\n \"In this embodiment is it particularly provided that the first connector elements and/or the second connector elements are arranged in a common connector plane which is, for example, elevationally displaced upwardly relative to the first capacitor electrodes provided on the sensor mass and/or relative to the second capacitor electrodes provided on the frame structure.\",\n \"Further, the coupling arrangement comprises particularly means for bridging the elevational displacement between the first capacitor electrodes and/or the second capacitor electrodes and the common connector plane.\",\n \"The essential advantage of this aspect of the micromechanical capacitive acceleration sensor according to the invention is seen in that it is particularly simple to produce.\",\n \"This advantage is particularly due to the fact that all electrical connections in the form of the connector elements for the first and second capacitor electrodes are positioned in a single plane.\",\n \"According to a preferred embodiment of the acceleration sensor according to the invention it is provided that the second capacitor electrodes provided at the frame structure and the first and second connector elements are arranged in the common connector plane and that the coupling arrangement comprises means for bridging the elevational displacement to the common connector plane.\",\n \"This embodiment is especially advantageous because the arrangement of the first and second connector elements together with the second capacitor electrodes in a common plane makes the production especially simple.\",\n \"According to a further aspect of the invention it is provided that the coupling arrangement forms a galvanic connection that bridges the elevational displacement to the common connector plane.\",\n \"According to a still other aspect of the invention it is provided that the coupling arrangement forms a capacitive connection that bridges the elevational displacement to the common connector plane.\",\n \"According to an especially preferred embodiment of the invention it is provided that the frame structure comprises a central section laterally surrounding the sensor mass and a first cover section elevationally displaced relative to the sensor mass and arranged on said central section, whereby the second capacitor electrodes are provided on the side of the first cover section that faces the sensor mass and wherein the first capacitor electrodes are provided on the sensor mass opposite said second capacitor electrodes.\",\n \"The advantage of this arrangement is seen in the simple contacting and in small stray capacities.\",\n \"An advantageous further development of the just mentioned embodiment provides that the common connector plane is formed by the side of the first cover section which faces the sensor mass and on which side said first connector elements and said second connector elements are arranged in said common connector-plane and in which side the second capacitor electrodes are also provided.\",\n \"A preferred embodiment hereof provides that the first connector elements, the second connector elements, and the second capacitor electrodes which are connected with the second connector elements, are produced of a single conducting layer which is formed on the side of the first cover section facing the sensor mass.\",\n \"In connection with embodiments in which the coupling arrangements comprises a capacitive connection it is advantageously provided that a second coupling electrode is formed on the side of the first cover section facing the sensor mass and that a first coupling electrode is provided on the sensor mass opposite the second coupling electrode.\",\n \"In this embodiment the first and the second coupling electrodes form a capacitive connection of the coupling arrangement for coupling the first capacitor electrodes provided on the sensor mass with a first connector element provided on the frame structure.\",\n \"This embodiment is advantageously further developed in that the second coupling electrode together with the first connector element are provided in the common connector plane which is formed by that side of the first cover section which faces the sensor mass.\",\n \"Furthermore, it is preferably provided that the second coupling electrode and the first connector element connected therewith are produced together with the second capacitor electrodes and the second connector element connected with the second capacitor electrodes, of a single conducting layer formed on that side of the first cover section which faces the sensor mass.\",\n \"In those embodiments in which the frame structure comprises a central section laterally surrounding the sensor mass and a first cover section elevationally displaced relative to the central section and arranged on the central section, it is advantageously provided that a second cover section is provided on the central section opposite the first cover section and elevationally displaced relative to the sensor mass.\",\n \"The first cover section, the central section and the second cover section form a housing that hermetically encloses the sensor mass, the first capacitor electrodes and to the second capacitor electrodes.\",\n \"The housing that hermetically seals the acceleration sensor provides on the one hand reliable protection relative to environmental influences and on the other hand makes it possible to adjust inside the housing a small gas pressure for reducing the damping.\",\n \"According to a type of embodiment of the acceleration sensor according to the invention it is provided that the bearing device is formed by torsion elements to which the sensor mass is mounted in a torsion elastic manner relative to the frame structure and about said starting position.\",\n \"A preferred embodiment of this version provides that two torsion elements forming the bearing device are positioned on a common rotation axis and wherein the two torsion elements are effective on two opposite sides of the sensor mass whereby the sensor mass is movable only in a single direction relative to the frame structure.\",\n \"A preferred embodiment of the acceleration sensor according the invention provides that the torsion elements are secured on the one hand to the sensor mass and on the other hand to the central section of the frame structure.\",\n \"According to an aspect of the invention it is provided that the coupling arrangement is formed by a galvanic connection which comprises the torsion elements.\",\n \"Another type of the embodiment of the acceleration sensor according to the invention provides that the bearing device is formed by one or more bending elements on which the sensor mass is supported in a bending elastic manner about said starting position and relative to the frame structure.\",\n \"A preferred embodiment hereof provides that two bending elements are positioned in a common plane, spaced from each other and connected to the sensor mass whereby the sensor mass is movable only in a single direction relative to the frame structure.\",\n \"According to an embodiment it is provided that the bending elements are connected on the one hand to the sensor mass and on the other hand to the first cover section of the frame structure.\",\n \"Another aspect of the invention provides that the coupling arrangement is formed by a galvanic connection that includes the bending elements.\",\n \"Another type of embodiment of the acceleration sensor according to the invention provides that a single bending element is centrally secured to the sensor mass whereby the sensor mass is movable relative to two different directions and relative to the frame structure.\",\n \"This embodiment further includes the pick-up unit for producing two capacitive output signals which are representing the position of the sensor mass relative to the frame structure.\",\n \"The advantage of this type of embodiment is seen in that with a single sensor mass it is possible to pick up accelerations in two different directions for example in the two directions of the horizontal plane.\",\n \"According to a preferred embodiment hereof it is provided that the pick-up unit comprises four first capacitor electrodes which are secured to the sensor mass in pairs positioned opposite each other and on opposite sides of the bending element.\",\n \"Four second capacitor electrodes are secured to the first cover section of the frame structure whereby the second capacitor electrodes are arranged in pairs positioned opposite each other on opposite sides of the bending element and opposite the first capacitor electrodes.\",\n \"In case a capacitive connection is to be formed in the last mentioned embodiment by the coupling arrangement, it is particularly advantageous to provide that the coupling arrangement comprises a first coupling electrode secured to the sensor mass and surrounding the bending element and a second coupling electrode secured to the first cover section of the frame structure said second coupling electrode being positioned opposite the first coupling electrode and surrounding the bending element.\",\n \"In connection with the embodiments in which the coupling arrangement forms a galvanic connection it is advantageous to provide that the coupling arrangement is formed by a galvanic connection which comprises the bending element.\",\n \"According to a further type of embodiment of the acceleration sensor according to the invention it is provided that for picking up the acceleration of an object in several directions, several sensor masses are provided each equipped with a capacitive pick-up unit.\",\n \"These several sensor masses are mounted independently of one another each by means of a corresponding bearing device in a frame structure.\",\n \"Each sensor mass is provided with a capacitive pick-up unit for producing an output signal representing the position of the corresponding sensor mass relative to the frame structure.\",\n \"Hereby, it is advantageously provided that the sensor masses are mounted in a common plane in the frame structure and that the first and second connector elements of all sensor masses are provided in a common connector plane.\",\n \"According to an embodiment of the acceleration sensor according to the invention it is provided that a sensor mass is provided for picking up the acceleration of the object in a horizontal direction, wherein the sensor mass comprises a bearing device which is elevationally displaced upwardly relative to the mass center of gravity whereby the sensor mass performs, in response to an acceleration of the object, a rotational motion about a rotation axis defined by the bearing device.\",\n \"According to another embodiment of the acceleration sensor according to the invention it is provided that a sensor mass for picking up the acceleration of the object in a vertical direction is provided which comprises a bearing device laterally offset relative to the mass center of gravity whereby the sensor mass performs, in response to an acceleration of the object, a rotational motion about a rotation axis defined by the bearing device.\",\n \"In this connection it is particularly advantageous to provide that the first capacitor electrodes are respectively provided in pairs on the sensor mass on opposite sides of the rotational axis defined by the bearing device and in that the second capacitor electrodes are provided on the frame structure also in pairs and respectively positioned opposite the first capacitor electrodes, and that the differential capacity of the capacitor electrodes forming pairs is used for producing the capacitive output signal of the pick-up unit.\",\n \"Picking up the difference capacity for producing the output signal is particularly advantageous for minimizing interfering influences.\",\n \"Advantageously it is provided that the first capacitor electrodes provided on the sensor mass are formed by a conducting material of which the sensor mass is made.\",\n \"In the embodiments in which the frame structure comprises a center section laterally surrounding the sensor mass and a first cover section secured thereto, it is advantageous if the first cover section of the frame structure is made of a glass containing an alkali metal.\",\n \"It is also advantageous to provide that the second cover section of the frame structure is made of a glass containing an alkali metal.\",\n \"Finally there is provided a method for producing an acceleration sensor in accordance with one of the preceding embodiments whereby each of the sensor masses is first produced as a single sensor with its own and wherein the single sensors are then assembled to a complete sensor.\",\n \"In the following example embodiments of the invention are described with reference to the drawing wherein: FIG.\",\n \"1a shows a perspective partially sectional view of a micromechanical capacitive acceleration sensor for picking up the acceleration of an object in one direction according to a first example embodiment of the invention; FIG.\",\n \"1b shows an electrical equivalent circuit diagram of the capacitive acceleration sensor according to FIG.\",\n \"1a; FIG.\",\n \"2 is a cross sectional view of a micromechanical capacitive acceleration sensor according to the first example embodiment shown in FIG.\",\n \"1a; FIG.\",\n \"3 shows the perspective views of three sensor masses of a micromechanical capacitive acceleration sensor for picking up the acceleration of an object in three directions (x, y, z) as an expansion of the first example embodiment of the invention shown in FIG.\",\n \"1.FIG.\",\n \"4a is a perspective view partially in section of a micromechanical capacitive acceleration sensor for picking up the acceleration of an object in one direction (x) according to a second example embodiment of the invention; FIG.\",\n \"4b shows an electrical equivalent circuit diagram of the capacitive acceleration sensor of FIG.\",\n \"4 according to a variant with a capacitive coupling; FIG.\",\n \"4c is an electrical equivalent circuit diagram of the capacitive acceleration sensor of FIG.\",\n \"4a according to a variant with a galvanic coupling; FIG.\",\n \"5 shows a cross sectional view of the micromechanical capacitive acceleration sensor according to the example embodiment of FIG.\",\n \"4a; FIG.\",\n \"6 shows a sectional view of a micromechanical capacitive acceleration sensor according to a third example embodiment of the invention; FIG.\",\n \"7 is a perspective view of a sensor mass of a micromechanical capacitive acceleration sensor for picking up the acceleration of an object in two directions (x, y) according to a fourth and according to a fifth example embodiment of the invention; and FIGS.\",\n \"8a and 8b respectively show plan views of arrangements of capacitor and connector elements of a micromechanical capacitive acceleration sensor for picking up the acceleration of an object in two directions (x, y) according to the fourth and fifth example embodiment of the invention.\",\n \"In FIGS.\",\n \"1a and 2 a first example embodiment of the micromechanical capacitive acceleration sensor for picking up the acceleration of an object in one direction namely the direction x is altogether designated with the reference character 100.The acceleration sensor 100 comprises a frame structure 110 including a central section 111, a first cover section 112 and a second cover section 113 whereby the latter is shown only in FIG.\",\n \"2.The frame structure 110 is arranged in a fixed position relative to the object the acceleration of which is to be picked up.\",\n \"The acceleration sensor 100 comprises a sensor inertia mass 101 which is movable relative to the frame structure 110 of the acceleration sensor 100 and which is elastically mounted about a starting position by a bearing device 105 relative to the frame structure 110.The sensor inertia mass 101 has, in the preferred example embodiment shown here, a center of gravity which is arranged offset relative to the rotation axis of the sensor mass 101 in a direction extending perpendicularly to the wafer plane.\",\n \"That is, in this example embodiment the center of gravity of the sensor mass 101 is supported outside of the rotation axis, whereby the rotation axis is positioned near to the surface of the wafer or of the sensor mass 101, and the center of gravity is positioned below the rotation axis when the wafer surface is horizontally aligned.\",\n \"Thus, an acceleration F oriented in parallel to the wafer surface can be measured.\",\n \"The acceleration F causes a deflection of the sensor inertia mass 101 about the rotation axis.\",\n \"The central section 111 of the frame structure 110 laterally surrounds the sensor mass 101 and the first cover section 112 is elevationally offset relative to the sensor mass 101 and arranged on the central section 111.The bearing device 105 in the example embodiment as shown in FIGS.\",\n \"1a and 2 is formed by torsion elements to which the sensor mass 101 is mounted in a torque elastic manner relative to the frame structure 110 i.e.\",\n \"relative to the central portion 111 for movement about said starting position.\",\n \"The torsion elements that form the bearing device 105 are provided on sides of the sensor mass 110 positioned opposite each other on a common rotation axis.\",\n \"Thus, the sensor mass 101 is only movable relative to a single direction, namely the coordinate direction designated by x in FIG.\",\n \"1a relative to the frame structure 110.The sensor mass 101 moves in the direction of the x-axis about its rotation axis in response to the occurrence of an acceleration force Fx whereby the deflection at the edge of the sensor mass 101 is directed upwardly and downwardly.\",\n \"The torsion elements 105 are connected on the one hand to the central section 111 of the frame structure 110 and on the other hand to the sensor mass 101 in a rotationally stable manner.\",\n \"Furthermore the acceleration sensor 100 comprises a capacitive pick-up unit 120 for producing a capacitive output signal which represents the position of the sensor mass 101 relative to the frame structure 110.The pick-up unit comprises first capacitor electrodes 121, 122 provided on the sensor mass 101 and second capacitor electrodes 125, 126 provided at the frame structure 110 opposite the first capacitor electrodes 121, 122.The first capacitor electrodes 121, 122 provided on the sensor mass 101 are connected to a first connector element 142 provided on the first cover section 112.The connection is a galvanic connection formed by a coupling arrangement which is formed by the torsion elements 105.The galvanic connection also passes through a metallization 141 on the central section 111 of the frame structure 110.Second connector elements 145, 146 are coupled with the second capacitor electrodes.\",\n \"The second connector elements are also provided on the first cover section 112 of the frame structure 110.The first capacitor electrodes 121, 122 provided on the sensor mass 101 are formed by a conducting material of which is the sensor mass 101 is made.\",\n \"In this manner one obtains an equivalent electrical circuit diagram for the first and second capacitor electrodes 121, 122 and 125, 126 as shown in FIG.\",\n \"1b.\",\n \"Two capacities E1 and E3 are connected in series between two reference voltages +Vref (connector element 145) and −Vref (connector element 146).\",\n \"The center junction of the capacitor series connection is connected to a terminal K1 (connector element 142) at which the output signal Vout can be tapped off.\",\n \"The first connector element 142 and the second connector elements 145, 146 are arranged in a common connector plane 150.In this first example embodiment this common connector plane 150 is offset relative to the first capacitor electrodes 121, 122 provided on the sensor mass 101.This elevational offset is bridged by the coupling arrangement formed by the torsion elements 105, the metallization 141 and the first connector element 142.The second capacitor electrodes 125,126 are also arranged in said common connector plane 150 together with the first and second connector elements 142 and 145, 146.The connector plane 150 is formed by one side of the first cover section 112.The first connector element 142, the second connector elements 145, 146 and the second capacitor electrodes 125, 126, which are connected with the latter, are made of a single conductive layer formed on that side of the first cover section 112 which faces the sensor mass 101.As seen in FIG.\",\n \"2, the central section 111, the first cover section 112 and the second cover section 113 form together a hermetically sealed housing in which the sensor mass 101 is arranged together with the first capacitor electrodes 121, 122 and the second capacitor electrodes 125, 126.The first cover section 112 and the second cover section 113 of the frame structure 110 are made of a glass containing an alkaline metal.\",\n \"FIG.\",\n \"3 shows the sensor masses 101, 102, 103 which are provided for picking up the acceleration of an object in several directions namely the directions x, y, z of the coordinate system shown in FIG.\",\n \"3.The sensor mass 101 (partial Fig.\",\n \"A) corresponds to the sensor mass 101 of the acceleration sensor shown in FIGS.\",\n \"1a and 2.The sensor mass 101 is mounted in a torsion elastic manner by means of torsion elements 105 whereby it is movable about a starting position.\",\n \"The torsion element 105 defines a rotation axis which is offset relative to the mass center of gravity Mx of the sensor mass 101 so that an acceleration force Fx effective in the x-direction causes a deflection Sx.\",\n \"The sensor mass 102 (partial Fig.\",\n \"B) corresponds in its configuration substantially to the sensor mass 101 and also has torsion elements 106 which form a bearing device for supporting the sensor mass in a torsion or rotational elastic manner.\",\n \"However, the rotation axis defined by the torsion elements 106 is angularly displaced by 90 degrees in the horizontal plane relative to the rotation axis of the sensor mass 101.The rotation axis formed by the torsion elements 106 is again displaced relative to the mass center of gravity My of the sensor mass 102 in elevation so that an acceleration force Fy effective in the y-direction causes a deflection Sy of the sensor mass about its starting position.\",\n \"The sensor masses 101 and 102 are provided, corresponding to the sensor mass 101 of FIGS.\",\n \"1a and 2 with first capacitor electrodes 121 and 122 of a capacitive pick-up unit not particularly shown in FIG.\",\n \"3.The capacitive pick-up unit produces a capacitive output signal representing the position of the sensor mass 102 relative to a frame structure also not shown in particular in FIG.\",\n \"3.A third sensor mass 103 (partial Fig.\",\n \"C of FIG.\",\n \"3) is mounted by means of a bearing device formed by torsion elements 107 for rotation about a rotation axis.\",\n \"The mass center of gravity Mz is laterally offset relative to the rotation axis formed by the torsion elements 107.A vertical acceleration Fz caused by an acceleration in the vertical z-direction causes a deflection Sz of the end of the sensor mass 103 as shown in partial Fig.\",\n \"C. Each of the sensor masses 101, 102, 103 is provided with a capacitive pick-up unit for producing a respective output signal representing the corresponding position of the sensor mass relative to a frame structure not shown.\",\n \"This capacitive pick-up unit comprises respectively first capacitor electrodes (not shown in FIG.\",\n \"3).\",\n \"These first capacitive electrodes are provided in pairs respectively on opposite sides of the rotation axis Ax, Ay, Az.\",\n \"These rotation axis are defined by the torsion elements 105, 106 and 107 respectively.\",\n \"Second capacitor electrodes of the capacitive pick-up unit are provided on the frame structure, not shown, of the acceleration sensor.\",\n \"The second capacitor electrodes are positioned opposite the first capacitor electrodes.\",\n \"For the sensor masses 101 and 102 shown in the partial Figs.\",\n \"A & B, this means an arrangement similar to that shown in FIGS.\",\n \"1a and 2.In connection with the sensor mass 103 shown in partial Fig.\",\n \"C of FIG.\",\n \"3, the first capacitor electrode is arranged to the left of the rotation axis defined by the torsion elements 107 on the short piece of the sensor mass 103.The other capacitor electrode is arranged symmetrically to the first capacitor electrode and to the right hand side of the rotation axis defined by the torsion elements 107.Here again the difference capacity of the capacitor electrodes arranged in pairs is used for producing the capacitive output signal of the capacitive pick-up units.\",\n \"This utilization of the difference capacity is similar to that explained with reference to the example embodiment of FIGS.\",\n \"1 & 2.The first capacitor electrodes provided on the sensor mass 101, 102 or 103 are formed in the just described example embodiment by a conducting material of which the sensor mass 101, 102, 103 is made.\",\n \"Each of the sensor masses 101, 102 and 103 shown in FIG.\",\n \"3 is initially and preferably produced as an individual sensor with its own frame structure.\",\n \"These individual sensors are then assembled to form a complete sensor capable of picking up accelerations in all three directions of space x, y, z.\",\n \"The acceleration sensor shown in FIGS.\",\n \"4a, 5 and 6 according to a second and a third example embodiment of the invention is designated altogether with the reference characters 200; 300.The acceleration sensor 200; 300 serves for picking up the acceleration of an object in the x-direction shown in the coordinate system illustrated in FIG.\",\n \"4a.\",\n \"The sensor comprises a frame structure 210, 310 which is mounted in a fixed position relative to the object the acceleration of which is to be picked up.\",\n \"The acceleration sensor further comprises a sensor inertia mass 201, 301 which is movable relative to the frame structure 210, 310 of the acceleration sensor 200, 300.The sensor mass 201, 301 is elastically mounted about a starting position by means of a bearing device 205, 305 relative to the frame structure 210, 310.The frame structure 210, 310 comprises a center section 211, 311 laterally surrounding the sensor mass 201, 301 and a first cover section 212, 312 arranged on the center section 211, 311 elevationally displaced relative to the sensor mass 201, 301.The bearing device of the second and third example embodiments shown in FIGS.\",\n \"4 to 6 is formed by bending elements 205, 305.The sensor mass 201, 301 is mounted to the bending elements in a bending elastic manner about the starting position and relative to the frame structure 210, 310.As shown in FIG.\",\n \"4a, two of these bending elements which are provided in a common plane and spaced from one another and connected to the sensor mass 201, 301.These bending elements 205, 305 define a rotation axis about which the sensor mass 201, 301 moves in response to the occurrence of an acceleration force Fx in the direction of the x-axis whereby the deflection Sx at an edge of the sensor mass 201, 301 is directed upwardly and downwardly.\",\n \"The bending elements 205, 305 are connected on the one hand to the sensor mass 201, 301 and on the other hand to the first cover section 212, 312 of the frame structure 210, 310.The acceleration sensor 200, 300 comprises a capacitive pick-up unit altogether designated by the reference character 220, 320.This pick-up unit 220, 320 serves for producing an output signal representing the position of the sensor mass 201, 301 relative to the frame structure 210, 310 and thus the size of the acceleration force Fx that deflects the sensor mass 201, 301 out of its starting position.\",\n \"This output signal is a measure for the acceleration of the object to which the acceleration sensor is attached.\",\n \"The capacitive pick-up unit 220, 320 comprises first capacitor electrodes 221, 222, 321, 322 which are provided on the sensor mass 201, 301.The pick-up unit further comprises second capacitor electrodes 225, 226; 325, 326 which are secured to the frame structure 210, 310 more specifically on the first cover section 212, 312 in positions opposite the first capacitor electrodes 221, 222; 321, 322.Generally, a coupling arrangement is provided for coupling the first capacitor electrodes 221, 222 or 321, 322 provided on the sensor mass 201, 301 with first connector elements.\",\n \"Of these connector elements, the first connector element 242 of the acceleration sensor 200 is seen in FIG.\",\n \"4a according to the second example embodiment.\",\n \"This first connector element 242 is arranged on the frame structure 210, more specifically on the first cover section 212 thereof.\",\n \"The acceleration sensor 300 shown in FIG.\",\n \"6 according to the third example embodiment, is arranged correspondingly, however, the connector element corresponding to the first connector element 242 of FIG.\",\n \"4a is not visible in FIG.\",\n \"6.The second capacitor electrodes 225, 226 or 325, 326 provided on the first cover section 212, 312 are coupled with the second connector elements.\",\n \"Here again the second connector elements 245, 246 which are connected with the second capacitor electrodes 225, 226 of the acceleration sensor 200, are seen in FIG.\",\n \"4a.\",\n \"The acceleration sensors 300 of the third example embodiment shown in FIG.\",\n \"6 has a corresponding arrangement.\",\n \"The first connector elements, compare the connector element 242 in FIG.\",\n \"4a, and the second connector elements, compare the connector elements 245, 246 in FIG.\",\n \"4a, are arranged in a common connector plane 250, 350.This common connector plane is formed by that side of the first cover section 212, 312 which faces the sensor mass 201, 301.Further, this common connector plane is elevationally offset relative to the first capacitor electrodes 221, 222; 321, 322 provided on the sensor mass 201, 301.The coupling arrangement which connects the first capacitor electrodes 221, 222 or 321, 322 provided on the sensor mass 201 or 301, with the first connector elements, compare 242 in FIG.\",\n \"4a, forms a bridging of the elevational offset between the sensor mass 201, 301 and said common connector plane 250, 350.The coupling arrangement in the acceleration sensor 200 of the second example embodiment shown in FIGS.\",\n \"4a and 5 is formed by a capacitive connection.\",\n \"This capacitive connection comprises first and second coupling electrodes 231 and 232 positioned opposite each other.\",\n \"The first coupling electrode 231 is provided on the sensor mass 201.The second coupling electrode is provided on the first cover section 212.The second coupling element 232 is connected with the above mentioned first connector element 242.The second coupling electrode is provided on that side of the first cover 212 which faces the sensor mass 201, compare FIG.\",\n \"4a.\",\n \"FIG.\",\n \"4b shows an electrical equivalent circuit diagram of the acceleration sensor 200 according to the second example embodiment as shown in FIGS.\",\n \"4a and 5.As can be seen the capacities E1 and E3 formed by the first capacitor electrodes 221, 222 and by the second capacitor electrodes 225, 226, are connected in series between a positive reference voltage +Vref (at the terminal 245) and a negative reference voltage −Vref (at the terminal 246).\",\n \"The output signal Vout is provided at the center junction through a capacity E2 formed by the capacitive coupling.\",\n \"Contrary to the above, in the third example embodiment of the acceleration sensor 300 shown in FIG.\",\n \"6, the first capacitor electrodes 321, 322 are galvanically connected with the first connector element corresponding to the connector element 242 in FIG.\",\n \"4a.\",\n \"Specifically, the connection extends through the bending elements 305 which are made of a conductive material.\",\n \"In this manner a galvanic connection is formed between the capacities E1 and E3 formed by the capacitor electrodes 321, 322 and 325, 326.The output voltage Vout can be tapped off at this galvanic connection, compare FIG.\",\n \"4c.\",\n \"As in the first example embodiment of the acceleration sensor shown in FIG.\",\n \"1a and FIG.\",\n \"2, the first cover section 212, 312 and the second cover section 213, 313 in the acceleration sensors 200 and 300 according to the second and third example embodiment of FIGS.\",\n \"4a, 5 and 6 are also made of a glass containing an alkali metal.\",\n \"The first cover section 212, 312, the central section 211, 311 and the second cover section 213, 313 form together a hermetically sealed housing for the sensor mass 201, 301, for the first capacitor electrodes 221, 222; 321, 322 and for the second capacitor electrodes 225, 226; 325, 326.The second cover section 213, 313 is connected to the center section 211, 311 of the frame structure 210, 310 and positioned opposite the first cover section 212, 312.The first connector elements, shown at 242 in FIG.\",\n \"4a, the second connector elements shown at 245, 246 in FIG.\",\n \"4a and the second capacitor electrodes 225, 226 or 325, 326 are formed of a single conducting layer in the example embodiments shown in FIGS.\",\n \"4a, 5 and 6.This single conducting layer is formed on that side of the first cover section 212, 312 that faces the sensor mass 201, 301.FIG.\",\n \"7 shows a sensor mass 401, 501 of fourth and fifth example embodiments of the acceleration sensor capable of picking up acceleration in two directions namely the x-direction and the y-direction in the horizontal plane.\",\n \"A single bending element 405, 505 is secured centrally to the sensor mass 401, 501 so that the sensor mass is movable in two different directions in response to acceleration forces Fx or Fy relative to a frame structure not shown in FIG.\",\n \"7.In order to obtain output signals representing the position of the sensor mass 401, 501, capacitive pick-up units are provided which will be described with reference to FIGS.\",\n \"8a and b.\",\n \"FIG.\",\n \"8 shows a portion of capacitive pick-up units 420, 520 for picking up the position of the sensor mass 401, 501 in two directions.\",\n \"The pick-up unit comprises four second capacitor electrodes 425, 426, 427, 428; 525, 526, 527 and 528 which are provided in pairs facing each other on opposite sides of the bending element 405, 505 on a first cover section 412, 512 of a frame structure similar to the example embodiments illustrated in FIG.\",\n \"1a and FIG.\",\n \"2 or in FIGS.\",\n \"4a to 6.These second capacitor electrodes 425, 426, 427, 428; 525, 526, 527, 528 are electrically connected with second connector elements 445, 446, 447, 448; 545, 546, 547 and 548.Similar first capacitor electrodes are provided on the sensor mass 401, 501 and positioned opposite the second capacitor electrodes 425, 426, 427, 428; 525, 526, 527 and 528.These first capacitor electrodes are not particularly shown in FIG.\",\n \"7.FIG.\",\n \"8a illustrates a fourth example embodiment including a coupling arrangement which connects the first capacitor electrodes provided on the sensor mass 401 with a first connector element 442 on the first cover section 412.The fourth example embodiment further comprises a first coupling electrode surrounding the bending element 405.The first coupling electrode is not shown but provided on the sensor mass 401, and a second coupling electrode 432 surrounding the bending element 405 and positioned opposite the first coupling electrode.\",\n \"The second coupling electrode 432 is secured to and provided on the first cover section 412 of the frame structure.\",\n \"Thus, a capacitive connection is formed between the first capacitor electrodes provided on the sensor mass 401 and the first connector element 442.Contrary to the above, in the example embodiment shown in FIG.\",\n \"8b, the coupling arrangement is formed by a galvanic connection comprising the bending element 505 so that the first capacitor electrodes provided on the sensor mass 501, please compare FIG.\",\n \"7, are connected galvanically through the bending element 505 with the first connector element 542.As in the above example embodiments, the first capacitor electrodes provided on the sensor mass 401, 501 are formed by a conducting material of which the sensor mass 401, 501 is made.\",\n \"The sensor mass used in particularly preferred embodiments of the invention is made of a single crystal silicon.\",\n \"Thereby the capacitor electrodes of the sensor mass are formed directly by the sensor mass itself.\",\n \"In these cases the sensor mass is electrically connected with the frame structure whereby the torsion beams or the suspensions of the sensor mass form conducting connections to the sensor mass.\",\n \"Thus, the sensor mass is maintained at a certain electrical potential and forms with the oppositely positioned capacitor electrodes capacitive elements for the measurement of the deflection of the sensor mass.\",\n \"The electrical contacting of the sensor mass takes place for example at a location of the frame which is readily accessible and which is connected in an electrically conducting manner with the sensor mass through the torsion beams.\",\n \"The contacting of all electrical connections can be achieved on a single side for example on the back side of the sensor or from its top side with the wafer alignment shown in FIG.\",\n \"1a.\",\n \"It is thereby possible to provide holes in the wafer which contain conducting material or are filled with conducting material in order to electrically contact from the outside the connector electrodes 142, 145 and 146 positioned on the inside for example on the side 150 of the cover section 112 positioned opposite the sensor mass 101.Instead of the coupling arrangement 141, 142 shown in FIG.\",\n \"1a, with one connecting electrode 142 of the sensor mass 101 at the cover section 112, it is possible to arrange the connector electrodes of the sensor mass 101 at the lower frame structure 110.Thus, the connector electrode may for example be positioned on the bottom side of the shoulder or projection of the frame structure 110, more specifically, below the coupling arrangement 141 shown in FIG.\",\n \"1a.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471296"}}},{"rowIdx":586,"cells":{"text":{"kind":"list like","value":[["Metal sheet coated with thermoplastic resin and can obtained therefrom","A thermoplastic-resin-coated metal sheet in which the thermoplastic resin has been applied to the metal sheet without through an adhesive primer and which, even after having been formed into a can, is excellent in impact resistance at low temperatures and resistance to corrosion by acid contents; and a can obtained from the coated metal sheet.","The thermoplastic-resin-coated metal sheet is obtained by coating a metal sheet with a thermoplastic resin composition comprising a blend of a polyester resin with a polyolefin resin or polyolefin elastomer.","This metal sheet is formed into a can through drawing/ironing with thickness reduction."],["1.A thermoplastic resin-coated metal sheet in which a thermoplastic resin composition comprising a blend of polybutylene terephthalate and a polyolefin ingredient comprising at least one member selected from the group consisting of polyolefin resins and polyolefin elastomers is coated in a substantially non-orientation state on at least one surface of a metal sheet.","2.A thermoplastic resin-coated metal sheet according to claim 1, wherein a polyolefin resin is used as the polyolefin ingredient.","3.A thermoplastic resin-coated metal sheet according to claim 1, wherein a polyolefin elastomer is used as the polyolefin ingredient.","4.A thermoplastic resin-coated metal sheet according to claim 1, wherein a polyolefin resin and a polyolefin elastomer are used as the polyolefin ingredient.","5.A thermoplastic resin-coated metal sheet according to any one of claim 1, wherein the ratio of a melt index of the polyolefin ingredient upon heat melting (VPOL) to that of the polybutylene terephthalate upon heat melting (VPES), that is, VPOL/VPES is 1.2 or less in the thermoplastic resin composition.","6.A thermoplastic resin-coated metal sheet according to claim 1, wherein a polybutylene terephthalate layer formed by coating the polybutylene terephthalate in a substantially non-orientation state is formed on the coating layer of the thermoplastic resin composition.","7.A thermoplastic resin-coated metal sheet according to claim 1, wherein a polybutylene terephthalate layer coating in which the polybutylene terephthalate is in a substantially non oriented state is formed on the surface of the metal sheet, and a coating layer of the thermoplastic resin composition is formed on the polyester resin layer.","8.A thermoplastic resin-coated metal sheet according to claim 1 wherein the polyolefin resin is a resin comprising one or more of 1-alkene polymer resins having a number or carbon atoms of 2 to 8.9.A thermoplastic resin-coated metal sheet according to claim 8, wherein the 1-alkene polymer resin is one of polyethylene, polypropylene, and an ethylene-propylene copolymer.","10.A thermoplastic resin-coated metal sheet according to claim 8, wherein the polyolefin resin is a polyolefin resin polymerized by means of a metallocene catalyst.","11.A thermoplastic resin-coated metal sheet according to claim 1, wherein at least a portion of the polyolefin resin is a modified polyolefin resin modified with one of maleic acid anhydride, acrylic acid, acrylic acid ester and diglycidyl methacrylate.","12.A thermoplastic resin-coated metal sheet according to claim 1 wherein the polyolefin elastomer is an in-plant produced ethylene-propylene copolymerized elastomer having a melt flow rate (MFR, 230° C.) of 0.4 to 30 g/10 min).","13-15.","(canceled) 16.A thermoplastic resin-coated metal sheet according to claims 1 to 12, wherein the thermoplastic resin composition contains 1 to 30% by weight of a polyolefin ingredient.","17.A thermoplastic resin-coated metal sheet according to claim 1, wherein the thermoplastic resin composition contains 70 to 95% by weight of the polybutylene terephthalate.","18.A thermoplastic resin-coated metal sheet according to claim 1, wherein the thermoplastic resin composition is heat melted and directly extruded from a T-die to a metal sheet for coating.","19.A thermoplastic resin-coated metal sheet according to claim 1, wherein the thermoplastic resin composition is heat melted, extruded from a T-die onto a casting roll, cooled to solidify into a film and then the film is hot press bonded on a metal sheet.","20.A thermoplastic resin-coated metal sheet according to claim 18, wherein, wherein the polyolefin ingredient in the coating layer comprising a thermoplastic resin composition is present being dispersed with a size of 1 to 10 μm in the extruding direction and 0.1 to 2 μm in the direction perpendicular to the extruding direction.","21.A thermoplastic resin-coated metal sheet according to claim 1, wherein the metal sheet is one of electrolytic chromic acid treated steel sheet, tin-plated sheet and aluminum alloy sheet.","22.A can using a thermoplastic resin-coated metal sheet according to claim 1."],["<SOH> BACKGROUND ART <EOH>In recent years, cans formed by coating a polyester resin on a metal sheet and improved with thickness reduction ratio for side wall portions by applying severe forming such as thickness-reducing drawing or thickness-reducing ironing have been used as cans mainly for drink applications.","In a case of forming a polyester resin coated metal sheet by applying severe such as wall reducing drawing or wall reducing ironing, it is necessary to coat in a non-orientation state capable of attaining excellent fabricability such that the resin is not peeled upon forming or cracks are not formed to the resin.","However, when applying thickness-reducing drawing or thickness-reducing ironing is applied to a resin-coated metal sheet with a non-orientation state of a polyester resin, then applying coating printing to the outer surface of the can and heating for baking, since the upper portion of the can is thermally set in a state where it is molecularly oriented in the direction of a height of the can by the fabrication, elongation of the not oriented resin in the circumferential direction of the can is extremely small to become brittle.","Particularly, cracks tend to be formed to the resin layer by mere collision between cans to each other, particularly, at low temperature.","Further, since the portion for the can bottom scarcely undergoes the forming, the resin crystals are grown into a coarse state and brittled upon heating for baking after the coating printing also tending to cause cracks upon receiving impacts particularly at low temperature.","That is, a polyester resin-coated metal sheet in a non-orientation state is poor in the impact resistance, particularly, the impact resistance at low temperature after formed into the can.","Poor impact resistance after forming can be improved by interposing an adhesive primer between the polyester resin and the metal sheet upon coating the polyester resin on the metal sheet.","However, a method of interposing the adhesive primer may possibly cause undesired effects on the environment due to evaporation of organic solvents and, further, requires surplus steps of coating and drying to increase the cost.","Further, in a case where acidic contents at pH of 5 or less are filled, the underlying metal sheet may sometimes be corroded in the upper portion of the can undergoing high degree of fabrication after lapse of a long time.","The present invention intends to provide a thermoplastic resin-coated metal sheet in which a resin layer, after applying severe forming to a thermoplastic resin-coated metal sheet where the resin layer after coating to the metal sheet is in a non-oriented state, is excellent in impact resistance, particularly, impact resistance at low temperature, coated to the metal sheet without interposing an adhesive primer and is excellent in corrosion resistance to acidic contents even in a case where it is formed into a can, as well as a can using the same."],["TECHNICAL FIELD The present invention concerns a thermoplastic resin-coated metal sheet and, particularly, a thermoplastic resin-coated metal sheet in which the coating resin is excellent in impact resistance at low temperature, as well as a can obtained by forming the same.","BACKGROUND ART In recent years, cans formed by coating a polyester resin on a metal sheet and improved with thickness reduction ratio for side wall portions by applying severe forming such as thickness-reducing drawing or thickness-reducing ironing have been used as cans mainly for drink applications.","In a case of forming a polyester resin coated metal sheet by applying severe such as wall reducing drawing or wall reducing ironing, it is necessary to coat in a non-orientation state capable of attaining excellent fabricability such that the resin is not peeled upon forming or cracks are not formed to the resin.","However, when applying thickness-reducing drawing or thickness-reducing ironing is applied to a resin-coated metal sheet with a non-orientation state of a polyester resin, then applying coating printing to the outer surface of the can and heating for baking, since the upper portion of the can is thermally set in a state where it is molecularly oriented in the direction of a height of the can by the fabrication, elongation of the not oriented resin in the circumferential direction of the can is extremely small to become brittle.","Particularly, cracks tend to be formed to the resin layer by mere collision between cans to each other, particularly, at low temperature.","Further, since the portion for the can bottom scarcely undergoes the forming, the resin crystals are grown into a coarse state and brittled upon heating for baking after the coating printing also tending to cause cracks upon receiving impacts particularly at low temperature.","That is, a polyester resin-coated metal sheet in a non-orientation state is poor in the impact resistance, particularly, the impact resistance at low temperature after formed into the can.","Poor impact resistance after forming can be improved by interposing an adhesive primer between the polyester resin and the metal sheet upon coating the polyester resin on the metal sheet.","However, a method of interposing the adhesive primer may possibly cause undesired effects on the environment due to evaporation of organic solvents and, further, requires surplus steps of coating and drying to increase the cost.","Further, in a case where acidic contents at pH of 5 or less are filled, the underlying metal sheet may sometimes be corroded in the upper portion of the can undergoing high degree of fabrication after lapse of a long time.","The present invention intends to provide a thermoplastic resin-coated metal sheet in which a resin layer, after applying severe forming to a thermoplastic resin-coated metal sheet where the resin layer after coating to the metal sheet is in a non-oriented state, is excellent in impact resistance, particularly, impact resistance at low temperature, coated to the metal sheet without interposing an adhesive primer and is excellent in corrosion resistance to acidic contents even in a case where it is formed into a can, as well as a can using the same.","DISCLOSURE OF THE INVENTION According to the present invention, it is provided a thermoplastic resin-coated metal sheet in which a thermoplastic resin composition comprising a blend of one or more of polyester resins and at least one polyolefin ingredient selected from the group consisting of polyolefin resins and polyolefin elastomers is coated on at least one surface of a metal sheet in a substantially non-orientation state.","Further, according to the present invention, a can using a thermoplastic resin-coated metal sheet is provided.","That is, in the present invention, as the polyolefin ingredient in the thermoplastic resin composition forming the coating layer, a polyolefin resin, a polyolefin elastomer, or a combination of a polyolefin resin and a polyolefin elastomer can be used.","Further, a polyolefin resin layer formed substantially in a not-yet oriented state can be disposed above the coating layer of the thermoplastic resin composition or in the lower layer to the coating layer (surface of metal sheet).","In the thermoplastic resin composition used in the present invention, it is preferred that the ratio of a melt viscosity upon heat melting the polyolefin ingredient (polyolefin resin or polyolefin elastomer, or a combination of the polyolefin resin and a polyolefin elastomer) (VPOL) and the melt viscosity upon heat melting the polyester resin (VPES), that is, VPOL/VPES is 1.2 or less.","The polyolefin resin used as the polyolefin ingredient in the present invention is preferably a resin comprising one or more of 1-alkene polymer resins having a number of carbon atoms of 2 to 8, and the 1-alkene polymer resin is preferably one of polyethylene, polypropylene and ethylene-propylene copolymer.","Further, as the polyolefin resin, a polyolefin resin polymerized by a metallocene catalyst can also be used.","Further, a modified polyolefin resin modified with one of maleic acid hydride, acrylic acid, acrylic acid ester, or diglycidyl methacrylate may be used also as the polyolefin resin.","Further, as the polyolefin elastomer used for the polyolefin ingredient, an in-plant produced ethylene-propylene copolymerized elastomer with a melt flow rate (MFR, 230° C.) of from 0.4 to 30 (g/10 min) is suitable.","In the present invention, the polyester used for the thermoplastic resin composition is a polyester resin comprising ester repetitive units of at least one of ethylene terephthalate, ethylene isophthalate, ethylene naphthalate, ethylene adipate, butylenes terephthalate, butylene isophthalate, butylene naphthalate and butylene adipate, with the intrinsic viscosity being preferably from 0.5 to 1.5 and, particularly, ethylene terephthalate/ethylene isophthalate copolymer or polybutylene terephthalate is preferred.","The polyester resin in the polyester resin layer capable of forming in an upper layer or a lower layer to the coating layer of the thermoplastic resin composition is preferably ethylene terephthalate/ethylene isophthalate copolymer.","Further, the thermoplastic resin composition preferably contains 1 to 30% by weight of the polyolefin ingredient and preferably contains 70 to 95% by weight of the polyester resin.","The thermoplastic resin-coated metal sheet according to the present invention is produced by heat melting a thermoplastic resin composition, directly extruding the same from a T-die onto a metal sheet for coating, or heat melting a thermoplastic resin composition, extruding the same from a T-die to a casting roll, cooling to solidify the same into a film and then hot press bonding the film on the metal sheet.","In the thermoplastic resin-coated metal sheet according to the present invention, it is preferred that the olefin ingredient in the coating layer comprised of the thermoplastic resin composition is present being dispersed with a size of 1 to 10 μm in the extruding direction of the resin and 0.1 to 2 μm in the direction perpendicular to the resin extruding direction.","Further, in the thermoplastic resin-coated metal sheet, the metal sheet is preferably one of electrolytic chromic acid treated steel sheet, tin-plated steel sheet or aluminum alloy sheet.","BEST MODE FOR PRACTICING THE INVENTION The present invention provides a thermoplastic resin-coated metal sheet having a coating layer of a thermoplastic resin composition comprising a blend of a polyester resin and a polyolefin ingredient (at least one member selected from the group consisting of polyolefin resins and polyolefin elastomers) at least on one surface of a metal sheet and, optionally, a polyester resin layer formed to an upper layer and/or lower layer of the coating layer, as well as a can obtained by forming the same, and the resin layer is excellent in impact resistance and, particularly, impact resistance at low temperature and also excellent in corrosion resistance to acidic contents even in a case of coating a thermoplastic resin coating without interposing an adhesive primer to a metal sheet and applying severe forming such as thickness reducing-drawing or thickness-reducing ironing.","The present invention is to be described specifically.","In the thermoplastic resin composition comprising the blend of the polyester resin and the polyolefin ingredient, a polyolefin resin and a polyolefin elastomer are used as the polyolefin ingredient each alone or in combination.","In the polyolefin ingredient and the polyester resin, it is preferred that the ratio of the melt viscosity in a case of melting the respective resin pellets is in a predetermined range.","That is, it is preferred that VPOL/VPES is 1.2 or less where VPOL represents the melt viscosity upon heat melting of the polyolefin ingredient (polyolefin resin, polyolefin elastomer or a combination of both of them) and VPES represents a melt viscosity upon heat melting of polyester resin.","The reason will be described below.","The coating layer of the thermoplastic resin composition (heareinafter sometimes simply referred to as a thermoplastic resin film) is formed by heat melting resin pellets and extruding the blended resin from a T-die of an extruder directly onto a long strip-like metal sheet unwound from an uncoiler, or heat melting a resin heat melted from a polyester resin pellets separately and heat melted and blended resin from respective separate extruders, then coextruding them from a T-die having a plurality of die nozzles directly onto a long and strip-like metal sheet unwound from an uncoiler, and pressing the same by pressing rolls to a metal sheet.","Alternatively, it is formed by extruding a heat melted resin in the same manner as described above from a T-die of an extruder onto a casting roll, cooling to solidify the same to form a film, then abutting the film against the heated long strip-like metal sheet which is unwound from an uncoiler and heated, and press bonding by sandwiching the same between a pair of laminate rolls by using an known laminater.","In each of the preparation method, when the molten resin is extended downwardly, that is, in the longitudinal direction when the heat-melted blend resin is extruded from the T-die onto the metal sheet or the casting roll, since the molten resin is stretched downwardly, that is, in the longitudinal direction gravitationally, anisotropy occurs although little in the resin film.","In a case where VPOL/VPES exceeds 1.2, anisotropy of the film of the blend resin increases to lower the rigidity of the film in the lateral direction compared with that of the longitudinal direction, and tend to cause lateral cracking upon exertion of impact after coating to the metal sheet.","With a view point described above, it is preferred that VPOL/VPES is 1.2 or less.","The polyolefin resin used as the polyolefin ingredient can include resins comprising one or more of 1-alkene copolymer resins with a number of carbon atoms of 2 to 8.The 1-alkene copolymer resin with the number of carbon atoms of 2 to 8 can include, for example, low density polyethylene, medium density polyethylene, high density polyethylene, polypropylene, polybutene-1, polypentent-1, polyhexene-1, polyheptene-1, polyoctene-1, ethylene-propylene copolymer, ethylene-1-butene copolymer, and ethylene-hexene copolymer.","Among them, in-plant produced ethylene-propylene copolymer is preferred.","Further, as the polyolefin resins, use of a polyolefin resin by a metallocene catalyst is preferred since it forms less oligomers that may give effects on the flavor of contents.","Further, modified polyolefin resins formed by modifying the polyolefin resin, for example, polyethylene or polypropylene, for example, with one of maleic acid anhydride, acrylic acid, acrylic acid ester, acrylic acid ionomer, and diglycidyl methacrylate may also be used.","The modified polyolefin resin blended at a ratio of 1 to 100% by weight to the not-modified polyolefin resin is used preferably as the polyolefin resin since the polyolefin ingredient is finely dispersed in the polyester resin.","As the polyolefin elastomer, use of an ethylene-propylene copolymer elastomer having a melt flow rate (MFR, 230° C.) of 0.4 to 30 (g/10 min), particularly, 0.8 to 25 g/10 min is preferred.","In a case where MFR is less than the range described above, the melt viscosity is excessively higher relative to the polyester resin when the resin is heat melted and the grains of the polyolefin resin dispersed in the polyester resin is grown excessively to deteriorate the impact resistance.","On the other hand, in a case where it exceeds the range described above, the impact resistance of the polyolefin elastomer per se is poor, and the resin blended with the polyester resin is also poor in the impact resistance.","Further, it is preferred that the ethylene-propylene copolymer elastomer is in-plant produced.","The blending ratio of the polyolefin ingredient to the thermoplastic resin composition (polyolefin ingredient content in the thermoplastic composition) is preferably 1 to 30% by weight.","In a case where the amount of the polyolefin ingredient is small, the thermoplastic resin composition after coating on the metal sheet lacks in the impact resistance.","In a case where the amount of the polyolefin ingredient is unnecessarily large, the transparency of the thermoplastic resin composition decreases to make the anisotropy larger and the hardness is also decreased, so that the resin surface tends to be injured.","As the polyester resin which is blended with the polyester resin to form a thermoplastic resin composition, those comprising ester repetitive units of at least one of ethylene terephthalate, ethylene isophthalate, ethylene naphthalate, ethylene adipate, butylenes terephthalate, butylenes isophthalate, butylenes naphthalate and butylenes adipate can be used alone or as a blend of two or more them.","Further, it is also possible to use those other than described above that use sebasic acid, trimellitic acid or azelaic acid as the acid ingredient for the ester unit, or those of three or more carbon atoms, for example, propylene glycol, diethylene glycol, neopentyl glycol, and pentaerythritol as the alcohol ingredient for the ester unit.","In the present invention, ethylene terephthalate/ethylene isophthalate copolymer or polybutylene terephthalate is used suitably as the polyester resin.","Particularly, a polyester resin comprising 5 to 15 mol % of ethylene isophthalate and 85 to 95 mol % of ethylene terephthalate is excellent in fabricability and cause no cracks in the resin layer even when severe forming such as thickness-reducing drawing is applied after blending with the polyolefin ingredient and lamination onto the metal sheet and is also excellent in adhesion with the metal sheet.","Further, in a case where it is in contact with contents filled in the can, it does not deteriorate flavor or taste of the contents and show favorable flavor property.","Further, polybutylene terephthalate is easily mixed with the polyolefin ingredient (particularly, polyolefin resin) and in a case where it is blended, for example, with a polyolefin resin, the polyolefin resin is dispersed more finely in the blend resin, which is effective for the improvement of the impact resistance and the fabricability.","Further, polybutylene terephthalate has high crystallization rate and in a case where a coated metal sheet having a coating layer of a thermoplastic resin composition is formed into a can and then the can is heated, for example, by outer surface coating, it has a feature of suppressing the grow of brittle and cause crystals.","Accordingly, use of polybutylene terephthalate is extremely effective for the improvement of the impact resistance at low temperature as an object of the present invention.","In addition, a thermoplastic resin composition blended with polybutylene terephthalate is excellent in water degradation resistance (hydrolysis resistance), and the can having the coating layer comprising the thermoplastic resin composition shows less lowering of the molecular weight of the resin even when left for long time with aqueous contents being filled therein and, accordingly, can maintain stable favorable impact resistance for a long time.","In the present invention, since it is on the premise that the thermoplastic resin composition comprising a blend of the polyester resin and the polyolefin ingredient described above is used in a non-orientation state excellent in the fabricability in order to enable severe fabrication such as thickness-reducing drawing- or thickness-reducing drawing and ironing without causing cracking, chipping, scraping and peeling of the resin, it is necessary to increase the intrinsic viscosity of the resin and strengthen the resin.","Accordingly, the intrinsic viscosity of the polyester resin preferably ranges from 0.5 to 1.5 and, more preferably, ranges from 0.8 to 1.2.In a case where polyester resin with an intrinsic viscosity of less than 0.5 is used, the strength of the resin is lowered extremely making it difficult for application use of thickness reduced drawn can or thickness reduced drawn and ironed can.","In addition, flavor property of contents tends to be deteriorated.","On the other hand, in a case where the intrinsic viscosity of the resin exceeds 1.5, melt viscosity upon heat melting the resin increases extremely making it extremely difficult for the operation of coating the thermoplastic resin composition on the metal sheet.","The blend ratio of the polyester resin to the thermoplastic resin composition (content of the polyester resin in the thermoplastic resin) is preferably from 70 to 95% by weight.","In a case where the amount of the polyester resin is small, the transparency is reduced and the anisotropy increases in the thermoplastic resin composition and, further, the hardness is decreased as well, so that the resin surface tends to be injured.","Further, if the amount of the polyester resin is unnecessarily large, the transparency is reduced and the anisotropy increases in the thermoplastic resin composition and, further, the hardness is decreased as well, so that the resin surface tends to be injured.","Particularly, in a case where the ethylene terephthalate/ethylene isophthalate copolymer described above is used as the polyester resin, when the blend ratio is less than 70% by weight, the fabricability of the thermoplastic resin composition after coating on the metal sheet is poor, whereas when the blend ratio exceeds 95% by weight, dispersed particles of the polyolefin ingredient in the resin composition become coarser, causing a worry of deteriorating the impact resistance and the workability.","Further, in a case where polybutylene terephthalate described above is used as the polyester resin, when the blend ratio is less than 70% by weight, the dispersed particles of polyolefin ingredient in the resin composition become coarser making it difficult to obtain a sufficient impact resistance.","When the blend ratio exceeds 95% by weight, the resin layer is clouded to deteriorate the appearance by heating of the can such as outer surface coating after forming the resin coated metal sheet into a can.","Accordingly, it is most suitable in the present invention that the ethylene terephthalate/ethylene isophthalate copolymer and polybutylene terephthalate is used together such that the total amount is within the range of the blend ratio described above.","In the present invention, a polyester resin layer can be formed for the upper layer and/or lower layer to the coating layer comprising the thermoplastic resin composition described above and a coating of a two-layered or three-layered structure as a whole can be formed on at least one surface of the metal sheet.","As the polyester resin for forming the upper layer or the lower layer, the same polyester resin as used in the formation of the thermoplastic resin composition can be used, and ethylene terephthalate/ethylene isophthalate copolymer is suitable since this is excellent in the flavor property to contents and the fabricability and is also excellent in the adhesion to the metal sheet as described previously.","In the copolymer described above, a copolymer comprising 3 to 15 mol % of ethylene isophthalate and 85 to 97 mol % of ethylene terephthalate is preferred for the upper layer, and a copolymer comprising 10 to 25 mol % of ethylene isophthalate and 75 to 90 mol % of ethylene terephthalate is preferred for the lower layer.","Further as described above, the resin coating in the thermoplastic resin-coated metal sheet according to the present invention has a coating layer of a thermoplastic resin composition at least comprising a polyester resin and a polyolefin ingredient and includes a case where the coating layer comprises a single-layered structure, a case of a 2-layered structure in which the polyester resin layer is formed to either one of the upper layer or the lower layer of the coating layer and a case of a three-layered structure in which the polyester resin layer is formed to the upper layer and the lower layer of the coating layer.","In the case of the single-layered structure comprising only the coating layer of the thermoplastic resin composition, the thickness is preferably within a range from 5 to 50 μm.","In a case of the two-layered structure of the coating layer and the polyester resin layer, it is preferred that the thickness of the upper layer ranges from 3 to 15 μm, the thickness of the lower layer ranges from 2 to 47 μm and, further, the entire thickness ranges from 5 to 50 μm.","In a case of the three-layered structure comprising the coating layer and the polyester resin layers, it is preferred that the thickness of the polyester resin layer for the upper layer ranges from 2 to 10 μm, the thickness of the coating layer as the intermediate layer ranges from 5 to 30 μm and the thickness of the polyester resin layer for the lower layer ranges from 3 to 10 μm.","The thermoplastic resin film constituting the resin coating described above may be formed by extruding a resin obtained by heat melting and blending resin pellets to be blended at a temperature higher by 20 to 40° C. than the melting point of the polyester resin having the highest melting point among the resins to be used (about 200 to 300° C.) from a T-die of an extruder directly onto a long strip-like metal sheet re-wound from a uncoiler, or heat melting a resin obtained by separately heat melting polyester resin pellets at a temperature higher by 20 to 40° C. than the melting point and the heat melted and blend resin described above by separate extruders respectively, then co-extruding them from a T-die having a plurality of die nozzles directly on a long strip-like metal sheet unwound from an uncoiler, pressing them against the metal sheet by press bonding rolls and then quenching directly in water thereby preparing a thermoplastic resin film-coated metal sheet, or by extruding the heat melted resin in the same manner as described above from a T-die of an extruder onto a casting roll, cooling to solidify the same into a film, then re-winding from an uncoiler and abutting the film to a long strip-like metal sheet heated to a temperature higher by 20 to 40° C. than the melting point of the polyester resin, press bonding them while sandwiching between a pair of laminate rolls by means of a known laminator and immediately quenching them in water thereby preparing a thermoplastic resin film-coated metal sheet.","Further, in a case of application use not applied with severe forming as in the present invention, heat melted resin may be extruded from a T-die of an extruder onto a casting roll, then stretched in a uni-axial direction (longitudinal direction) or bi-axial direction (longitudinal direction and lateral direction) and then heat set to form a film in which crystal molecules are oriented, which may be laminated onto the metal sheet.","In the thermoplastic resin composition in which the polyester resin and the polyolefin ingredient are heat melted as described above (blend resin), the polyolefin resin or the polyolefin ingredient is finely dispersed in the matrix of the polyester resin.","When the heat melted blend resin in this state is extruded from the T-die onto the metal sheet or the casting roll, the molten resin is gravitationally stretched downward although little.","In this process, the polyolefin ingredient (polyolefin resin or polyolefin elastomer) dispersed finely in the matrix of the polyester resin is stretched in the direction of gravitational force and dispersed in the matrix of the polyester resin being dispersed in a fibrous state after the blend resin has been cooled to solidify to the metal sheet on the casting roll.","In the dispersion state, when the resin coated metal sheet is applied with thickness-reducing drawing or thickness reducing drawing and ironing to form a can, a dispersed fibrous blend resin is sometimes visible with naked eyes.","The fibrous blend resin is no more visible with naked eyes when it is 10 μm or less in the extruding direction and 2 μm or less in the direction perpendicular to the extruding direction when extruded from the T-die.","Accordingly, it is preferred in the present invention that the olefin ingredient in the thermoplastic resin composition is present being dispersed with a size of 1 to 10 μm in the extruding direction of the resin (length in the film direction) and 0.1 to 2 μm in the direction perpendicular to the extruding direction of the resin (length in the lateral direction of the film) in the coating layer of the thermoplastic resin composition after preparing the thermoplastic resin-coated metal sheet.","As the substrate for the thermoplastic resin-coated metal sheet of the present invention, various kinds of surface treated steel sheets such as electrolytic chromic acid treated steel sheet (tin free steel, hereinafter represented by TFS) or tin-plated steel sheet (tin sheet hereinafter represented by tin sheet), and aluminum alloy sheet which are usually used generally as materials for cans can be used.","For the surface treated steel sheets, TFS having, on a steel sheet, a 2-layered film comprising a lower layer composed of metallic chromium in a membrane amount of 10 to 200 mg/m2 and an upper layer composed of chromium hydrate oxides in a membrane amount of 1 to 30 mg/m2 based on the chromium content is preferred, which has a sufficient adhesive property with the thermoplastic resin film according to the present invention and also has corrosion resistance.","Preferred film sheets are those obtained by applying plating in a plating amount of 0.1 to 11.2 g/m2 on the surface of the steel sheet and, forming thereon, a 2-layered film comprising metallic chromium and chromium-hydrate oxides in an amount of membrane from 1 to 30 mg/m2 based on chromium content, or forming a single layered film comprising only chromium hydrate oxides.","In any of the cases, the steel sheets as the substrates is preferably a cold rolled low carbon steel sheet used generally as the material for cans.","The thickness of the steel sheet is preferably from 0.1 to 0.32 mm.","For the aluminum alloy sheet, JIS 3000 series or 5000 series is preferred, and preferred are those applied with an electrolytic chromic acid treatment to the surface to form a 2-layered film comprising a lower layer composed of metallic chromium in a film amount of 0 to 200 mg/m2 and an upper layer comprising chromium hydrate oxides in a membrane amount of 1 to 30 mg/m2 based on the chromium content, or those applied with a chromium phosphate treatment to deposit 1 to 30 mg/m2 of a chromic ingredient based on the chromium content and 0 to 30 mg/m2 of phosphoric ingredient based on the phosphorus content.","The thickness of the aluminum alloy sheet is preferably from 0.15 to 0.4 mm.","EXAMPLE The present invention is to be described more specifically by way of examples.","Example 1 (Specimen Nos.","1-6, 11-17, 26-32) A long strip-like electrolytic chromic acid treated steel plate (hereinafter referred to as TFS) was re-wound at a rate of 150 m/min from an uncoiler, and pellets comprising a polyester resin shown in Table 1 (represented by PES in the table here and hereinafter), a polyolefin resin (represented by POL in the table here and hereinafter) and/or a polyolefin elastomer (represented by PEL in the table here and hereinafter), or only comprising the polyester resin were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using an extruder to form a blend resin, then fed to a T-die and extruded from a die nozzle on one surface of TFS, that is, one surface to be an inner surface of a can (represented by inner surface in the table herein and hereinafter).","Simultaneously, pellets of a polyester resin incorporated with 20% by weight of white titanium oxide shown in Table 1 were heat melted at a temperature higher by about 30° C. than the melting temperature of the polyester resin using an extruder, then fed into a T-die and extruded from the die nozzle on the other surface of TFS, that is, one surface to be an outer surface of the can (represented by outer surface in the table here and hereinafter).","Then, after sandwiching the TFS to which the resin layer was extruded on both surface thereof by using a pair of press bonding rolls, they were directly quenched in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.","1-6, 11-17, 26-0.32 in Tables 2 and 3.","(Specimen Nos.","18, 19) A long strip-like tin sheet was re-wound from an uncoiler at a rate of 150 m/min and heated, and pellets of the polyester resin and the polyolefin resin shown in Table 1 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using an extruder to form a blend resin, the polyester resin shown in Table 1 was heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using a separate extruder, then they were fed to a T-die having two die nozzles and co-extruded on one surface of a tin sheet from the die nozzles such that the blend resin layer containing the polyolefin resin was in contact with the tin sheet.","Simultaneously, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Table 1 was heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using an extruder, fed to a T-die and then extruded from the die nozzle to the other surface of the heated tin sheet to be an outer surface of a can.","Then, sandwiching the tin sheet to which the resin layer was extruded on both surface thereof by using a pair of press bonding rolls and, they were directly quenched in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.","18 and 19 in Table 3.","(Specimen Nos.","7-10, 20-23) Pellets of the polyester resin and the polyolefin resin or polyolefin elastomer shown in Table 1 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using an extruder to form a blend resin, then fed into a T-die, extruded from a die nozzle, followed by trimming and taken-up to a coiler as a non-orientation film.","Further, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Table 1 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using an extruder, fed into a T-die, extruded from a die nozzle, followed by trimming and then taken up as a non-orientation film to a coiler.","Then, a long strip-like aluminum alloy sheet was rewound at rate of 150 m/min from an uncoiler and heated, a blend resin film was abutted while being re-wound from the coiler to one surface to be an inner surface of a can and, at the same time, a white polyester film was abutted while being re-wound from the coiler to the other surface to be an outer surface of the can.","Then, after sandwiching the aluminum alloy sheet to which the resin layer was extruded on both surface thereof by using a pair of press bonding rolls, they were directly quenched in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.","7-10 and 20-23 in Tables 2 and 3.","(Specimen Nos.","24, 25) Pellets of the polyester resin and the polyolefin resin shown in Table 1 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using an extruder to form a blend resin, the polyester resin shown in Table 1 was heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using a separate extruder, then fed into a T-dye having two die nozzles, co-extruded from the die nozzles, followed by trimming and then taken up as two layers of a non-orientation film to a coiler.","Further, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Table 2 and 3 were heat melted at a temperature higher by about 30° C. than the melting temperature of the polyester resin by using an extruder, fed into a T-die, extruded from the die nozzle, followed by trimming and then taken-up as a non-orientation film to a coiler.","Then, a long strip-like tin sheet was re-wound at a rate of 150 m/min from an uncoiler, and heated, and the two-layered resin film was abutted against one surface thereof to be an inner surface of a can while being re-wound from the coiler such that the blend resin layer containing the polyolefin resin was in contact with the tin sheet.","At the same time, the white polyester film was abutted, while being re-wound from the coiler to the outer surface of the can.","Then, after sandwiching the tin sheet to which the resin layer was extruded on both surface thereof by using a pair of press bonding rolls, they were directly quenched in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.","24 and 25 in Table 3.The metal sheets applied with the surface treatment shown below were used as the three types of the metal sheets described above.","(1) Electrolytic chromic acid treated steel sheet (indicated as TFS in the table) Sheet thickness: 0.18 mm Amount of metallic chromium: 160 mg/m2 Amount of chromium hydrate oxide: (as chromium) 19 mg/m2 Heating temperature: temperature higher by about 30° C. than the melting point of the polyester resin (2) Tin sheet (indicated as ET in the table) Sheet thickness: 0.18 mm Amount of tin plating: 0.2 g/m2 Amount of chromium hydrate oxide: (as chromium) 9 mg/m2 Heating temperature: 200° C. (3) Aluminum alloy sheet (JIS 5052H 39) (indicated as Al in the table) Sheet thickness: 0.26 mm Amount of membrane: (as phosphorus) 11 mg/m2 (as chromium) 7 mg/m2 Heating temperature: temperature higher by about 30° C. than the melting point of the polyester resin As each of the polyolefin resins described above, polyolefin resins using metallocene catalysts were used for any of the cases.","TABLE 1 Resin composition Resin No.","Resin Composition PES1 Polyethylene terephthalate (IV value: 0.75) PES2 Polyethylene terephthalate (IV value: 0.82) PES3 Polyethylene terephthalate (IV value: 1.1) PES4 Ethylene terephthalate - ethylene isophthalate copolymer (ethylene isophthalate:: 5 mol %) (IV value: 0.9) PES5 Ethylene terephthalate - ethylene isophthalate copolymer (ethylene isophthalate:: 10 mol %) (IV value: 0.9) PES6 Ethylene terephthalate - ethylene isophthalate copolymer (ethylene isophthalate:: 15 mol %) (IV value: 1.5) PES7 Ethylene terephthalate - ethylene adiipate copolymer (ethylene adipate:: 10 mol %) (IV value: 0.6) PES8 Ethylene terephthalate - ethylene naphthalate copolymer (ethylene-naphthalate: 10 mol %) (IV value: 0.9) PES9 Polybutylene terephthalate (IV value: 0.5) POL1 Polyethylene POL2 Polypropylene POL3 Polybutene-1 POL4 Polyoctene-1 POL5 Ethylene-propylene copolymer POL6 Ethylene-butene-1 copolymer POL7 Ethylene-hexene copolymer POL8 Maleic acid anhydride copolymerized polyethylene POL9 Acrylic acid copolymerized polypropylene POL10 Methylacrylate copolymerized polyethylene POL11 Diglycidyl methacrylate copolymerized polyethylene PEL1 Ethylene-propylene polymerized elastomer (MFR: 0.4 g/10 min) PEL2 Ethylene-propylene polymerized elastomer (MFR: 1 g/10 min) PEL3 Ethylene-propylene polymerized elastomer (MFR: 5 g/10 min) PEL4 Ethylene-propylene polymerized elastomer (MFR: 10 g/10 min) PEL5 Ethylene-propylene polymerized elastomer (MFR: 30 g/10 min) PEL6 Ethylene-propylene polymerized elastomer (MFR: 35 g/10 min) TABLE 2 Thermoplastic resin-coated metal sheet Inner surface resin layer Outer surface Lower layer (blend resin) Melt Upper resin (TiO2: POL Grain viscosity layer (PES) WT % content) Specimen Blend ratio Thickness size ratio Thickness Thickness No.","Kind (%) (μm) (μm) Vpol/Vpes Type (μm) Type (μm) Metal sheet Section 1 PES1/POL8/PEL4 80/1/19 25 7 × 1.5 0.60 — — PES3 15 TFS Invention 2 PES1/POL8/PEL4 80/5/15 25 5 × 1.0 0.63 — — PES3 15 TFS Invention 3 PES2/POL9/PEL3 80/5/15 25 3 × 0.5 0.86 — — PES3 15 TFS Invention 4 PES2/POL9/PEL2 85/5/10 28 8 × 1.0 1.10 — — PES3 15 TFS Invention 5 PES3/POL9/PEL1 80/10/10 30 6 × 0.8 0.71 — — PES3 15 TFS Invention 6 PES3/POL9/PEL1 75/10/15 20 14 × 3.1 1.25 — — PES3 15 TFS Comp.","Ex 7 PES4/POL10/POL1 80/7/13 35 10 × 2.0 0.80 — — PES3 15 AL Invention 8 PES3/POL11/POL2 80/15/5 25 4 × 0.8 0.61 — — PES3 15 AL Invention 9 PES6/POL8/POL3 80/5/15 20 6 × 0.9 0.19 — — PES3 15 AL Invention 10 PES7/POL8/POL1 80/10/10 25 5 × 0.6 0.88 — — PES3 15 AL Invention 11 PES9/POL9/PEL3 90/5/5 35 3 × 0.3 0.67 — — PES3 15 TFS Invention 12 PES9/POL10/PEL5 80/10/10 25 8 × 1.6 1.13 — — PES3 15 TFS Invention 13 PES2/POL9/POL4 80/10/10 25 5 × 0.8 0.72 — — PES3 15 TFS Invention 14 PES4/POL8/POL5 75/10/15 30 4 × 1.2 0.59 — — PES3 15 TFS Invention 15 PES2/POL1 99/1 30 8 × 1.8 0.62 — — PES3 15 TFS Invention 16 PES3/POL1 95/5 20 7 × 1.5 0.23 — — PES3 15 TFS Invention TABLE 3 Thermoplastic resin-coated metal sheet Inner surface resin layer Outer surface Lower layer (blend resin) Melt Upper resin (TiO2: POL grain viscosity layer (PES) WT % content Specimen Blend ratio Thickness size ratio Thickness Thickness Metal No.","Kind (%) (μm) (μm) Vpol/Vpes Kind (μm) Kind (μm) sheet Section 17 PES2/POL2 85/15 30 7 × 1.6 0.65 — — PES3 15 TFS Invention 18 PES4/POL3 90/10 20 6 × 1.2 0.58 PES3 15 PES3 15 ET Invention 19 PES5/POL1 90/10 20 5 × 1.5 0.62 PES3 15 PES3 15 ET Invention 20 PES2/POL9 85/15 25 4.5 × 0.2 0.30 — — PES3 15 AL Invention 21 PES3/POL8 80/20 25 4 × 0.3 0.32 — — PES3 15 AL Invention 22 PES4/POL8 85/15 28 3.5 × 0.2 0.32 — — PES3 15 AL Invention 23 PES5/PEL3 70/30 28 3 × 0.4 0.28 — — PES3 15 AL Invention 24 PES2/POL7 80/20 15 7 × 0.8 0.35 PES3 20 PES3 15 ET Invention 25 PES8/POL6 90/10 15 6 × 0.9 0.30 PES3 20 PES3 15 ET Invention 26 PES2/PEL4 90/10 20 6 × 0.8 0.26 — — PES3 15 TFS Invention 27 PES1 — 25 — — — — PES3 15 TFS Comp.","Ex 28 PES2/POL1 99.5/0.5 25 7 × 1.5 0.62 — — PES3 16 TFS Comp.","Ex 29 PES5/POL2 65/35 25 12 × 3.0 0.57 — — PES3 16 TFS Comp.","Ex 30 PES4 — 25 — — — — PES3 16 TFS Comp.","Ex 31 PES2/PEL6 80/20 30 5 × 1.2 0.05 — — PES3 16 TFS Comp.","Ex 32 PES1/PEL1 85/15 28 15 × 3.0 4.20 — — PES3 16 TFS Comp.","Ex The thermoplastic resin-coated metal sheet obtained as described above was formed into a can of a bottomed cylindrical shape by using a thickness-reducing drawing and ironing as described below.","After punching a thermoplastic resin-coated metal sheet into a blank of 160 mm diameter, a drawn can with 100 mm can bottom diameter was formed such that the white polyester resin-coated surface was on the outer surface of the can.","Then, it was re-drawn into a re-drawn can with 80 mm can bottom diameter.","Further, the re-drawn can was applied with stretching and, at the same time, ironing by composite fabrication to form a drawn and ironed can with 65 mm can bottom diameter.","The composite fabrication was conducted under the conditions that the distance between the re-drawn portion and the upper end of the can was 20 mm, the R portion at the shoulder of a re-drawing dice was 1.5 times the sheet thickness, a clearance between the re-drawing dice and a punch was 1.0 times the sheet thickness and the clearance at the ironed portion was 50% of the original thickness.","Then, an upper portion of the can was trimmed by a known method and applied with neck-in fabrication and flange fabrication.","Then, methods of evaluating the thermoplastic resin and the thermoplastic resin-coated metal sheet are to be explained.","(Size of Polyolefin Resin Dispersed in Polyester Resin) A Blend resin layer of the thermoplastic resin-coated metal sheet was observed under a scanning type electron microscope to measure the size of the fibrous resin.","The measured value shows an average value measured at three time points, that is, during coating or during film preparation, completion of coating or five min before completion of film preparation, at three positions, that is, 5 cm from both ends and at the central portion in view of the lateral direction of the resin-coated metal sheet.","In Tables 2 and 3, it is represented by “length in the resin extruding direction” (μm)דlength in the direction perpendicular to resin extruding direction” (μm).","(Appearance for the can Inner Surface) The can inner surface formed by the thickness-reducing drawing and ironing fabrication method was observed with naked eyes, and presence or absence of fibrous unevenness of the polyolefin resin in the resin layer and the transparency of the resin layer were evaluated by the following standards.","◯: no fibrous unevenness was observed and it had sufficient transparency Δ: fibrous unevenness was observed slightly, or slight clouding of the resin layer was observed X: fibrous unevenness is observed, clouding of the resin layer was observed.","(Melt Flow Rate of Polyolefin Elastomer) Melt flow rate (230° C.) was measured by a customary method.","(Thickness of Resin Film) The thermoplastic resin-coated metal sheet was buried in an epoxy type embedding resin, sliced to a thickness of 5 μm and the cross section was measured by microscopic observation.","(Intrinsic Viscosity (IV Value)) After dissolving the polyester resin in a 1:1 mixed solution of phenol/tetrachloroethane, the specific viscosity was measured in a thermostable bath at 30° C. by a Ubbelohde's viscometer to determine an intrinsic viscosity.","(Melt Viscosity) The melt viscosity was determined by a capillary rheometer according to JIS K 7199.","(Fabricability) Can formed by using a thickness-reducing drawing and ironing method was observed with naked eyes and the fabricability was evaluated based on the following standards.","⊚: fine crack or film breakage was not recognized ◯: slight fine crack to an extent not causing practical problems was recognized Δ: crack and film breakage to an extent causing practical problem was recognized X: broken upon forming (Impact Resistance at Low Temperature) After forming by using the thickness-reducing drawing and ironing method, trimming an upper portion of a can, and applying necking-in and flange fabrication, acidic drinks (Acerola drink, trade name of products: manufactured by Nichirei Co.) at pH 2.6 were filled, aged at 37° C. for one month and then seal was opened.","The can was cut out from the upper end thereof in a circumferential direction at a width of 30 mm to form a specimen.","After immersing the specimen in iced water for 5 min, it was taken out and a steel rod attached with steel balls each of {fraction (1/2)} inch diameter at the top end at 15 mm distance in the circumferential direction (weight: 1 kg) was dropped from the height of 40 mm, a sponge impregnated with an aqueous 3% sodium chloride solution was abutted against generated convex portions on the can inner surface, a DC current at 6.3 V was applied to the specimen, the value of the flowing current was measured and the impact resistance at low temperature was measured by the following standards depending on the magnitude of the measured current value.","⊚: less than 0.05 mA ◯: 0.05 mA or more and less than 0.1 mA Δ: 0.1 mA or more and less than 0.3 mA X: 0.3 mA or more (Corrosion Resistance) After forming by using a thickness-reducing drawing and ironing method, an upper portion of a can was trimmed and applied with neck-in and flange fabrication.","Then after filling acidic drinks (Acelora drink, trade name of products: manufactured by Nichirei Co.), impact was applied at a low temperature from the outside of the can to the wall of the can in the same manner as practiced in the evaluation of the impact resistance at low temperature to form a concave portion.","Then, after aging at 37° C. for one month, seal was opened and concentration of leached metal was measured by atomic absorption spectroscopy and the corrosion resistance was evaluated in accordance with the following standards depending on the concentration.","⊚: less than 0.3 ppm, ◯: more than 0.3 ppm and less than 0.5 ppm, Δ: more than 0.5 ppm and less than 1.0 ppm, X: 1.0 ppm or more The result of evaluation was shown in Tables 4 and 5.As shown in Table 4 and Table 5, it can be seen that any of the thermoplastic resin-coated metal sheets of the present invention is excellent in the fabricability and shows favorable impact resistance at low temperature and corrosion resistance.","TABLE 4 Result of characteristic evaluation Result of characteristic evaluation Impact Spec- resistance imen at low Corrosion Resin No.","Fabricability temperature resistance unevenness Section 1 ⊚ ⊚ ⊚ ◯ Invention 2 ⊚ ⊚ ⊚ ◯ Invention 3 ⊚ ⊚ ⊚ ◯ Invention 4 ⊚ ◯ ◯ ◯ Invention 5 ⊚ ◯ ⊚ ◯ Invention 6 ⊚ X ◯ X Comp.","Ex 7 ⊚ ⊚ ⊚ ◯ Invention 8 ⊚ ◯ ⊚ ◯ Invention 9 ⊚ ⊚ ⊚ ◯ Invention 10 ⊚ ⊚ ⊚ ◯ Invention 11 ⊚ ⊚ ⊚ □ Invention 12 ⊚ ◯ ◯ ◯ Invention 13 ⊚ ⊚ ⊚ ◯ Invention 14 ⊚ ⊚ ⊚ ◯ Invention 15 ⊚ ◯ ◯ ◯ Invention 16 ⊚ ⊚ ◯ ◯ Invention TABLE 5 Result of characteristic evaluation Result of characteristic evaluation Impact Spec- resistance imen at low Corrosion Resin No.","Fabricability temperature resistance unevenness Section 17 ⊚ ⊚ ◯ ◯ Invention 18 ⊚ ⊚ ⊚ ◯ Invention 19 ⊚ ⊚ ⊚ ◯ Invention 20 ⊚ ⊚ ⊚ ◯ Invention 21 ⊚ ⊚ ⊚ ◯ Invention 22 ⊚ ⊚ ⊚ ◯ Invention 23 ◯ ◯ ◯ ◯ Invention 24 ⊚ ⊚ ⊚ ◯ Invention 25 ⊚ ⊚ ⊚ ◯ Invention 26 ⊚ ⊚ ⊚ ◯ Invention 27 ⊚ X Δ ◯ Comp.","Ex 28 ⊚ X Δ ◯ Comp.","Ex 29 ◯ Δ ◯ Δ Comp.","Ex 30 ⊚ X Δ ◯ Comp.","Ex 31 ⊚ X Δ ◯ Comp.","Ex 32 ◯ X Δ Δ Comp.","Ex Example 2 (Specimen Nos.","33-38, 43-53) A long strip-like electrolytic chromic acid treated steel sheet (TFS) was re-wound from an uncoiler at a rate of 150 m/min and heated, and pellets of the polyester resin (PES), the polyolefin resin (POL) and/or polyolefin elastomer (PEL) or only of the polyester resin shown in Table 6 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using an extruder to form a blend resin, fed to a T-die and extruded on one surface of TFS to be an inner surface of a can (inner surface).","Simultaneously, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Tables 7 and 8 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using an extruder, then fed into a T-die and extruded on the other surface of the heated TFS to be an outer surface of the can (outer surface) from the die nozzle.","Then, a pair of press bonding rolls were used and after sandwiching TFS having resin layers extruded on both surfaces thereof, it was quenched directly in water to prepare thermoplastic resin-coated metal sheets of Specimens Nos.","33-38, 43-53 in Table 7 and Table 8.","(Specimen Nos.","39-42) A long strip-like tin sheet (ET) was re-wound from an uncoiler at a rate of 150 m/min and heated, and pellets of the polyester resin and the polyolefin resin shown in Table 6 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using extruder to form a blend resin, the polyester resin shown in Table 6 was heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using a separate extruder, fed to a T-die having two die nozzles, and coextruded on one surface of a tin sheet to be an inner surface of a can from the die nozzles such that the blend resin layer containing the polyolefin resin was in contact with the tin sheet.","Simultaneously, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Table 7 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using an extruder, fed into a T-die and extruded on the other surface of the heated tin sheet to be an outer surface of the can from a die nozzle.","Then, a pair of press bonding rolls were used and after sandwiching the tin sheet having resin layers extruded on both surfaces, it was quenched directly in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.","39-42 in Table 7.","(Specimen Nos.","54-58) Pellets of the polyester resin and the polyolefin resin or the polyolefin elastomer shown in Table 6 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying kneading time by using an extruder to form a blend resin, then fed to a T-die, extruded from a die nozzle, followed by trimming and taken up as a non-orientation film to a coiler.","Further, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Table 8 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using an extruder, fed into a T-die, extruded from a die nozzle, followed by trimming and was taken up as a non-orientation film to an uncoiler.","Then, a long strip-like aluminum alloy sheet (AL) was re-wound at a rate of 150 m/min from the uncoiler and the blend resin film was abutted while rewinding from the coiler to one surface thereof to be the inner surface of a can and, simultaneously, white polyester film was abutted while being re-wound from the coiler against the other surface to be the outer surface of the can.","Then, a pair of press bonding rolls were used and after sandwiching the aluminum alloy sheet having resin layers extruded on both surfaces, it was quenched directly in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.","54-58 in Table 8.","(Specimen Nos.","59-61) Pellets of the polyester resin and the polyolefin resin and/or the polyolefin elastomer shown in Table 9 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using an extruder to form a blend resin, pellets of the polyester (PES4) in Table 6 were heat melted to a temperature higher by about 30° C. than the melting point of the respective polyester resins by using separate extruders respectively, then fed to a die having two nozzles, co-extruded from the die nozzles such that the upper layer comprised of PES4 and the lower layer comprised of the blend resin, followed by trimming and then taken up to a coiler as a two-layered non-orientation film.","Further, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Table 9 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using an extruder, fed to a T-die, extruded from a die nozzle, followed by trimming and taken up as a non-orientation film to a coiler.","Then, a long strip-like TFS was re-wound from an uncoiler at a rate of 150 m/min and heated, and the two-layered resin film was abutted while being re-wound from the coiler to one surface to be an inner surface of a can and, at the same time, the white polyester film was abutted while being re-wound from the coiler against the other surface to be an outer surface of the can.","Then, after sandwiching the TFS having resin layers extruded on both surfaces by using a pair of press bonding rolls, it was quenched directly in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.","59-61 in Table 9.","(Specimen Nos.","62-64) Pellets of the polyester resin and the polyolefin resin and/or the polyolefin elastomer shown in Table 10 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using an extruder to form a blend resin, pellets of the polyester PES4 and PES6 in Table 6 were heat melted to a temperature higher by about 30° C. than the melting point of the respective polyester resins by using separated extruders respectively, then fed to a die having three nozzles, co-extruded from the die nozzles such that the upper layer comprised of PES4, the intermediate layer comprised of the blend resin, and the lower layer comprised of the PES6, followed by trimming and then taken up to a coiler as a three-layered non-orientation film.","Further, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Table 9 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using an extruder, fed to a T-die, extruded from die nozzle, followed by trimming and taken up as a non-orientation film to a coiler.","Then, a long strip-like TFS was re-wound from an uncoiler at a rate of 150 m/min and heated, and the three-layered resin film was abutted while being re-wound from the coiler to one surface to be an inner surface of a can and, at the same time, the white polyester film was abutted while being re-wound from the coiler against the other surface to be an outer surface of the can.","Then, after sandwiching the TFS having resin layers extruded on both surfaces by using a pair of press bonding rolls, it was quenched directly in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.","62-64 in Table 10.In Tables 7-10, the outer surface resin layer contains 20% by weight of TiO2 as a white pigment.","TABLE 6 Resin composition Resin No.","Resin Composition PES1 Polyethylene terephthalate (IV value: 0.75) PES2 Polyethylene terephthalate (IV value: 0.82) PES3 Polyethylene terephthalate (IV value: 1.1) PES4 Ethylene terephthalate/ethylene isophthalate copolymer (ethylene-isophthalate:: 5 mol %) (IV value: 0.9) PES5 Ethylene terephthalate/ethylene isophthalate copolymer (ethylene isophthalate:: 10 mol %) (IV value: 0.9) PES6 Ethylene terephthalate/ethylene isophthalate copolymer (ethylene isophthalate:: 15 mol %) (IV value: 1.5) PES7 Ethylene terephthalate/ethylene adipate copolymer (ethylene adipate:: 10 mol %) (IV value: 0.5) PES8 Ethylene terephthalate - ethylene naphthalate copolymer (ethylene-naphthalate: 10 mol %) (IV value: 0.9) PES9 Polybutylene terephthalate (IV value: 1.35) POL1 Polyethylene POL2 Polypropylene POL3 Polybutene-1 POL4 Polyoctene-1 POL5 Ethylene-propylene copolymer POL6 Ethylene-butene-1 copolymer POL7 Ethylene-hexene copolymer PEL1 Ethylene/propylene polymerized elastomer (MFR: 0.45 g/10 min) PEL2 Ethylene/propylene polymerized elastomer (MFR: 0.8 g/10 min) PEL3 Ethylene/propylene polymerized elastomer (MFR: 6 g/10 min) PEL4 Ethylene/propylene polymerized elastomer (MFR: 12 g/10 min) PEL5 Ethylene/propylene polymerized elastomer (MFR: 25 g/10 min) PEL6 Ethylene/propylene polymerized elastomer (MFR: 30 g/10 min) TABLE 7 Thermoplastic resin-coated metal sheet Outer surface Inner surface resin layer resin layer Specimen Blend ratio Thickness Thickness Metal No.","Type (wt %) (μm) Type (μm) sheet Section 33 PES4/PES9/POL5 30/50/20 25 PES4 15 TFS Invention 34 PES5/PES9/POL5 50/47/3 25 PES4 15 TFS Comp.","Ex 35 PES5/PES9/POL5 50/45/5 25 PES4 15 TFS Invention 36 PES5/PES9/POL5 30/50/20 25 PES4 15 TFS Invention 37 PES5/PES9/POL5 25/50/30 25 PES4 15 TFS Invention 38 PES5/PES9/POL5 20/45/35 25 PES4 15 TFS Comp.","Ex 39 PES6/PES9/POL5 30/50/20 25 PES4 15 ET Invention 40 PES6/PES9/POL1/POL5 30/50/10/10 25 PES4 15 ET Invention 41 PES6/PES9/PEL3 30/50/20 25 PES4 15 ET Invention 42 PES6/PES9/POL5/PEL3 30/50/10/10 25 PES4 15 ET Invention 43 PES4/POL5 80/20 25 PES4 15 TFS Invention 44 PES5/PES9/POL2 30/50/20 25 PES4 15 TFS Invention 45 PES5/PES9/POL3 50/30/20 25 PES4 15 TFS Invention 46 PES5/PES9/POL4 30/50/20 25 PES4 15 TFS Invention TABLE 8 Thermoplastic resin-coated metal sheet Outer surface Inner surface resin layer resin layer Specimen Blend ratio Thickness Thickness Metal No Type (wt %) (μm) Type (μm) sheet Section 47 PES5/PES9/POL6 30/50/20 25 PES4 15 TFS Invention 48 PES5/PES9/POL7 30/50/20 25 PES4 15 TFS Invention 49 PES5/PES9/PEL1 30/50/20 25 PES4 15 TFS Comp.","ex 50 PES5/PES9/PEL2 30/50/20 25 PES4 15 TFS Invention 51 PES5/PES9/PEL4 30/50/20 25 PES4 15 TFS Invention 52 PES5/PES9/PEL5 30/50/20 25 PES4 15 TFS Invention 53 PES5/PES9/PEL6 30/50/20 25 PES4 15 TFS Comp.","ex 54 PES1/PES9/PEL5 30/50/20 25 PES4 15 AL Invention 55 PES2/PES9/POL1/PEL4 30/50/10/10 25 PES4 15 AL Invention 56 PES3/PES9/POL5/PEL4 30/50/5/15 25 PES4 15 AL Invention 57 PES7/PES9/POL5/PEL5 30/50/10/10 25 PES4 15 AL Invention 58 PES8/PES9/POL6/PEL3 30/50/10/10 25 PES4 15 AL Invention TABLE 9 Thermoplastic resin-coated metal sheet Inner surface resin layer Upper layer Lower layer Outer surface Specimen Thickness Blend ratio Thickness resin layer No.","Type (μm) Type (wt %) (μm) Type Thickness Metal sheet Section 59 PES 5 PES6/PES9/POL5 30/50/20 25 PES4 15 TFS Invention 60 PES 5 PES6/PES9/POL1/PEL3 30/50/10/10 25 PES4 15 TFS Invention 61 PES 5 PES6/PES9/PEL2 30/50/20 25 PES4 15 TFS Invention TABLE 10 Thermoplastic resin-coated metal sheet Inner surface resin layer Outer surface Upper layer Intermediate layer Lower layer resin layer Specimen Thickness Blend ratio Thickness Thickness Thickness No.","Type (μm) Type (wt %) (μm) Type (μM) Type (μm) Metal sheet Section 62 PES4 5 PES6/PES9/PEL4 30/50/20 20 PES6 5 PES4 15 TFS Inveniton 63 PES4 5 PES6/PES9/POL1/PEL2 30/50/10/10 20 PES6 5 PES4 15 TFS Inveniton 64 PES4 5 PES6/PES9/PEL3 30/50/20 20 PES6 5 PES4 15 TFS Inveniton TABLE 11 Result of characteristic evaluation Result of characteristic evaluation Specimen Low temperature Corrosion No.","Fabricability impact resistance resistance Section 33 ⊚ ⊚ ⊚ Invention 34 ◯ X Δ Comp.","Ex 35 ⊚ ⊚ ⊚ Invention 36 ⊚ ⊚ ⊚ Invention 37 ⊚ ⊚ ⊚ Invention 38 ⊚ X Δ Comp.","Ex 39 ⊚ ⊚ ⊚ Invention 40 ⊚ ⊚ ⊚ Invention 41 ⊚ ⊚ ⊚ Invention 42 ⊚ ⊚ ⊚ Invention 43 ⊚ ◯ ◯ Invention 44 ⊚ ⊚ ⊚ Invention 45 ⊚ ⊚ ⊚ Invention 46 ⊚ ⊚ ⊚ Invention 47 ⊚ ⊚ ⊚ Invention 48 ⊚ ⊚ ⊚ Invention TABLE 12 Result of characteristic evaluation Result of characteristic evaluation Specimen Low temperature Corrosion No.","Fabricability impact resistance resistance Section 49 Δ X ◯ Comp.","Ex 50 ⊚ ⊚ ⊚ Invention 51 ⊚ ⊚ ⊚ Invention 52 ⊚ ◯ ◯ Invention 53 ⊚ X Δ Comp.","Ex 54 ⊚ ⊚ ⊚ Invention 55 ⊚ ⊚ ⊚ Invention 56 ⊚ ⊚ ⊚ Invention 57 ⊚ ⊚ ⊚ Invention 58 ⊚ ⊚ ⊚ Invention 59 ⊚ ⊚ ⊚ Invention 60 ⊚ ⊚ ⊚ Invention 61 ⊚ ⊚ ⊚ Invention 62 ⊚ ⊚ ⊚ Invention 63 ⊚ ⊚ ⊚ Invention 64 ⊚ ⊚ ⊚ Invention Further, the three types of the metal sheets described above were identical with those used in Experiment Example 1 and any of the polyolefin resins used was synthesized by means of metallocene catalyst.","Cans each of a bottomed cylindrical shape were formed using the thermoplastic resin-coated metal sheets obtained above in the same manner as in Experiment Example 1 and thermoplastic resin coating and the thermoplastic resin-coated metal sheet were evaluated in the same manner as in Experimental Example 1 and the results are shown in Table 11 and Table 12.As shown in Table 11 and Table 12, it can be seen that any of the thermoplastic resin-coated metal sheets according to the present invention is excellent in the fabricability and shows favorable impact resistance at low temperature and corrosion resistance.","INDUSTRIAL APPLICABILITY The present invention provides a thermoplastic resin-coated metal sheet in which a coating layer of a thermoplastic resin composition comprising a blend a polyester resin and at least one of polyolefin ingredients selected from the group consisting of polyolefin resins and polyolefin elastomers is formed on at least one surface of a metal sheet, and a can formed obtained by forming the same, the thermoplastic resin-coated metal sheet described above is excellent in the workability, and shows excellent workability causing no crackings and peeling to the resin layer even after being applied with severe forming such as thickness-reducing drawing or thickness-reducing drawing and ironing.","Further, it shows excellent impact resistance at low temperature with no occurrence of cracks in the resin layer even when applied with impact at low temperature and, further, leaches less metals when the formed can is left for a long time while being filled with acidic contents and shows excellent corrosion resistance."]],"string":"[\n [\n \"Metal sheet coated with thermoplastic resin and can obtained therefrom\",\n \"A thermoplastic-resin-coated metal sheet in which the thermoplastic resin has been applied to the metal sheet without through an adhesive primer and which, even after having been formed into a can, is excellent in impact resistance at low temperatures and resistance to corrosion by acid contents; and a can obtained from the coated metal sheet.\",\n \"The thermoplastic-resin-coated metal sheet is obtained by coating a metal sheet with a thermoplastic resin composition comprising a blend of a polyester resin with a polyolefin resin or polyolefin elastomer.\",\n \"This metal sheet is formed into a can through drawing/ironing with thickness reduction.\"\n ],\n [\n \"1.A thermoplastic resin-coated metal sheet in which a thermoplastic resin composition comprising a blend of polybutylene terephthalate and a polyolefin ingredient comprising at least one member selected from the group consisting of polyolefin resins and polyolefin elastomers is coated in a substantially non-orientation state on at least one surface of a metal sheet.\",\n \"2.A thermoplastic resin-coated metal sheet according to claim 1, wherein a polyolefin resin is used as the polyolefin ingredient.\",\n \"3.A thermoplastic resin-coated metal sheet according to claim 1, wherein a polyolefin elastomer is used as the polyolefin ingredient.\",\n \"4.A thermoplastic resin-coated metal sheet according to claim 1, wherein a polyolefin resin and a polyolefin elastomer are used as the polyolefin ingredient.\",\n \"5.A thermoplastic resin-coated metal sheet according to any one of claim 1, wherein the ratio of a melt index of the polyolefin ingredient upon heat melting (VPOL) to that of the polybutylene terephthalate upon heat melting (VPES), that is, VPOL/VPES is 1.2 or less in the thermoplastic resin composition.\",\n \"6.A thermoplastic resin-coated metal sheet according to claim 1, wherein a polybutylene terephthalate layer formed by coating the polybutylene terephthalate in a substantially non-orientation state is formed on the coating layer of the thermoplastic resin composition.\",\n \"7.A thermoplastic resin-coated metal sheet according to claim 1, wherein a polybutylene terephthalate layer coating in which the polybutylene terephthalate is in a substantially non oriented state is formed on the surface of the metal sheet, and a coating layer of the thermoplastic resin composition is formed on the polyester resin layer.\",\n \"8.A thermoplastic resin-coated metal sheet according to claim 1 wherein the polyolefin resin is a resin comprising one or more of 1-alkene polymer resins having a number or carbon atoms of 2 to 8.9.A thermoplastic resin-coated metal sheet according to claim 8, wherein the 1-alkene polymer resin is one of polyethylene, polypropylene, and an ethylene-propylene copolymer.\",\n \"10.A thermoplastic resin-coated metal sheet according to claim 8, wherein the polyolefin resin is a polyolefin resin polymerized by means of a metallocene catalyst.\",\n \"11.A thermoplastic resin-coated metal sheet according to claim 1, wherein at least a portion of the polyolefin resin is a modified polyolefin resin modified with one of maleic acid anhydride, acrylic acid, acrylic acid ester and diglycidyl methacrylate.\",\n \"12.A thermoplastic resin-coated metal sheet according to claim 1 wherein the polyolefin elastomer is an in-plant produced ethylene-propylene copolymerized elastomer having a melt flow rate (MFR, 230° C.) of 0.4 to 30 g/10 min).\",\n \"13-15.\",\n \"(canceled) 16.A thermoplastic resin-coated metal sheet according to claims 1 to 12, wherein the thermoplastic resin composition contains 1 to 30% by weight of a polyolefin ingredient.\",\n \"17.A thermoplastic resin-coated metal sheet according to claim 1, wherein the thermoplastic resin composition contains 70 to 95% by weight of the polybutylene terephthalate.\",\n \"18.A thermoplastic resin-coated metal sheet according to claim 1, wherein the thermoplastic resin composition is heat melted and directly extruded from a T-die to a metal sheet for coating.\",\n \"19.A thermoplastic resin-coated metal sheet according to claim 1, wherein the thermoplastic resin composition is heat melted, extruded from a T-die onto a casting roll, cooled to solidify into a film and then the film is hot press bonded on a metal sheet.\",\n \"20.A thermoplastic resin-coated metal sheet according to claim 18, wherein, wherein the polyolefin ingredient in the coating layer comprising a thermoplastic resin composition is present being dispersed with a size of 1 to 10 μm in the extruding direction and 0.1 to 2 μm in the direction perpendicular to the extruding direction.\",\n \"21.A thermoplastic resin-coated metal sheet according to claim 1, wherein the metal sheet is one of electrolytic chromic acid treated steel sheet, tin-plated sheet and aluminum alloy sheet.\",\n \"22.A can using a thermoplastic resin-coated metal sheet according to claim 1.\"\n ],\n [\n \"<SOH> BACKGROUND ART <EOH>In recent years, cans formed by coating a polyester resin on a metal sheet and improved with thickness reduction ratio for side wall portions by applying severe forming such as thickness-reducing drawing or thickness-reducing ironing have been used as cans mainly for drink applications.\",\n \"In a case of forming a polyester resin coated metal sheet by applying severe such as wall reducing drawing or wall reducing ironing, it is necessary to coat in a non-orientation state capable of attaining excellent fabricability such that the resin is not peeled upon forming or cracks are not formed to the resin.\",\n \"However, when applying thickness-reducing drawing or thickness-reducing ironing is applied to a resin-coated metal sheet with a non-orientation state of a polyester resin, then applying coating printing to the outer surface of the can and heating for baking, since the upper portion of the can is thermally set in a state where it is molecularly oriented in the direction of a height of the can by the fabrication, elongation of the not oriented resin in the circumferential direction of the can is extremely small to become brittle.\",\n \"Particularly, cracks tend to be formed to the resin layer by mere collision between cans to each other, particularly, at low temperature.\",\n \"Further, since the portion for the can bottom scarcely undergoes the forming, the resin crystals are grown into a coarse state and brittled upon heating for baking after the coating printing also tending to cause cracks upon receiving impacts particularly at low temperature.\",\n \"That is, a polyester resin-coated metal sheet in a non-orientation state is poor in the impact resistance, particularly, the impact resistance at low temperature after formed into the can.\",\n \"Poor impact resistance after forming can be improved by interposing an adhesive primer between the polyester resin and the metal sheet upon coating the polyester resin on the metal sheet.\",\n \"However, a method of interposing the adhesive primer may possibly cause undesired effects on the environment due to evaporation of organic solvents and, further, requires surplus steps of coating and drying to increase the cost.\",\n \"Further, in a case where acidic contents at pH of 5 or less are filled, the underlying metal sheet may sometimes be corroded in the upper portion of the can undergoing high degree of fabrication after lapse of a long time.\",\n \"The present invention intends to provide a thermoplastic resin-coated metal sheet in which a resin layer, after applying severe forming to a thermoplastic resin-coated metal sheet where the resin layer after coating to the metal sheet is in a non-oriented state, is excellent in impact resistance, particularly, impact resistance at low temperature, coated to the metal sheet without interposing an adhesive primer and is excellent in corrosion resistance to acidic contents even in a case where it is formed into a can, as well as a can using the same.\"\n ],\n [\n \"TECHNICAL FIELD The present invention concerns a thermoplastic resin-coated metal sheet and, particularly, a thermoplastic resin-coated metal sheet in which the coating resin is excellent in impact resistance at low temperature, as well as a can obtained by forming the same.\",\n \"BACKGROUND ART In recent years, cans formed by coating a polyester resin on a metal sheet and improved with thickness reduction ratio for side wall portions by applying severe forming such as thickness-reducing drawing or thickness-reducing ironing have been used as cans mainly for drink applications.\",\n \"In a case of forming a polyester resin coated metal sheet by applying severe such as wall reducing drawing or wall reducing ironing, it is necessary to coat in a non-orientation state capable of attaining excellent fabricability such that the resin is not peeled upon forming or cracks are not formed to the resin.\",\n \"However, when applying thickness-reducing drawing or thickness-reducing ironing is applied to a resin-coated metal sheet with a non-orientation state of a polyester resin, then applying coating printing to the outer surface of the can and heating for baking, since the upper portion of the can is thermally set in a state where it is molecularly oriented in the direction of a height of the can by the fabrication, elongation of the not oriented resin in the circumferential direction of the can is extremely small to become brittle.\",\n \"Particularly, cracks tend to be formed to the resin layer by mere collision between cans to each other, particularly, at low temperature.\",\n \"Further, since the portion for the can bottom scarcely undergoes the forming, the resin crystals are grown into a coarse state and brittled upon heating for baking after the coating printing also tending to cause cracks upon receiving impacts particularly at low temperature.\",\n \"That is, a polyester resin-coated metal sheet in a non-orientation state is poor in the impact resistance, particularly, the impact resistance at low temperature after formed into the can.\",\n \"Poor impact resistance after forming can be improved by interposing an adhesive primer between the polyester resin and the metal sheet upon coating the polyester resin on the metal sheet.\",\n \"However, a method of interposing the adhesive primer may possibly cause undesired effects on the environment due to evaporation of organic solvents and, further, requires surplus steps of coating and drying to increase the cost.\",\n \"Further, in a case where acidic contents at pH of 5 or less are filled, the underlying metal sheet may sometimes be corroded in the upper portion of the can undergoing high degree of fabrication after lapse of a long time.\",\n \"The present invention intends to provide a thermoplastic resin-coated metal sheet in which a resin layer, after applying severe forming to a thermoplastic resin-coated metal sheet where the resin layer after coating to the metal sheet is in a non-oriented state, is excellent in impact resistance, particularly, impact resistance at low temperature, coated to the metal sheet without interposing an adhesive primer and is excellent in corrosion resistance to acidic contents even in a case where it is formed into a can, as well as a can using the same.\",\n \"DISCLOSURE OF THE INVENTION According to the present invention, it is provided a thermoplastic resin-coated metal sheet in which a thermoplastic resin composition comprising a blend of one or more of polyester resins and at least one polyolefin ingredient selected from the group consisting of polyolefin resins and polyolefin elastomers is coated on at least one surface of a metal sheet in a substantially non-orientation state.\",\n \"Further, according to the present invention, a can using a thermoplastic resin-coated metal sheet is provided.\",\n \"That is, in the present invention, as the polyolefin ingredient in the thermoplastic resin composition forming the coating layer, a polyolefin resin, a polyolefin elastomer, or a combination of a polyolefin resin and a polyolefin elastomer can be used.\",\n \"Further, a polyolefin resin layer formed substantially in a not-yet oriented state can be disposed above the coating layer of the thermoplastic resin composition or in the lower layer to the coating layer (surface of metal sheet).\",\n \"In the thermoplastic resin composition used in the present invention, it is preferred that the ratio of a melt viscosity upon heat melting the polyolefin ingredient (polyolefin resin or polyolefin elastomer, or a combination of the polyolefin resin and a polyolefin elastomer) (VPOL) and the melt viscosity upon heat melting the polyester resin (VPES), that is, VPOL/VPES is 1.2 or less.\",\n \"The polyolefin resin used as the polyolefin ingredient in the present invention is preferably a resin comprising one or more of 1-alkene polymer resins having a number of carbon atoms of 2 to 8, and the 1-alkene polymer resin is preferably one of polyethylene, polypropylene and ethylene-propylene copolymer.\",\n \"Further, as the polyolefin resin, a polyolefin resin polymerized by a metallocene catalyst can also be used.\",\n \"Further, a modified polyolefin resin modified with one of maleic acid hydride, acrylic acid, acrylic acid ester, or diglycidyl methacrylate may be used also as the polyolefin resin.\",\n \"Further, as the polyolefin elastomer used for the polyolefin ingredient, an in-plant produced ethylene-propylene copolymerized elastomer with a melt flow rate (MFR, 230° C.) of from 0.4 to 30 (g/10 min) is suitable.\",\n \"In the present invention, the polyester used for the thermoplastic resin composition is a polyester resin comprising ester repetitive units of at least one of ethylene terephthalate, ethylene isophthalate, ethylene naphthalate, ethylene adipate, butylenes terephthalate, butylene isophthalate, butylene naphthalate and butylene adipate, with the intrinsic viscosity being preferably from 0.5 to 1.5 and, particularly, ethylene terephthalate/ethylene isophthalate copolymer or polybutylene terephthalate is preferred.\",\n \"The polyester resin in the polyester resin layer capable of forming in an upper layer or a lower layer to the coating layer of the thermoplastic resin composition is preferably ethylene terephthalate/ethylene isophthalate copolymer.\",\n \"Further, the thermoplastic resin composition preferably contains 1 to 30% by weight of the polyolefin ingredient and preferably contains 70 to 95% by weight of the polyester resin.\",\n \"The thermoplastic resin-coated metal sheet according to the present invention is produced by heat melting a thermoplastic resin composition, directly extruding the same from a T-die onto a metal sheet for coating, or heat melting a thermoplastic resin composition, extruding the same from a T-die to a casting roll, cooling to solidify the same into a film and then hot press bonding the film on the metal sheet.\",\n \"In the thermoplastic resin-coated metal sheet according to the present invention, it is preferred that the olefin ingredient in the coating layer comprised of the thermoplastic resin composition is present being dispersed with a size of 1 to 10 μm in the extruding direction of the resin and 0.1 to 2 μm in the direction perpendicular to the resin extruding direction.\",\n \"Further, in the thermoplastic resin-coated metal sheet, the metal sheet is preferably one of electrolytic chromic acid treated steel sheet, tin-plated steel sheet or aluminum alloy sheet.\",\n \"BEST MODE FOR PRACTICING THE INVENTION The present invention provides a thermoplastic resin-coated metal sheet having a coating layer of a thermoplastic resin composition comprising a blend of a polyester resin and a polyolefin ingredient (at least one member selected from the group consisting of polyolefin resins and polyolefin elastomers) at least on one surface of a metal sheet and, optionally, a polyester resin layer formed to an upper layer and/or lower layer of the coating layer, as well as a can obtained by forming the same, and the resin layer is excellent in impact resistance and, particularly, impact resistance at low temperature and also excellent in corrosion resistance to acidic contents even in a case of coating a thermoplastic resin coating without interposing an adhesive primer to a metal sheet and applying severe forming such as thickness reducing-drawing or thickness-reducing ironing.\",\n \"The present invention is to be described specifically.\",\n \"In the thermoplastic resin composition comprising the blend of the polyester resin and the polyolefin ingredient, a polyolefin resin and a polyolefin elastomer are used as the polyolefin ingredient each alone or in combination.\",\n \"In the polyolefin ingredient and the polyester resin, it is preferred that the ratio of the melt viscosity in a case of melting the respective resin pellets is in a predetermined range.\",\n \"That is, it is preferred that VPOL/VPES is 1.2 or less where VPOL represents the melt viscosity upon heat melting of the polyolefin ingredient (polyolefin resin, polyolefin elastomer or a combination of both of them) and VPES represents a melt viscosity upon heat melting of polyester resin.\",\n \"The reason will be described below.\",\n \"The coating layer of the thermoplastic resin composition (heareinafter sometimes simply referred to as a thermoplastic resin film) is formed by heat melting resin pellets and extruding the blended resin from a T-die of an extruder directly onto a long strip-like metal sheet unwound from an uncoiler, or heat melting a resin heat melted from a polyester resin pellets separately and heat melted and blended resin from respective separate extruders, then coextruding them from a T-die having a plurality of die nozzles directly onto a long and strip-like metal sheet unwound from an uncoiler, and pressing the same by pressing rolls to a metal sheet.\",\n \"Alternatively, it is formed by extruding a heat melted resin in the same manner as described above from a T-die of an extruder onto a casting roll, cooling to solidify the same to form a film, then abutting the film against the heated long strip-like metal sheet which is unwound from an uncoiler and heated, and press bonding by sandwiching the same between a pair of laminate rolls by using an known laminater.\",\n \"In each of the preparation method, when the molten resin is extended downwardly, that is, in the longitudinal direction when the heat-melted blend resin is extruded from the T-die onto the metal sheet or the casting roll, since the molten resin is stretched downwardly, that is, in the longitudinal direction gravitationally, anisotropy occurs although little in the resin film.\",\n \"In a case where VPOL/VPES exceeds 1.2, anisotropy of the film of the blend resin increases to lower the rigidity of the film in the lateral direction compared with that of the longitudinal direction, and tend to cause lateral cracking upon exertion of impact after coating to the metal sheet.\",\n \"With a view point described above, it is preferred that VPOL/VPES is 1.2 or less.\",\n \"The polyolefin resin used as the polyolefin ingredient can include resins comprising one or more of 1-alkene copolymer resins with a number of carbon atoms of 2 to 8.The 1-alkene copolymer resin with the number of carbon atoms of 2 to 8 can include, for example, low density polyethylene, medium density polyethylene, high density polyethylene, polypropylene, polybutene-1, polypentent-1, polyhexene-1, polyheptene-1, polyoctene-1, ethylene-propylene copolymer, ethylene-1-butene copolymer, and ethylene-hexene copolymer.\",\n \"Among them, in-plant produced ethylene-propylene copolymer is preferred.\",\n \"Further, as the polyolefin resins, use of a polyolefin resin by a metallocene catalyst is preferred since it forms less oligomers that may give effects on the flavor of contents.\",\n \"Further, modified polyolefin resins formed by modifying the polyolefin resin, for example, polyethylene or polypropylene, for example, with one of maleic acid anhydride, acrylic acid, acrylic acid ester, acrylic acid ionomer, and diglycidyl methacrylate may also be used.\",\n \"The modified polyolefin resin blended at a ratio of 1 to 100% by weight to the not-modified polyolefin resin is used preferably as the polyolefin resin since the polyolefin ingredient is finely dispersed in the polyester resin.\",\n \"As the polyolefin elastomer, use of an ethylene-propylene copolymer elastomer having a melt flow rate (MFR, 230° C.) of 0.4 to 30 (g/10 min), particularly, 0.8 to 25 g/10 min is preferred.\",\n \"In a case where MFR is less than the range described above, the melt viscosity is excessively higher relative to the polyester resin when the resin is heat melted and the grains of the polyolefin resin dispersed in the polyester resin is grown excessively to deteriorate the impact resistance.\",\n \"On the other hand, in a case where it exceeds the range described above, the impact resistance of the polyolefin elastomer per se is poor, and the resin blended with the polyester resin is also poor in the impact resistance.\",\n \"Further, it is preferred that the ethylene-propylene copolymer elastomer is in-plant produced.\",\n \"The blending ratio of the polyolefin ingredient to the thermoplastic resin composition (polyolefin ingredient content in the thermoplastic composition) is preferably 1 to 30% by weight.\",\n \"In a case where the amount of the polyolefin ingredient is small, the thermoplastic resin composition after coating on the metal sheet lacks in the impact resistance.\",\n \"In a case where the amount of the polyolefin ingredient is unnecessarily large, the transparency of the thermoplastic resin composition decreases to make the anisotropy larger and the hardness is also decreased, so that the resin surface tends to be injured.\",\n \"As the polyester resin which is blended with the polyester resin to form a thermoplastic resin composition, those comprising ester repetitive units of at least one of ethylene terephthalate, ethylene isophthalate, ethylene naphthalate, ethylene adipate, butylenes terephthalate, butylenes isophthalate, butylenes naphthalate and butylenes adipate can be used alone or as a blend of two or more them.\",\n \"Further, it is also possible to use those other than described above that use sebasic acid, trimellitic acid or azelaic acid as the acid ingredient for the ester unit, or those of three or more carbon atoms, for example, propylene glycol, diethylene glycol, neopentyl glycol, and pentaerythritol as the alcohol ingredient for the ester unit.\",\n \"In the present invention, ethylene terephthalate/ethylene isophthalate copolymer or polybutylene terephthalate is used suitably as the polyester resin.\",\n \"Particularly, a polyester resin comprising 5 to 15 mol % of ethylene isophthalate and 85 to 95 mol % of ethylene terephthalate is excellent in fabricability and cause no cracks in the resin layer even when severe forming such as thickness-reducing drawing is applied after blending with the polyolefin ingredient and lamination onto the metal sheet and is also excellent in adhesion with the metal sheet.\",\n \"Further, in a case where it is in contact with contents filled in the can, it does not deteriorate flavor or taste of the contents and show favorable flavor property.\",\n \"Further, polybutylene terephthalate is easily mixed with the polyolefin ingredient (particularly, polyolefin resin) and in a case where it is blended, for example, with a polyolefin resin, the polyolefin resin is dispersed more finely in the blend resin, which is effective for the improvement of the impact resistance and the fabricability.\",\n \"Further, polybutylene terephthalate has high crystallization rate and in a case where a coated metal sheet having a coating layer of a thermoplastic resin composition is formed into a can and then the can is heated, for example, by outer surface coating, it has a feature of suppressing the grow of brittle and cause crystals.\",\n \"Accordingly, use of polybutylene terephthalate is extremely effective for the improvement of the impact resistance at low temperature as an object of the present invention.\",\n \"In addition, a thermoplastic resin composition blended with polybutylene terephthalate is excellent in water degradation resistance (hydrolysis resistance), and the can having the coating layer comprising the thermoplastic resin composition shows less lowering of the molecular weight of the resin even when left for long time with aqueous contents being filled therein and, accordingly, can maintain stable favorable impact resistance for a long time.\",\n \"In the present invention, since it is on the premise that the thermoplastic resin composition comprising a blend of the polyester resin and the polyolefin ingredient described above is used in a non-orientation state excellent in the fabricability in order to enable severe fabrication such as thickness-reducing drawing- or thickness-reducing drawing and ironing without causing cracking, chipping, scraping and peeling of the resin, it is necessary to increase the intrinsic viscosity of the resin and strengthen the resin.\",\n \"Accordingly, the intrinsic viscosity of the polyester resin preferably ranges from 0.5 to 1.5 and, more preferably, ranges from 0.8 to 1.2.In a case where polyester resin with an intrinsic viscosity of less than 0.5 is used, the strength of the resin is lowered extremely making it difficult for application use of thickness reduced drawn can or thickness reduced drawn and ironed can.\",\n \"In addition, flavor property of contents tends to be deteriorated.\",\n \"On the other hand, in a case where the intrinsic viscosity of the resin exceeds 1.5, melt viscosity upon heat melting the resin increases extremely making it extremely difficult for the operation of coating the thermoplastic resin composition on the metal sheet.\",\n \"The blend ratio of the polyester resin to the thermoplastic resin composition (content of the polyester resin in the thermoplastic resin) is preferably from 70 to 95% by weight.\",\n \"In a case where the amount of the polyester resin is small, the transparency is reduced and the anisotropy increases in the thermoplastic resin composition and, further, the hardness is decreased as well, so that the resin surface tends to be injured.\",\n \"Further, if the amount of the polyester resin is unnecessarily large, the transparency is reduced and the anisotropy increases in the thermoplastic resin composition and, further, the hardness is decreased as well, so that the resin surface tends to be injured.\",\n \"Particularly, in a case where the ethylene terephthalate/ethylene isophthalate copolymer described above is used as the polyester resin, when the blend ratio is less than 70% by weight, the fabricability of the thermoplastic resin composition after coating on the metal sheet is poor, whereas when the blend ratio exceeds 95% by weight, dispersed particles of the polyolefin ingredient in the resin composition become coarser, causing a worry of deteriorating the impact resistance and the workability.\",\n \"Further, in a case where polybutylene terephthalate described above is used as the polyester resin, when the blend ratio is less than 70% by weight, the dispersed particles of polyolefin ingredient in the resin composition become coarser making it difficult to obtain a sufficient impact resistance.\",\n \"When the blend ratio exceeds 95% by weight, the resin layer is clouded to deteriorate the appearance by heating of the can such as outer surface coating after forming the resin coated metal sheet into a can.\",\n \"Accordingly, it is most suitable in the present invention that the ethylene terephthalate/ethylene isophthalate copolymer and polybutylene terephthalate is used together such that the total amount is within the range of the blend ratio described above.\",\n \"In the present invention, a polyester resin layer can be formed for the upper layer and/or lower layer to the coating layer comprising the thermoplastic resin composition described above and a coating of a two-layered or three-layered structure as a whole can be formed on at least one surface of the metal sheet.\",\n \"As the polyester resin for forming the upper layer or the lower layer, the same polyester resin as used in the formation of the thermoplastic resin composition can be used, and ethylene terephthalate/ethylene isophthalate copolymer is suitable since this is excellent in the flavor property to contents and the fabricability and is also excellent in the adhesion to the metal sheet as described previously.\",\n \"In the copolymer described above, a copolymer comprising 3 to 15 mol % of ethylene isophthalate and 85 to 97 mol % of ethylene terephthalate is preferred for the upper layer, and a copolymer comprising 10 to 25 mol % of ethylene isophthalate and 75 to 90 mol % of ethylene terephthalate is preferred for the lower layer.\",\n \"Further as described above, the resin coating in the thermoplastic resin-coated metal sheet according to the present invention has a coating layer of a thermoplastic resin composition at least comprising a polyester resin and a polyolefin ingredient and includes a case where the coating layer comprises a single-layered structure, a case of a 2-layered structure in which the polyester resin layer is formed to either one of the upper layer or the lower layer of the coating layer and a case of a three-layered structure in which the polyester resin layer is formed to the upper layer and the lower layer of the coating layer.\",\n \"In the case of the single-layered structure comprising only the coating layer of the thermoplastic resin composition, the thickness is preferably within a range from 5 to 50 μm.\",\n \"In a case of the two-layered structure of the coating layer and the polyester resin layer, it is preferred that the thickness of the upper layer ranges from 3 to 15 μm, the thickness of the lower layer ranges from 2 to 47 μm and, further, the entire thickness ranges from 5 to 50 μm.\",\n \"In a case of the three-layered structure comprising the coating layer and the polyester resin layers, it is preferred that the thickness of the polyester resin layer for the upper layer ranges from 2 to 10 μm, the thickness of the coating layer as the intermediate layer ranges from 5 to 30 μm and the thickness of the polyester resin layer for the lower layer ranges from 3 to 10 μm.\",\n \"The thermoplastic resin film constituting the resin coating described above may be formed by extruding a resin obtained by heat melting and blending resin pellets to be blended at a temperature higher by 20 to 40° C. than the melting point of the polyester resin having the highest melting point among the resins to be used (about 200 to 300° C.) from a T-die of an extruder directly onto a long strip-like metal sheet re-wound from a uncoiler, or heat melting a resin obtained by separately heat melting polyester resin pellets at a temperature higher by 20 to 40° C. than the melting point and the heat melted and blend resin described above by separate extruders respectively, then co-extruding them from a T-die having a plurality of die nozzles directly on a long strip-like metal sheet unwound from an uncoiler, pressing them against the metal sheet by press bonding rolls and then quenching directly in water thereby preparing a thermoplastic resin film-coated metal sheet, or by extruding the heat melted resin in the same manner as described above from a T-die of an extruder onto a casting roll, cooling to solidify the same into a film, then re-winding from an uncoiler and abutting the film to a long strip-like metal sheet heated to a temperature higher by 20 to 40° C. than the melting point of the polyester resin, press bonding them while sandwiching between a pair of laminate rolls by means of a known laminator and immediately quenching them in water thereby preparing a thermoplastic resin film-coated metal sheet.\",\n \"Further, in a case of application use not applied with severe forming as in the present invention, heat melted resin may be extruded from a T-die of an extruder onto a casting roll, then stretched in a uni-axial direction (longitudinal direction) or bi-axial direction (longitudinal direction and lateral direction) and then heat set to form a film in which crystal molecules are oriented, which may be laminated onto the metal sheet.\",\n \"In the thermoplastic resin composition in which the polyester resin and the polyolefin ingredient are heat melted as described above (blend resin), the polyolefin resin or the polyolefin ingredient is finely dispersed in the matrix of the polyester resin.\",\n \"When the heat melted blend resin in this state is extruded from the T-die onto the metal sheet or the casting roll, the molten resin is gravitationally stretched downward although little.\",\n \"In this process, the polyolefin ingredient (polyolefin resin or polyolefin elastomer) dispersed finely in the matrix of the polyester resin is stretched in the direction of gravitational force and dispersed in the matrix of the polyester resin being dispersed in a fibrous state after the blend resin has been cooled to solidify to the metal sheet on the casting roll.\",\n \"In the dispersion state, when the resin coated metal sheet is applied with thickness-reducing drawing or thickness reducing drawing and ironing to form a can, a dispersed fibrous blend resin is sometimes visible with naked eyes.\",\n \"The fibrous blend resin is no more visible with naked eyes when it is 10 μm or less in the extruding direction and 2 μm or less in the direction perpendicular to the extruding direction when extruded from the T-die.\",\n \"Accordingly, it is preferred in the present invention that the olefin ingredient in the thermoplastic resin composition is present being dispersed with a size of 1 to 10 μm in the extruding direction of the resin (length in the film direction) and 0.1 to 2 μm in the direction perpendicular to the extruding direction of the resin (length in the lateral direction of the film) in the coating layer of the thermoplastic resin composition after preparing the thermoplastic resin-coated metal sheet.\",\n \"As the substrate for the thermoplastic resin-coated metal sheet of the present invention, various kinds of surface treated steel sheets such as electrolytic chromic acid treated steel sheet (tin free steel, hereinafter represented by TFS) or tin-plated steel sheet (tin sheet hereinafter represented by tin sheet), and aluminum alloy sheet which are usually used generally as materials for cans can be used.\",\n \"For the surface treated steel sheets, TFS having, on a steel sheet, a 2-layered film comprising a lower layer composed of metallic chromium in a membrane amount of 10 to 200 mg/m2 and an upper layer composed of chromium hydrate oxides in a membrane amount of 1 to 30 mg/m2 based on the chromium content is preferred, which has a sufficient adhesive property with the thermoplastic resin film according to the present invention and also has corrosion resistance.\",\n \"Preferred film sheets are those obtained by applying plating in a plating amount of 0.1 to 11.2 g/m2 on the surface of the steel sheet and, forming thereon, a 2-layered film comprising metallic chromium and chromium-hydrate oxides in an amount of membrane from 1 to 30 mg/m2 based on chromium content, or forming a single layered film comprising only chromium hydrate oxides.\",\n \"In any of the cases, the steel sheets as the substrates is preferably a cold rolled low carbon steel sheet used generally as the material for cans.\",\n \"The thickness of the steel sheet is preferably from 0.1 to 0.32 mm.\",\n \"For the aluminum alloy sheet, JIS 3000 series or 5000 series is preferred, and preferred are those applied with an electrolytic chromic acid treatment to the surface to form a 2-layered film comprising a lower layer composed of metallic chromium in a film amount of 0 to 200 mg/m2 and an upper layer comprising chromium hydrate oxides in a membrane amount of 1 to 30 mg/m2 based on the chromium content, or those applied with a chromium phosphate treatment to deposit 1 to 30 mg/m2 of a chromic ingredient based on the chromium content and 0 to 30 mg/m2 of phosphoric ingredient based on the phosphorus content.\",\n \"The thickness of the aluminum alloy sheet is preferably from 0.15 to 0.4 mm.\",\n \"EXAMPLE The present invention is to be described more specifically by way of examples.\",\n \"Example 1 (Specimen Nos.\",\n \"1-6, 11-17, 26-32) A long strip-like electrolytic chromic acid treated steel plate (hereinafter referred to as TFS) was re-wound at a rate of 150 m/min from an uncoiler, and pellets comprising a polyester resin shown in Table 1 (represented by PES in the table here and hereinafter), a polyolefin resin (represented by POL in the table here and hereinafter) and/or a polyolefin elastomer (represented by PEL in the table here and hereinafter), or only comprising the polyester resin were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using an extruder to form a blend resin, then fed to a T-die and extruded from a die nozzle on one surface of TFS, that is, one surface to be an inner surface of a can (represented by inner surface in the table herein and hereinafter).\",\n \"Simultaneously, pellets of a polyester resin incorporated with 20% by weight of white titanium oxide shown in Table 1 were heat melted at a temperature higher by about 30° C. than the melting temperature of the polyester resin using an extruder, then fed into a T-die and extruded from the die nozzle on the other surface of TFS, that is, one surface to be an outer surface of the can (represented by outer surface in the table here and hereinafter).\",\n \"Then, after sandwiching the TFS to which the resin layer was extruded on both surface thereof by using a pair of press bonding rolls, they were directly quenched in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.\",\n \"1-6, 11-17, 26-0.32 in Tables 2 and 3.\",\n \"(Specimen Nos.\",\n \"18, 19) A long strip-like tin sheet was re-wound from an uncoiler at a rate of 150 m/min and heated, and pellets of the polyester resin and the polyolefin resin shown in Table 1 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using an extruder to form a blend resin, the polyester resin shown in Table 1 was heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using a separate extruder, then they were fed to a T-die having two die nozzles and co-extruded on one surface of a tin sheet from the die nozzles such that the blend resin layer containing the polyolefin resin was in contact with the tin sheet.\",\n \"Simultaneously, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Table 1 was heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using an extruder, fed to a T-die and then extruded from the die nozzle to the other surface of the heated tin sheet to be an outer surface of a can.\",\n \"Then, sandwiching the tin sheet to which the resin layer was extruded on both surface thereof by using a pair of press bonding rolls and, they were directly quenched in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.\",\n \"18 and 19 in Table 3.\",\n \"(Specimen Nos.\",\n \"7-10, 20-23) Pellets of the polyester resin and the polyolefin resin or polyolefin elastomer shown in Table 1 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using an extruder to form a blend resin, then fed into a T-die, extruded from a die nozzle, followed by trimming and taken-up to a coiler as a non-orientation film.\",\n \"Further, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Table 1 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using an extruder, fed into a T-die, extruded from a die nozzle, followed by trimming and then taken up as a non-orientation film to a coiler.\",\n \"Then, a long strip-like aluminum alloy sheet was rewound at rate of 150 m/min from an uncoiler and heated, a blend resin film was abutted while being re-wound from the coiler to one surface to be an inner surface of a can and, at the same time, a white polyester film was abutted while being re-wound from the coiler to the other surface to be an outer surface of the can.\",\n \"Then, after sandwiching the aluminum alloy sheet to which the resin layer was extruded on both surface thereof by using a pair of press bonding rolls, they were directly quenched in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.\",\n \"7-10 and 20-23 in Tables 2 and 3.\",\n \"(Specimen Nos.\",\n \"24, 25) Pellets of the polyester resin and the polyolefin resin shown in Table 1 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using an extruder to form a blend resin, the polyester resin shown in Table 1 was heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using a separate extruder, then fed into a T-dye having two die nozzles, co-extruded from the die nozzles, followed by trimming and then taken up as two layers of a non-orientation film to a coiler.\",\n \"Further, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Table 2 and 3 were heat melted at a temperature higher by about 30° C. than the melting temperature of the polyester resin by using an extruder, fed into a T-die, extruded from the die nozzle, followed by trimming and then taken-up as a non-orientation film to a coiler.\",\n \"Then, a long strip-like tin sheet was re-wound at a rate of 150 m/min from an uncoiler, and heated, and the two-layered resin film was abutted against one surface thereof to be an inner surface of a can while being re-wound from the coiler such that the blend resin layer containing the polyolefin resin was in contact with the tin sheet.\",\n \"At the same time, the white polyester film was abutted, while being re-wound from the coiler to the outer surface of the can.\",\n \"Then, after sandwiching the tin sheet to which the resin layer was extruded on both surface thereof by using a pair of press bonding rolls, they were directly quenched in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.\",\n \"24 and 25 in Table 3.The metal sheets applied with the surface treatment shown below were used as the three types of the metal sheets described above.\",\n \"(1) Electrolytic chromic acid treated steel sheet (indicated as TFS in the table) Sheet thickness: 0.18 mm Amount of metallic chromium: 160 mg/m2 Amount of chromium hydrate oxide: (as chromium) 19 mg/m2 Heating temperature: temperature higher by about 30° C. than the melting point of the polyester resin (2) Tin sheet (indicated as ET in the table) Sheet thickness: 0.18 mm Amount of tin plating: 0.2 g/m2 Amount of chromium hydrate oxide: (as chromium) 9 mg/m2 Heating temperature: 200° C. (3) Aluminum alloy sheet (JIS 5052H 39) (indicated as Al in the table) Sheet thickness: 0.26 mm Amount of membrane: (as phosphorus) 11 mg/m2 (as chromium) 7 mg/m2 Heating temperature: temperature higher by about 30° C. than the melting point of the polyester resin As each of the polyolefin resins described above, polyolefin resins using metallocene catalysts were used for any of the cases.\",\n \"TABLE 1 Resin composition Resin No.\",\n \"Resin Composition PES1 Polyethylene terephthalate (IV value: 0.75) PES2 Polyethylene terephthalate (IV value: 0.82) PES3 Polyethylene terephthalate (IV value: 1.1) PES4 Ethylene terephthalate - ethylene isophthalate copolymer (ethylene isophthalate:: 5 mol %) (IV value: 0.9) PES5 Ethylene terephthalate - ethylene isophthalate copolymer (ethylene isophthalate:: 10 mol %) (IV value: 0.9) PES6 Ethylene terephthalate - ethylene isophthalate copolymer (ethylene isophthalate:: 15 mol %) (IV value: 1.5) PES7 Ethylene terephthalate - ethylene adiipate copolymer (ethylene adipate:: 10 mol %) (IV value: 0.6) PES8 Ethylene terephthalate - ethylene naphthalate copolymer (ethylene-naphthalate: 10 mol %) (IV value: 0.9) PES9 Polybutylene terephthalate (IV value: 0.5) POL1 Polyethylene POL2 Polypropylene POL3 Polybutene-1 POL4 Polyoctene-1 POL5 Ethylene-propylene copolymer POL6 Ethylene-butene-1 copolymer POL7 Ethylene-hexene copolymer POL8 Maleic acid anhydride copolymerized polyethylene POL9 Acrylic acid copolymerized polypropylene POL10 Methylacrylate copolymerized polyethylene POL11 Diglycidyl methacrylate copolymerized polyethylene PEL1 Ethylene-propylene polymerized elastomer (MFR: 0.4 g/10 min) PEL2 Ethylene-propylene polymerized elastomer (MFR: 1 g/10 min) PEL3 Ethylene-propylene polymerized elastomer (MFR: 5 g/10 min) PEL4 Ethylene-propylene polymerized elastomer (MFR: 10 g/10 min) PEL5 Ethylene-propylene polymerized elastomer (MFR: 30 g/10 min) PEL6 Ethylene-propylene polymerized elastomer (MFR: 35 g/10 min) TABLE 2 Thermoplastic resin-coated metal sheet Inner surface resin layer Outer surface Lower layer (blend resin) Melt Upper resin (TiO2: POL Grain viscosity layer (PES) WT % content) Specimen Blend ratio Thickness size ratio Thickness Thickness No.\",\n \"Kind (%) (μm) (μm) Vpol/Vpes Type (μm) Type (μm) Metal sheet Section 1 PES1/POL8/PEL4 80/1/19 25 7 × 1.5 0.60 — — PES3 15 TFS Invention 2 PES1/POL8/PEL4 80/5/15 25 5 × 1.0 0.63 — — PES3 15 TFS Invention 3 PES2/POL9/PEL3 80/5/15 25 3 × 0.5 0.86 — — PES3 15 TFS Invention 4 PES2/POL9/PEL2 85/5/10 28 8 × 1.0 1.10 — — PES3 15 TFS Invention 5 PES3/POL9/PEL1 80/10/10 30 6 × 0.8 0.71 — — PES3 15 TFS Invention 6 PES3/POL9/PEL1 75/10/15 20 14 × 3.1 1.25 — — PES3 15 TFS Comp.\",\n \"Ex 7 PES4/POL10/POL1 80/7/13 35 10 × 2.0 0.80 — — PES3 15 AL Invention 8 PES3/POL11/POL2 80/15/5 25 4 × 0.8 0.61 — — PES3 15 AL Invention 9 PES6/POL8/POL3 80/5/15 20 6 × 0.9 0.19 — — PES3 15 AL Invention 10 PES7/POL8/POL1 80/10/10 25 5 × 0.6 0.88 — — PES3 15 AL Invention 11 PES9/POL9/PEL3 90/5/5 35 3 × 0.3 0.67 — — PES3 15 TFS Invention 12 PES9/POL10/PEL5 80/10/10 25 8 × 1.6 1.13 — — PES3 15 TFS Invention 13 PES2/POL9/POL4 80/10/10 25 5 × 0.8 0.72 — — PES3 15 TFS Invention 14 PES4/POL8/POL5 75/10/15 30 4 × 1.2 0.59 — — PES3 15 TFS Invention 15 PES2/POL1 99/1 30 8 × 1.8 0.62 — — PES3 15 TFS Invention 16 PES3/POL1 95/5 20 7 × 1.5 0.23 — — PES3 15 TFS Invention TABLE 3 Thermoplastic resin-coated metal sheet Inner surface resin layer Outer surface Lower layer (blend resin) Melt Upper resin (TiO2: POL grain viscosity layer (PES) WT % content Specimen Blend ratio Thickness size ratio Thickness Thickness Metal No.\",\n \"Kind (%) (μm) (μm) Vpol/Vpes Kind (μm) Kind (μm) sheet Section 17 PES2/POL2 85/15 30 7 × 1.6 0.65 — — PES3 15 TFS Invention 18 PES4/POL3 90/10 20 6 × 1.2 0.58 PES3 15 PES3 15 ET Invention 19 PES5/POL1 90/10 20 5 × 1.5 0.62 PES3 15 PES3 15 ET Invention 20 PES2/POL9 85/15 25 4.5 × 0.2 0.30 — — PES3 15 AL Invention 21 PES3/POL8 80/20 25 4 × 0.3 0.32 — — PES3 15 AL Invention 22 PES4/POL8 85/15 28 3.5 × 0.2 0.32 — — PES3 15 AL Invention 23 PES5/PEL3 70/30 28 3 × 0.4 0.28 — — PES3 15 AL Invention 24 PES2/POL7 80/20 15 7 × 0.8 0.35 PES3 20 PES3 15 ET Invention 25 PES8/POL6 90/10 15 6 × 0.9 0.30 PES3 20 PES3 15 ET Invention 26 PES2/PEL4 90/10 20 6 × 0.8 0.26 — — PES3 15 TFS Invention 27 PES1 — 25 — — — — PES3 15 TFS Comp.\",\n \"Ex 28 PES2/POL1 99.5/0.5 25 7 × 1.5 0.62 — — PES3 16 TFS Comp.\",\n \"Ex 29 PES5/POL2 65/35 25 12 × 3.0 0.57 — — PES3 16 TFS Comp.\",\n \"Ex 30 PES4 — 25 — — — — PES3 16 TFS Comp.\",\n \"Ex 31 PES2/PEL6 80/20 30 5 × 1.2 0.05 — — PES3 16 TFS Comp.\",\n \"Ex 32 PES1/PEL1 85/15 28 15 × 3.0 4.20 — — PES3 16 TFS Comp.\",\n \"Ex The thermoplastic resin-coated metal sheet obtained as described above was formed into a can of a bottomed cylindrical shape by using a thickness-reducing drawing and ironing as described below.\",\n \"After punching a thermoplastic resin-coated metal sheet into a blank of 160 mm diameter, a drawn can with 100 mm can bottom diameter was formed such that the white polyester resin-coated surface was on the outer surface of the can.\",\n \"Then, it was re-drawn into a re-drawn can with 80 mm can bottom diameter.\",\n \"Further, the re-drawn can was applied with stretching and, at the same time, ironing by composite fabrication to form a drawn and ironed can with 65 mm can bottom diameter.\",\n \"The composite fabrication was conducted under the conditions that the distance between the re-drawn portion and the upper end of the can was 20 mm, the R portion at the shoulder of a re-drawing dice was 1.5 times the sheet thickness, a clearance between the re-drawing dice and a punch was 1.0 times the sheet thickness and the clearance at the ironed portion was 50% of the original thickness.\",\n \"Then, an upper portion of the can was trimmed by a known method and applied with neck-in fabrication and flange fabrication.\",\n \"Then, methods of evaluating the thermoplastic resin and the thermoplastic resin-coated metal sheet are to be explained.\",\n \"(Size of Polyolefin Resin Dispersed in Polyester Resin) A Blend resin layer of the thermoplastic resin-coated metal sheet was observed under a scanning type electron microscope to measure the size of the fibrous resin.\",\n \"The measured value shows an average value measured at three time points, that is, during coating or during film preparation, completion of coating or five min before completion of film preparation, at three positions, that is, 5 cm from both ends and at the central portion in view of the lateral direction of the resin-coated metal sheet.\",\n \"In Tables 2 and 3, it is represented by “length in the resin extruding direction” (μm)דlength in the direction perpendicular to resin extruding direction” (μm).\",\n \"(Appearance for the can Inner Surface) The can inner surface formed by the thickness-reducing drawing and ironing fabrication method was observed with naked eyes, and presence or absence of fibrous unevenness of the polyolefin resin in the resin layer and the transparency of the resin layer were evaluated by the following standards.\",\n \"◯: no fibrous unevenness was observed and it had sufficient transparency Δ: fibrous unevenness was observed slightly, or slight clouding of the resin layer was observed X: fibrous unevenness is observed, clouding of the resin layer was observed.\",\n \"(Melt Flow Rate of Polyolefin Elastomer) Melt flow rate (230° C.) was measured by a customary method.\",\n \"(Thickness of Resin Film) The thermoplastic resin-coated metal sheet was buried in an epoxy type embedding resin, sliced to a thickness of 5 μm and the cross section was measured by microscopic observation.\",\n \"(Intrinsic Viscosity (IV Value)) After dissolving the polyester resin in a 1:1 mixed solution of phenol/tetrachloroethane, the specific viscosity was measured in a thermostable bath at 30° C. by a Ubbelohde's viscometer to determine an intrinsic viscosity.\",\n \"(Melt Viscosity) The melt viscosity was determined by a capillary rheometer according to JIS K 7199.\",\n \"(Fabricability) Can formed by using a thickness-reducing drawing and ironing method was observed with naked eyes and the fabricability was evaluated based on the following standards.\",\n \"⊚: fine crack or film breakage was not recognized ◯: slight fine crack to an extent not causing practical problems was recognized Δ: crack and film breakage to an extent causing practical problem was recognized X: broken upon forming (Impact Resistance at Low Temperature) After forming by using the thickness-reducing drawing and ironing method, trimming an upper portion of a can, and applying necking-in and flange fabrication, acidic drinks (Acerola drink, trade name of products: manufactured by Nichirei Co.) at pH 2.6 were filled, aged at 37° C. for one month and then seal was opened.\",\n \"The can was cut out from the upper end thereof in a circumferential direction at a width of 30 mm to form a specimen.\",\n \"After immersing the specimen in iced water for 5 min, it was taken out and a steel rod attached with steel balls each of {fraction (1/2)} inch diameter at the top end at 15 mm distance in the circumferential direction (weight: 1 kg) was dropped from the height of 40 mm, a sponge impregnated with an aqueous 3% sodium chloride solution was abutted against generated convex portions on the can inner surface, a DC current at 6.3 V was applied to the specimen, the value of the flowing current was measured and the impact resistance at low temperature was measured by the following standards depending on the magnitude of the measured current value.\",\n \"⊚: less than 0.05 mA ◯: 0.05 mA or more and less than 0.1 mA Δ: 0.1 mA or more and less than 0.3 mA X: 0.3 mA or more (Corrosion Resistance) After forming by using a thickness-reducing drawing and ironing method, an upper portion of a can was trimmed and applied with neck-in and flange fabrication.\",\n \"Then after filling acidic drinks (Acelora drink, trade name of products: manufactured by Nichirei Co.), impact was applied at a low temperature from the outside of the can to the wall of the can in the same manner as practiced in the evaluation of the impact resistance at low temperature to form a concave portion.\",\n \"Then, after aging at 37° C. for one month, seal was opened and concentration of leached metal was measured by atomic absorption spectroscopy and the corrosion resistance was evaluated in accordance with the following standards depending on the concentration.\",\n \"⊚: less than 0.3 ppm, ◯: more than 0.3 ppm and less than 0.5 ppm, Δ: more than 0.5 ppm and less than 1.0 ppm, X: 1.0 ppm or more The result of evaluation was shown in Tables 4 and 5.As shown in Table 4 and Table 5, it can be seen that any of the thermoplastic resin-coated metal sheets of the present invention is excellent in the fabricability and shows favorable impact resistance at low temperature and corrosion resistance.\",\n \"TABLE 4 Result of characteristic evaluation Result of characteristic evaluation Impact Spec- resistance imen at low Corrosion Resin No.\",\n \"Fabricability temperature resistance unevenness Section 1 ⊚ ⊚ ⊚ ◯ Invention 2 ⊚ ⊚ ⊚ ◯ Invention 3 ⊚ ⊚ ⊚ ◯ Invention 4 ⊚ ◯ ◯ ◯ Invention 5 ⊚ ◯ ⊚ ◯ Invention 6 ⊚ X ◯ X Comp.\",\n \"Ex 7 ⊚ ⊚ ⊚ ◯ Invention 8 ⊚ ◯ ⊚ ◯ Invention 9 ⊚ ⊚ ⊚ ◯ Invention 10 ⊚ ⊚ ⊚ ◯ Invention 11 ⊚ ⊚ ⊚ □ Invention 12 ⊚ ◯ ◯ ◯ Invention 13 ⊚ ⊚ ⊚ ◯ Invention 14 ⊚ ⊚ ⊚ ◯ Invention 15 ⊚ ◯ ◯ ◯ Invention 16 ⊚ ⊚ ◯ ◯ Invention TABLE 5 Result of characteristic evaluation Result of characteristic evaluation Impact Spec- resistance imen at low Corrosion Resin No.\",\n \"Fabricability temperature resistance unevenness Section 17 ⊚ ⊚ ◯ ◯ Invention 18 ⊚ ⊚ ⊚ ◯ Invention 19 ⊚ ⊚ ⊚ ◯ Invention 20 ⊚ ⊚ ⊚ ◯ Invention 21 ⊚ ⊚ ⊚ ◯ Invention 22 ⊚ ⊚ ⊚ ◯ Invention 23 ◯ ◯ ◯ ◯ Invention 24 ⊚ ⊚ ⊚ ◯ Invention 25 ⊚ ⊚ ⊚ ◯ Invention 26 ⊚ ⊚ ⊚ ◯ Invention 27 ⊚ X Δ ◯ Comp.\",\n \"Ex 28 ⊚ X Δ ◯ Comp.\",\n \"Ex 29 ◯ Δ ◯ Δ Comp.\",\n \"Ex 30 ⊚ X Δ ◯ Comp.\",\n \"Ex 31 ⊚ X Δ ◯ Comp.\",\n \"Ex 32 ◯ X Δ Δ Comp.\",\n \"Ex Example 2 (Specimen Nos.\",\n \"33-38, 43-53) A long strip-like electrolytic chromic acid treated steel sheet (TFS) was re-wound from an uncoiler at a rate of 150 m/min and heated, and pellets of the polyester resin (PES), the polyolefin resin (POL) and/or polyolefin elastomer (PEL) or only of the polyester resin shown in Table 6 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using an extruder to form a blend resin, fed to a T-die and extruded on one surface of TFS to be an inner surface of a can (inner surface).\",\n \"Simultaneously, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Tables 7 and 8 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using an extruder, then fed into a T-die and extruded on the other surface of the heated TFS to be an outer surface of the can (outer surface) from the die nozzle.\",\n \"Then, a pair of press bonding rolls were used and after sandwiching TFS having resin layers extruded on both surfaces thereof, it was quenched directly in water to prepare thermoplastic resin-coated metal sheets of Specimens Nos.\",\n \"33-38, 43-53 in Table 7 and Table 8.\",\n \"(Specimen Nos.\",\n \"39-42) A long strip-like tin sheet (ET) was re-wound from an uncoiler at a rate of 150 m/min and heated, and pellets of the polyester resin and the polyolefin resin shown in Table 6 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using extruder to form a blend resin, the polyester resin shown in Table 6 was heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using a separate extruder, fed to a T-die having two die nozzles, and coextruded on one surface of a tin sheet to be an inner surface of a can from the die nozzles such that the blend resin layer containing the polyolefin resin was in contact with the tin sheet.\",\n \"Simultaneously, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Table 7 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using an extruder, fed into a T-die and extruded on the other surface of the heated tin sheet to be an outer surface of the can from a die nozzle.\",\n \"Then, a pair of press bonding rolls were used and after sandwiching the tin sheet having resin layers extruded on both surfaces, it was quenched directly in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.\",\n \"39-42 in Table 7.\",\n \"(Specimen Nos.\",\n \"54-58) Pellets of the polyester resin and the polyolefin resin or the polyolefin elastomer shown in Table 6 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying kneading time by using an extruder to form a blend resin, then fed to a T-die, extruded from a die nozzle, followed by trimming and taken up as a non-orientation film to a coiler.\",\n \"Further, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Table 8 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using an extruder, fed into a T-die, extruded from a die nozzle, followed by trimming and was taken up as a non-orientation film to an uncoiler.\",\n \"Then, a long strip-like aluminum alloy sheet (AL) was re-wound at a rate of 150 m/min from the uncoiler and the blend resin film was abutted while rewinding from the coiler to one surface thereof to be the inner surface of a can and, simultaneously, white polyester film was abutted while being re-wound from the coiler against the other surface to be the outer surface of the can.\",\n \"Then, a pair of press bonding rolls were used and after sandwiching the aluminum alloy sheet having resin layers extruded on both surfaces, it was quenched directly in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.\",\n \"54-58 in Table 8.\",\n \"(Specimen Nos.\",\n \"59-61) Pellets of the polyester resin and the polyolefin resin and/or the polyolefin elastomer shown in Table 9 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using an extruder to form a blend resin, pellets of the polyester (PES4) in Table 6 were heat melted to a temperature higher by about 30° C. than the melting point of the respective polyester resins by using separate extruders respectively, then fed to a die having two nozzles, co-extruded from the die nozzles such that the upper layer comprised of PES4 and the lower layer comprised of the blend resin, followed by trimming and then taken up to a coiler as a two-layered non-orientation film.\",\n \"Further, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Table 9 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using an extruder, fed to a T-die, extruded from a die nozzle, followed by trimming and taken up as a non-orientation film to a coiler.\",\n \"Then, a long strip-like TFS was re-wound from an uncoiler at a rate of 150 m/min and heated, and the two-layered resin film was abutted while being re-wound from the coiler to one surface to be an inner surface of a can and, at the same time, the white polyester film was abutted while being re-wound from the coiler against the other surface to be an outer surface of the can.\",\n \"Then, after sandwiching the TFS having resin layers extruded on both surfaces by using a pair of press bonding rolls, it was quenched directly in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.\",\n \"59-61 in Table 9.\",\n \"(Specimen Nos.\",\n \"62-64) Pellets of the polyester resin and the polyolefin resin and/or the polyolefin elastomer shown in Table 10 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin while varying the kneading time by using an extruder to form a blend resin, pellets of the polyester PES4 and PES6 in Table 6 were heat melted to a temperature higher by about 30° C. than the melting point of the respective polyester resins by using separated extruders respectively, then fed to a die having three nozzles, co-extruded from the die nozzles such that the upper layer comprised of PES4, the intermediate layer comprised of the blend resin, and the lower layer comprised of the PES6, followed by trimming and then taken up to a coiler as a three-layered non-orientation film.\",\n \"Further, pellets of the polyester resin incorporated with 20% by weight of white titanium dioxide shown in Table 9 were heat melted at a temperature higher by about 30° C. than the melting point of the polyester resin by using an extruder, fed to a T-die, extruded from die nozzle, followed by trimming and taken up as a non-orientation film to a coiler.\",\n \"Then, a long strip-like TFS was re-wound from an uncoiler at a rate of 150 m/min and heated, and the three-layered resin film was abutted while being re-wound from the coiler to one surface to be an inner surface of a can and, at the same time, the white polyester film was abutted while being re-wound from the coiler against the other surface to be an outer surface of the can.\",\n \"Then, after sandwiching the TFS having resin layers extruded on both surfaces by using a pair of press bonding rolls, it was quenched directly in water to prepare thermoplastic resin-coated metal sheets of Specimen Nos.\",\n \"62-64 in Table 10.In Tables 7-10, the outer surface resin layer contains 20% by weight of TiO2 as a white pigment.\",\n \"TABLE 6 Resin composition Resin No.\",\n \"Resin Composition PES1 Polyethylene terephthalate (IV value: 0.75) PES2 Polyethylene terephthalate (IV value: 0.82) PES3 Polyethylene terephthalate (IV value: 1.1) PES4 Ethylene terephthalate/ethylene isophthalate copolymer (ethylene-isophthalate:: 5 mol %) (IV value: 0.9) PES5 Ethylene terephthalate/ethylene isophthalate copolymer (ethylene isophthalate:: 10 mol %) (IV value: 0.9) PES6 Ethylene terephthalate/ethylene isophthalate copolymer (ethylene isophthalate:: 15 mol %) (IV value: 1.5) PES7 Ethylene terephthalate/ethylene adipate copolymer (ethylene adipate:: 10 mol %) (IV value: 0.5) PES8 Ethylene terephthalate - ethylene naphthalate copolymer (ethylene-naphthalate: 10 mol %) (IV value: 0.9) PES9 Polybutylene terephthalate (IV value: 1.35) POL1 Polyethylene POL2 Polypropylene POL3 Polybutene-1 POL4 Polyoctene-1 POL5 Ethylene-propylene copolymer POL6 Ethylene-butene-1 copolymer POL7 Ethylene-hexene copolymer PEL1 Ethylene/propylene polymerized elastomer (MFR: 0.45 g/10 min) PEL2 Ethylene/propylene polymerized elastomer (MFR: 0.8 g/10 min) PEL3 Ethylene/propylene polymerized elastomer (MFR: 6 g/10 min) PEL4 Ethylene/propylene polymerized elastomer (MFR: 12 g/10 min) PEL5 Ethylene/propylene polymerized elastomer (MFR: 25 g/10 min) PEL6 Ethylene/propylene polymerized elastomer (MFR: 30 g/10 min) TABLE 7 Thermoplastic resin-coated metal sheet Outer surface Inner surface resin layer resin layer Specimen Blend ratio Thickness Thickness Metal No.\",\n \"Type (wt %) (μm) Type (μm) sheet Section 33 PES4/PES9/POL5 30/50/20 25 PES4 15 TFS Invention 34 PES5/PES9/POL5 50/47/3 25 PES4 15 TFS Comp.\",\n \"Ex 35 PES5/PES9/POL5 50/45/5 25 PES4 15 TFS Invention 36 PES5/PES9/POL5 30/50/20 25 PES4 15 TFS Invention 37 PES5/PES9/POL5 25/50/30 25 PES4 15 TFS Invention 38 PES5/PES9/POL5 20/45/35 25 PES4 15 TFS Comp.\",\n \"Ex 39 PES6/PES9/POL5 30/50/20 25 PES4 15 ET Invention 40 PES6/PES9/POL1/POL5 30/50/10/10 25 PES4 15 ET Invention 41 PES6/PES9/PEL3 30/50/20 25 PES4 15 ET Invention 42 PES6/PES9/POL5/PEL3 30/50/10/10 25 PES4 15 ET Invention 43 PES4/POL5 80/20 25 PES4 15 TFS Invention 44 PES5/PES9/POL2 30/50/20 25 PES4 15 TFS Invention 45 PES5/PES9/POL3 50/30/20 25 PES4 15 TFS Invention 46 PES5/PES9/POL4 30/50/20 25 PES4 15 TFS Invention TABLE 8 Thermoplastic resin-coated metal sheet Outer surface Inner surface resin layer resin layer Specimen Blend ratio Thickness Thickness Metal No Type (wt %) (μm) Type (μm) sheet Section 47 PES5/PES9/POL6 30/50/20 25 PES4 15 TFS Invention 48 PES5/PES9/POL7 30/50/20 25 PES4 15 TFS Invention 49 PES5/PES9/PEL1 30/50/20 25 PES4 15 TFS Comp.\",\n \"ex 50 PES5/PES9/PEL2 30/50/20 25 PES4 15 TFS Invention 51 PES5/PES9/PEL4 30/50/20 25 PES4 15 TFS Invention 52 PES5/PES9/PEL5 30/50/20 25 PES4 15 TFS Invention 53 PES5/PES9/PEL6 30/50/20 25 PES4 15 TFS Comp.\",\n \"ex 54 PES1/PES9/PEL5 30/50/20 25 PES4 15 AL Invention 55 PES2/PES9/POL1/PEL4 30/50/10/10 25 PES4 15 AL Invention 56 PES3/PES9/POL5/PEL4 30/50/5/15 25 PES4 15 AL Invention 57 PES7/PES9/POL5/PEL5 30/50/10/10 25 PES4 15 AL Invention 58 PES8/PES9/POL6/PEL3 30/50/10/10 25 PES4 15 AL Invention TABLE 9 Thermoplastic resin-coated metal sheet Inner surface resin layer Upper layer Lower layer Outer surface Specimen Thickness Blend ratio Thickness resin layer No.\",\n \"Type (μm) Type (wt %) (μm) Type Thickness Metal sheet Section 59 PES 5 PES6/PES9/POL5 30/50/20 25 PES4 15 TFS Invention 60 PES 5 PES6/PES9/POL1/PEL3 30/50/10/10 25 PES4 15 TFS Invention 61 PES 5 PES6/PES9/PEL2 30/50/20 25 PES4 15 TFS Invention TABLE 10 Thermoplastic resin-coated metal sheet Inner surface resin layer Outer surface Upper layer Intermediate layer Lower layer resin layer Specimen Thickness Blend ratio Thickness Thickness Thickness No.\",\n \"Type (μm) Type (wt %) (μm) Type (μM) Type (μm) Metal sheet Section 62 PES4 5 PES6/PES9/PEL4 30/50/20 20 PES6 5 PES4 15 TFS Inveniton 63 PES4 5 PES6/PES9/POL1/PEL2 30/50/10/10 20 PES6 5 PES4 15 TFS Inveniton 64 PES4 5 PES6/PES9/PEL3 30/50/20 20 PES6 5 PES4 15 TFS Inveniton TABLE 11 Result of characteristic evaluation Result of characteristic evaluation Specimen Low temperature Corrosion No.\",\n \"Fabricability impact resistance resistance Section 33 ⊚ ⊚ ⊚ Invention 34 ◯ X Δ Comp.\",\n \"Ex 35 ⊚ ⊚ ⊚ Invention 36 ⊚ ⊚ ⊚ Invention 37 ⊚ ⊚ ⊚ Invention 38 ⊚ X Δ Comp.\",\n \"Ex 39 ⊚ ⊚ ⊚ Invention 40 ⊚ ⊚ ⊚ Invention 41 ⊚ ⊚ ⊚ Invention 42 ⊚ ⊚ ⊚ Invention 43 ⊚ ◯ ◯ Invention 44 ⊚ ⊚ ⊚ Invention 45 ⊚ ⊚ ⊚ Invention 46 ⊚ ⊚ ⊚ Invention 47 ⊚ ⊚ ⊚ Invention 48 ⊚ ⊚ ⊚ Invention TABLE 12 Result of characteristic evaluation Result of characteristic evaluation Specimen Low temperature Corrosion No.\",\n \"Fabricability impact resistance resistance Section 49 Δ X ◯ Comp.\",\n \"Ex 50 ⊚ ⊚ ⊚ Invention 51 ⊚ ⊚ ⊚ Invention 52 ⊚ ◯ ◯ Invention 53 ⊚ X Δ Comp.\",\n \"Ex 54 ⊚ ⊚ ⊚ Invention 55 ⊚ ⊚ ⊚ Invention 56 ⊚ ⊚ ⊚ Invention 57 ⊚ ⊚ ⊚ Invention 58 ⊚ ⊚ ⊚ Invention 59 ⊚ ⊚ ⊚ Invention 60 ⊚ ⊚ ⊚ Invention 61 ⊚ ⊚ ⊚ Invention 62 ⊚ ⊚ ⊚ Invention 63 ⊚ ⊚ ⊚ Invention 64 ⊚ ⊚ ⊚ Invention Further, the three types of the metal sheets described above were identical with those used in Experiment Example 1 and any of the polyolefin resins used was synthesized by means of metallocene catalyst.\",\n \"Cans each of a bottomed cylindrical shape were formed using the thermoplastic resin-coated metal sheets obtained above in the same manner as in Experiment Example 1 and thermoplastic resin coating and the thermoplastic resin-coated metal sheet were evaluated in the same manner as in Experimental Example 1 and the results are shown in Table 11 and Table 12.As shown in Table 11 and Table 12, it can be seen that any of the thermoplastic resin-coated metal sheets according to the present invention is excellent in the fabricability and shows favorable impact resistance at low temperature and corrosion resistance.\",\n \"INDUSTRIAL APPLICABILITY The present invention provides a thermoplastic resin-coated metal sheet in which a coating layer of a thermoplastic resin composition comprising a blend a polyester resin and at least one of polyolefin ingredients selected from the group consisting of polyolefin resins and polyolefin elastomers is formed on at least one surface of a metal sheet, and a can formed obtained by forming the same, the thermoplastic resin-coated metal sheet described above is excellent in the workability, and shows excellent workability causing no crackings and peeling to the resin layer even after being applied with severe forming such as thickness-reducing drawing or thickness-reducing drawing and ironing.\",\n \"Further, it shows excellent impact resistance at low temperature with no occurrence of cracks in the resin layer even when applied with impact at low temperature and, further, leaches less metals when the formed can is left for a long time while being filled with acidic contents and shows excellent corrosion resistance.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471317"}}},{"rowIdx":587,"cells":{"text":{"kind":"list like","value":[["Method for Protecting a Logic or Mathematical operator Installed in an Electronic Module with a Microprocessor, as well as the Associated Embarked Electronic Module and the System","The method for protecting a logic or mathematical operator of the NOR operator type, able to be used for executing a program in a microprocessor electronic module wherein the execution of the NOR operator is replaced by the execution (CAL-XORSEC(1) of a sequence Si operations having for final result a result identical to that of the XOR function.","The sequence of operations Si composed of elementary operations with AND, OR and NOT is selected at each XOR operator from a set of eight equivalent sequences (S1 to S8) after determination CAL-NDO) of an order number NDO=1 according to the parameters of the program and/or a random parameter R supplied by a pseudo-random number generator (14)."],["1.Method for protecting a logic or mathematical operator or a similar functional circuit able to be used for executing a program in an electronic module with a processor, wherein the execution of said operator is replaced by a sequence of replacement operations whose or final result is identical to that of the function of said operator.","2.Method according to claim 1, wherein said sequence of replacement operations is selected on each call from said operator from a set of equivalent sequences.","3.Method according to claim 2, wherein the order number in said set of the selected sequence is determined according to certain parameters of the program being executed and/or a random parameter obtained from a pseudo-random number generator.","4.Method according to claim 2, wherein certain sequences include at least one non-operative instruction intended for introducing a delay time in the execution of the sequences in question, said non-operative instruction being selected from non-operative instructions in relation to the microprocessor or from instructions which are normally operative but rendered ineffective via their positions in the in the sequence of operations.","5.Method according to claim 2, wherein at least one sequence of replacement operations is made up of elementary logic operators.","6.Method according to claim 5, wherein at least one sequence of replacement operations is made up of AND, OR and NOT elementary logic operators.","7.Electronic module including at least one microprocessor and one program to be executed and including at least one logic or mathematical operator or similar functional circuit to be protected, wherein the module includes means for replacing the execution of said operator by the execution of a sequence of operations whose final result is identical to that of the function of said operator.","8.Electronic module according to claim 7, wherein it includes means for selecting said sequence of operations on each call of said operator from a set of equivalent sequences.","9.Electronic module according to claim 8, wherein it includes data processing means so as to determine the order number in said set of the selected sequence according to certain parameters of the program being executed and/or a random parameter generated by a pseudo-random numbers generator.","10.Installed system, wherein it includes an electronic module according to claim 7."],["<SOH> BACKGROUND OF THE INVENTION <EOH>The multiplication of applications in the current life of smart cards and other embarked systems and the generalization of their use in certain fields, such as those relating to bank cards, health cards or pay television, has made it necessary to introduce protection procedures, such as cryptographic procedures and/or data scrambling procedures, for example the data transiting on the internal bus of the microcontroller.","These protective procedures concern in particular the authentication of the user, the authentication of the transaction and its validity, the retaining of the confidentiality of the data and the encryption/decryption of said data.","Although the fraudulent use of smart cards is not a new phenomenon, the increase of the volume and value of smart card transactions has led to fraudulent persons using increasingly sophisticated means and methods.","In particular, attacks by short radiations targeted on the chip have the result in modifying the data and/or the codes transiting from the ROM to the microprocessor on the internal bus which results in the non-execution or irregular execution of certain portions of the code, for example the execution of inoperable instructions instead of one or several protective operations.","It has been shown that the precise marking of the position of a sensitive data operation (for example relating to encryption and/or decryption) in a program encoded in a ROM memory makes it easier to target the attack and significantly increase the nuisance power of the latter.","So as to carry out this localization, fraudulent persons use the SPA method (Simple Power analysis) which consists of recording the current consumption at certain points of the microcontroller.","The SPA method can be completed by the DPA (Differential Power Analysis) method based on the comparative analysis of the signals.","From this point of view, it happens that the execution of certain programmed operations (such as the [OR exclusive] operation frequently used in encryption/decryption) reveals a sufficiently characteristic <<signature>> to allow identification of the operation in question and accordingly its localization in the program."],["<SOH> SUMMARY OF THE INVENTION <EOH>The object of the present invention is to protect certain vulnerable operations inside a program, especially logic or mathematical operators or similar operational circuits rendering them difficult, even more difficult, to identify.","With this purpose in mind, the invention concerns a method for protecting a logic or mathematical operator or a similar operational circuit able to be used in the execution of a program in an electronic module with a microprocessor, wherein in said method, execution of said operator is replaced by the execution of a sequence of replacement operations whose final result is identical to the result of the function of said operator.","Thus, the highly characteristic signature of a complex operator, such as the XOR operator, is replaced by the signal of a sequence of operations, often less complex but not necessarily, such as a sequence of elementary operations of less characterized adjacent or identical signatures, the identification of the operator being therefore rendered more difficult.","According to one preferred implementation of the method of the invention, the sequence of replacement operations is selected on each call from the operator to be protected from a set of equivalent sequences.","Thus, the difficulty of identification is further increased by the multiple changes of the sequence of operations replacing the operator to be protected during the execution of a given program.","Advantageously, the set of sequences comprises at least four equivalent sequences and preferably eight equivalent sequences, which makes even more difficult identification of the sequence as the operator sought by fraudulent persons.","According to a first variant of the invention, the order number in its entirety of the selected sequence is determined according to certain parameters of the current program being executed and/or a random parameter advantageously obtained from a generator of pseudo-random numbers.","This scrambling mechanism for scrambling sequences proves to be extremely effective when the operator to be protected is repeated several times in a program, as for example the XOR operator in an encryption/decryption processing.","According to a second variant of the invention (not exclusive of the first variant), the sequences of operations of a given set all have the same period of execution.","Advantageously, certain sequences comprise at least one non-operative instruction intended to introduce a delay time in the execution of the sequences concerned.","In particular, the non-operative sequence is selected from non-operative instructions in relation to the microprocessor or from normally operative instructions but rendered ineffective via their positions in the sequence of operations.","This mechanism for making uniform the periods of execution of the sequences makes it more difficult to distinguish these sequences with respect to one another.","According to one first application of the method of the invention, the operator to be protected is by way of example a non-restrictive logic operator, such as the XOR (or OR exclusive) logic operator.","Advantageously, at least one sequence of replacement operations is composed from elementary logic operators.","For example, at least one sequence of operations is composed from AND, OR and NOT elementary logic operators.","As regards the processing on protecting the XOR operator, the invention also concerns the sets of sequences of replacement operations S 1 to S 8 and S′ 1 to S′ 8 as given below: S 1 =(x OR y) AND NOT (x AND y) S 2 =(x OR y) AND (NOT x OR NOT y) S 3 =NOT (NOT x AND NOT y) AND NOT (x AND y) S 4 =NOT (NOT x AND NOT y) AND (NOT x OR NOT y) S 5 =NOT(NOT(x OR y) OR (x AND y)) S 6 =NOT ((NOT x AND NOT y) OR (x AND y)) S 7 =NOT ((NOT x AND NOT y) OR NOT (NOT x OR NOT y)) S 8 =NOT (NOT (x OR y) OR NOT (NOT x OR NOT y)) and with sequences of replacement operations with the same period of execution: S′ 1 =(x OR NOP y OR y) AND NOP NOT (x AND NOP y AND y) S′ 2 =(x OR y NOP OR y) AND NOP (NOT x OR NOP NOT y) S′ 3 =NOT (NOT x AND NOP NOT y) AND NOT (x AND y AND y) S′ 4 =NOT (NOT x AND NOP NOT y) AND (NOT x OR NOT y) S′ 5 =NOT(NOT(x NOP OR y OR y) OR (x NOP AND y AND y)) S′ 6 =NOT ((NOT x AND NOT y NOP) OR (x AND y NOP AND y)) S′ 7 =NOT ((NOT x AND NOT y) OR NOT (NOT x OR NOT y)) S′ 8 =NOT (NOT (x OR y OR y) OR NOT (NOT x OR NOT y)) in which the instruction NOP corresponds to a non-operative instruction in relation to the microprocessor.","According to other applications of the method of the invention, the operator to be protected is a mathematical operator, such as a one-digit adder, an adder, a subtractor or a multiplier, or a functional circuit similar to a logic or mathematical operator, such as a combinational circuit, especially a multiplexer and/or a demultiplexer, an encoder and/or decoder, a generator and/or parity detector or a comparator.","In particular, according to a second application of the method of the invention, the operator is a mathematical operator of the “multiplication by two” obtained by shifting to the left of one bit with resetting of the least significant bit, the notation at C ANSI of this operator being written (x<<1).","As regards processing protection of the operator (x<<1), the replacement sequences S′′ 1 to S′′ 4 following equivalents: S′′ 1 =(x ADD x) S′′ 2 =(x AND FOh) ADD x ADD (x AND OFh) S′′ 3 =(NOT ((NOT x) ADD (NOT x))) SUB 1 S′′ 4 =(y ADD x) SUB (y SUB x), in which the ADD operator is the standard addition operator on one octet, the SUB operator being the standard subtraction operator on one octet and the suffix <<h>> indicates a hexadecimal value.","The invention also concerns an electronic module with a protected operator and comprising at least one microprocessor and a program to be executed comprising at least one logic or mathematical operator or a similar operational circuit to be protected, wherein the module comprises means for replacing the execution of the operator by the execution of a sequence of operations whose final result is identical to the result of the function of the operator.","Advantageously, the electronic module of the invention comprises means for selecting the sequence of operations on each call of the operator from a set of equivalent sequences.","According to one extremely advantageous variant, the module comprises computer processing means for determining the order number concerning the sequence selected according to certain parameters of the program being executed and/or a random parameter generated by a generator of pseudo-random numbers.","The invention also concerns an electronic module with a protected operator and comprising at least one microprocessor and a program to be executed comprising at least one logic or mathematical operator or a similar circuit to be protected, wherein it comprises hardware and/or software means for implementing the method of the invention shown above.","The invention also concerns an embarked system or a microcircuit card comprising an electronic module with a protected operator as previously defined in its various variants."],["FIELD OF THE INVENTION The present invention concerns the protection of embarked systems comprising one or several electronic modules incorporating at least one microprocessor, a ROM type memory containing at least one program to be executed and input/output means for communicating with the outside world.","Certain modules also comprise other accessory circuits, such as RAM and EEPROM memories for more elaborate applications and are also known under the name of microcontrollers.","These modules are most often embodied in the form of a monolithic electronic microcircuit or chip.","These modules can be used firstly as such once physically protected and mounted for example on a portable smart card object, microcircuit card or similar element able to used in various domains, especially those relating to bank and/or commercial cards, mobile radiotelephony, paying television, health and transport, and secondly in other embarked systems ranging from relatively simple systems, such as sensors and circuit breakers, to more complex systems, such as industrial controllers, multiswitches and other sub-units and electrically controlled devices up to complete industrial systems electronically controlled.","BACKGROUND OF THE INVENTION The multiplication of applications in the current life of smart cards and other embarked systems and the generalization of their use in certain fields, such as those relating to bank cards, health cards or pay television, has made it necessary to introduce protection procedures, such as cryptographic procedures and/or data scrambling procedures, for example the data transiting on the internal bus of the microcontroller.","These protective procedures concern in particular the authentication of the user, the authentication of the transaction and its validity, the retaining of the confidentiality of the data and the encryption/decryption of said data.","Although the fraudulent use of smart cards is not a new phenomenon, the increase of the volume and value of smart card transactions has led to fraudulent persons using increasingly sophisticated means and methods.","In particular, attacks by short radiations targeted on the chip have the result in modifying the data and/or the codes transiting from the ROM to the microprocessor on the internal bus which results in the non-execution or irregular execution of certain portions of the code, for example the execution of inoperable instructions instead of one or several protective operations.","It has been shown that the precise marking of the position of a sensitive data operation (for example relating to encryption and/or decryption) in a program encoded in a ROM memory makes it easier to target the attack and significantly increase the nuisance power of the latter.","So as to carry out this localization, fraudulent persons use the SPA method (Simple Power analysis) which consists of recording the current consumption at certain points of the microcontroller.","The SPA method can be completed by the DPA (Differential Power Analysis) method based on the comparative analysis of the signals.","From this point of view, it happens that the execution of certain programmed operations (such as the [OR exclusive] operation frequently used in encryption/decryption) reveals a sufficiently characteristic <<signature>> to allow identification of the operation in question and accordingly its localization in the program.","SUMMARY OF THE INVENTION The object of the present invention is to protect certain vulnerable operations inside a program, especially logic or mathematical operators or similar operational circuits rendering them difficult, even more difficult, to identify.","With this purpose in mind, the invention concerns a method for protecting a logic or mathematical operator or a similar operational circuit able to be used in the execution of a program in an electronic module with a microprocessor, wherein in said method, execution of said operator is replaced by the execution of a sequence of replacement operations whose final result is identical to the result of the function of said operator.","Thus, the highly characteristic signature of a complex operator, such as the XOR operator, is replaced by the signal of a sequence of operations, often less complex but not necessarily, such as a sequence of elementary operations of less characterized adjacent or identical signatures, the identification of the operator being therefore rendered more difficult.","According to one preferred implementation of the method of the invention, the sequence of replacement operations is selected on each call from the operator to be protected from a set of equivalent sequences.","Thus, the difficulty of identification is further increased by the multiple changes of the sequence of operations replacing the operator to be protected during the execution of a given program.","Advantageously, the set of sequences comprises at least four equivalent sequences and preferably eight equivalent sequences, which makes even more difficult identification of the sequence as the operator sought by fraudulent persons.","According to a first variant of the invention, the order number in its entirety of the selected sequence is determined according to certain parameters of the current program being executed and/or a random parameter advantageously obtained from a generator of pseudo-random numbers.","This scrambling mechanism for scrambling sequences proves to be extremely effective when the operator to be protected is repeated several times in a program, as for example the XOR operator in an encryption/decryption processing.","According to a second variant of the invention (not exclusive of the first variant), the sequences of operations of a given set all have the same period of execution.","Advantageously, certain sequences comprise at least one non-operative instruction intended to introduce a delay time in the execution of the sequences concerned.","In particular, the non-operative sequence is selected from non-operative instructions in relation to the microprocessor or from normally operative instructions but rendered ineffective via their positions in the sequence of operations.","This mechanism for making uniform the periods of execution of the sequences makes it more difficult to distinguish these sequences with respect to one another.","According to one first application of the method of the invention, the operator to be protected is by way of example a non-restrictive logic operator, such as the XOR (or OR exclusive) logic operator.","Advantageously, at least one sequence of replacement operations is composed from elementary logic operators.","For example, at least one sequence of operations is composed from AND, OR and NOT elementary logic operators.","As regards the processing on protecting the XOR operator, the invention also concerns the sets of sequences of replacement operations S1 to S8 and S′1 to S′8 as given below: S1=(x OR y) AND NOT (x AND y) S2=(x OR y) AND (NOT x OR NOT y) S3=NOT (NOT x AND NOT y) AND NOT (x AND y) S4=NOT (NOT x AND NOT y) AND (NOT x OR NOT y) S5=NOT(NOT(x OR y) OR (x AND y)) S6=NOT ((NOT x AND NOT y) OR (x AND y)) S7=NOT ((NOT x AND NOT y) OR NOT (NOT x OR NOT y)) S8=NOT (NOT (x OR y) OR NOT (NOT x OR NOT y)) and with sequences of replacement operations with the same period of execution: S′1=(x OR NOP y OR y) AND NOP NOT (x AND NOP y AND y) S′2=(x OR y NOP OR y) AND NOP (NOT x OR NOP NOT y) S′3=NOT (NOT x AND NOP NOT y) AND NOT (x AND y AND y) S′4=NOT (NOT x AND NOP NOT y) AND (NOT x OR NOT y) S′5=NOT(NOT(x NOP OR y OR y) OR (x NOP AND y AND y)) S′6=NOT ((NOT x AND NOT y NOP) OR (x AND y NOP AND y)) S′7=NOT ((NOT x AND NOT y) OR NOT (NOT x OR NOT y)) S′8=NOT (NOT (x OR y OR y) OR NOT (NOT x OR NOT y)) in which the instruction NOP corresponds to a non-operative instruction in relation to the microprocessor.","According to other applications of the method of the invention, the operator to be protected is a mathematical operator, such as a one-digit adder, an adder, a subtractor or a multiplier, or a functional circuit similar to a logic or mathematical operator, such as a combinational circuit, especially a multiplexer and/or a demultiplexer, an encoder and/or decoder, a generator and/or parity detector or a comparator.","In particular, according to a second application of the method of the invention, the operator is a mathematical operator of the “multiplication by two” obtained by shifting to the left of one bit with resetting of the least significant bit, the notation at C ANSI of this operator being written (x<<1).","As regards processing protection of the operator (x<<1), the replacement sequences S′′1 to S′′4 following equivalents: S′′1=(x ADD x) S′′2=(x AND FOh) ADD x ADD (x AND OFh) S′′3=(NOT ((NOT x) ADD (NOT x))) SUB 1 S′′4=(y ADD x) SUB (y SUB x), in which the ADD operator is the standard addition operator on one octet, the SUB operator being the standard subtraction operator on one octet and the suffix <<h>> indicates a hexadecimal value.","The invention also concerns an electronic module with a protected operator and comprising at least one microprocessor and a program to be executed comprising at least one logic or mathematical operator or a similar operational circuit to be protected, wherein the module comprises means for replacing the execution of the operator by the execution of a sequence of operations whose final result is identical to the result of the function of the operator.","Advantageously, the electronic module of the invention comprises means for selecting the sequence of operations on each call of the operator from a set of equivalent sequences.","According to one extremely advantageous variant, the module comprises computer processing means for determining the order number concerning the sequence selected according to certain parameters of the program being executed and/or a random parameter generated by a generator of pseudo-random numbers.","The invention also concerns an electronic module with a protected operator and comprising at least one microprocessor and a program to be executed comprising at least one logic or mathematical operator or a similar circuit to be protected, wherein it comprises hardware and/or software means for implementing the method of the invention shown above.","The invention also concerns an embarked system or a microcircuit card comprising an electronic module with a protected operator as previously defined in its various variants.","BRIEF DESCRIPTION OF THE DRAWINGS Other aims, advantages and characteristics of the invention shall appear on a reading of the following description of the implementation of the method of the invention applied to the protection of the XOR operator and an embodiment of an electronic module with a microprocessor according to the invention and given by way of non-restrictive example with reference to the accompanying drawings on which: FIG.","1 shows a diagrammatic representation of an embodiment of an electronic module with a microprocessor and a protected XOR operator according to the invention, and FIG.","2 shows a diagrammatic representation of the equivalent execution of the XOR operator implementing the method of the invention in the module of FIG.","1.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The monolithic electronic module 10 with a microprocessor shown on FIG.","1 according to the present invention and described by way of non-restrictive example generally comprises one microprocessor or central unit CPU 11 connected bidirectionally by an internal bus 12 to a live RAM memory 14, a dead ROM memory 16, an EEPROM memory 18 and an I/O input/output interface 20.The module 10 also comprises a Timer 22 with automatic resetting (in an optional variant) and a generator of pseudo-random numbers GNPA 24 connected to the internal bus 12.Application programs are installed at ROM 16, such as applications of bank card transactions or medical card applications which for reasons of confidentiality and protection comprise encryption/decryption, operator authentication or transaction validation sub-program in which the XOR operator is frequently present, especially for carrying out comparisons octet by octet.","As regards execution of the XOR operator, this operator most commonly used generally forms part of the set of arithmetic instructions with two operands OP1 et OP2) of the central unit CPU or microprocessor 11.In the embodiment described here, the means for implementing the method for protecting the XOR operator are mainly software items in the form of a protected XOR calculation routine (or XORSEC routine) diagrammatically shown on FIG.","2.Thus, on each call from the XOR instruction OP1, OP2) the program is rerouted with the two operands OP1 and OP2 towards the XORSEC routine which shall be executed in place of the XOR instruction, it being understood that the execution of the XORSEC routine carries out a function equivalent to that of the original XOR instruction.","According to the main characteristic of the invention, the execution of the XOR instruction is replaced in the XORSEC routine by the execution of a sequence of operations, namely of the type but not exclusively of operations with a degree of less complexity, such as elementary operations, whose final result is identical to the result of the function of the XOR operator (condition easily verified amongst others by identical outlet tables).","By way of non-restrictive examples, a set of eight sequences S1 to S8 equivalent to the XOR instruction is given below: S1=(x OR y) AND NOT (x AND y) S2=(x OR y) AND (NOT x OR NOT y) S3=NOT (NOT x AND NOT y) AND NOT (x AND y) S4=NOT (NOT x AND NOT y) AND (NOT x OR NOT y) S5=NOT(NOT(x OR y) OR (x AND y)) S6=NOT ((NOT x AND NOT y) OR (x AND y)) S7=NOT ((NOT x AND NOT y) OR NOT (NOT x OR NOT y)) S8=NOT (NOT (x OR y) OR NOT (NOT x OR NOT y)) It shall be observed that all these sequences S1 to S8 are based on the use of at least two of three AND, NOT and OR logic elementary instructions and have the same truth table outlet as for the XOR instruction.","By using the traditional presentation of truth tables with two inlets x, y and one outlet s, it is possible to write for the XOR, AND and OR operators and for the sequence S5 (selected by way of non-restrictive example) the following four truth tables: XOR AND OR x y s(XOR) x y s(AND) x y s(OR) 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 0 1 1 1 0 1 1 0 0 1 0 1 1 1 0 1 1 1 1 1 1 and for S5=NOT(NOT(x OR y) OR (x AND y)) with A=(x OR y), B=NOT A, C=(x AND y) D=(NOT(x OR y) OR (x AND y)=B OR C, and s(S5)=E=NOT D x y A B C D E 0 0 0 1 0 1 0 0 1 1 0 0 0 1 1 0 1 0 0 0 1 1 1 1 0 1 1 0 It is thus verified that s(S5)=E is identical to s(XOR).","It shall be observed that the sequence automatically selected to replace the XOR operator is made up of five elementary operations whose signature shall be significantly different from the XOR operator.","Thus, it is possible to embody the simplest variant for implementing the method of the invention.","According to one optional, but extremely advantageous, characteristic of the invention used in the embodiment described here, the sequence of replacement operations, namely the set ES constituted by the eight sequences S1 to S8 given above.","Thus, the difficulty of identification of the XOR operator is further increased by the multiple changes of the sequence of operations replacing the XOR operator during execution of the program, the sequences S1 to S8 able to be used having all different signatures.","According to another optional characteristic of the invention, but also extremely advantageous, used in the embodiment described here, the order number NDO=i (i ranging from 1 to 8) in its entirety (S1 to S8) of the sequence S1 selected to be executed is determined according to certain parameters of the program currently being executed and/or of a random parameter.","Advantageously, said random parameter is obtained from a pseudo-random numbers generator.","This mechanism for scrambling sequences rendering random the sequence effectively selected to replace the XOR operator on each call proves to be extremely effective, especially in an encryption/decryption processing when the XOR operator is called several times in the program.","As shown on FIG.","2, the XORSEC routine comprises four main processing phases, namely in the order of execution: a start or initialization phase IN-XORSEC with in particular the storing of the program counter values, and the operands OP1 and OP2, a phase CAL-NDO for determining by calculation the order number NDO=i (i ranging from 1 to 8) of the sequence of operations Si to be executed to replace the instruction XOR with branching towards the corresponding sub-routine CAL-XORSEC(i) and present in the set CAL-XORSEC of eight sub-routines CAL-XORSEC(1) to CAL-XORSEC(8), a phase CAL-XORSEC(i) for executing the sub-routine CAL-XORSEC(i) by logic calculation with the operands OP1 and OP2 of the sequence of operations Si selected by the phase CAL_NDO, a phase OUT-XORSEC for return to the main program so as to resume its execution and transfer of the results of the CAL-XORSEC(i) equivalent to the calculation of an XOR between the two operands OP1 and OP2.It shall be noted that for the phase CAL-NDO, the random generator GNPA 24 supplies on demand one random octet R used as a calculation parameter alone or with other parameters extracted from the values of the operands OP1 and OP2, the final result of the calculation being one octet F(R).","By using for example an operation of the type NOD=i=F(R) AND 07 h, the three less significant bits are extracted from this octet so as to obtain the binary value of NOD=i (from 000 to 111 namely 00h to 07h), order number of the sequence Si to be executed.","It is to be noted that the value of the order number is a sensitive data element of the algorithm in question.","Finally, the routine XORSEC exhibits a further improved variant as regards the difficulty of identification in which the series of replacement sequences is constituted by sequences with a given period of execution (and owing to this more difficult to distinguish).","So as to achieve this, certain sequences comprise at least one non-operative instruction designed to introduce a delay time in the execution of the sequences concerned.","In particular, the non-operative instruction is selected from non-operative instructions in relation to the microprocessor or from normally operative instructions but rendered ineffective via their positions in the sequence of operations.","If it is assumed that the elementary operations AND, OR and NOT have approximately equal periods of execution (for example 4 cycle times of the clock of the central unit CPU 11), as for the non-operative <<blank>> operation NOP, the set ES of the sequences S1 to S8 is modified into a new set of sequences made uniform to 9 operations ES′ being written by way of non-restrictive example as follows (and in which the added operations appear in thick type): S′1=(x OR NOP y OR y) AND NOP NOT (x AND NOP y AND y) S′2=(x OR y NOP OR y) AND NOP (NOT x OR NOP NOT y) S′3=NOT (NOT x AND NOP NOT y) AND NOT (x AND y AND y) S′4=NOT (NOT x AND NOP NOT y) AND (NOT x OR NOT y) S′5=NOT(NOT(x NOP OR y OR y) OR (x NOP AND y AND y)) S′6=NOT ((NOT x AND NOT y NOP) OR (x AND y NOP AND y)) S′7=NOT ((NOT x AND NOT y) OR NOT (NOT x OR NOT y)) S′8=NOT (NOT (x OR y OR y) OR NOT (NOT x OR NOT y)) For example, for the sequence S′5: S′5=NOT(NOT(x NOP OR y OR y) OR (x NOP AND y AND y)) with x′=x NOP, A=(x′ OR y), A′=A OR y , B=NOT A′, C=(x′ AND y), C′=C AND y, D=B OR C′ and s(S′5)=E=NOT D It is possible to write the truth table of the sequence S′5 which also verifies s(S′5)=s(XOR) S′5 x y x′ A A′ B C C′ D E 0 0 0 0 0 1 0 0 1 0 0 1 0 1 1 0 0 0 0 1 1 0 1 1 1 0 0 0 0 1 1 1 1 1 1 0 1 1 1 0 It shall be observed that for sequences with nine elementary operations, the machine time remains quite reasonable.","The invention is not limited to its application concerning the protection of logic operators, but is also applicable to protection of mathematical operators, such as one-digit adders, adders, subtractors or multipliers or functional circuits similar to logic or mathematical operators, such as combinatory circuits, especially multiplexers and/or demultiplexers, coders and/or decoders, generators and/or parity detectors or comparators.","For example, according to another application of the method of the invention, the operator to be protected is the mathematical operator of the <<multiplication by two>> obtained by shifting left one bit with resetting of the low order bit.","In notation C ANSI, this operator is also denoted (x<<1).","Thus, on each call from the operator (x<<1), its execution shall be replaced by the execution of a sequence of operations selected from the following equivalent sequences: S′′1=(x ADD x) S′′2=(x AND FOh) ADD x ADD (x AND OFh) S′′3=(NOT ((NOT x) ADD (NOT x))) SUB 1 S′′4=(y ADD x) SUB (y SUB x), in which the operator ADD is the standard addition operator on one octet, the instruction SUB is the standard subtraction operator on one octet and the suffix <<h>> indicates a hexadecimal value.","Generally speaking, the choice of the equivalent sequence S′′i and its implementation are embodied similarly, sometimes identically, to what has been described in details as previously for the XOR operator.","It shall also be noted that, without departing from the context of the invention, the smart card accommodating the electronic module with a protected operator according to the invention can be replaced by any other embarked system.","One embodiment of the invention is shown below concerning its implementation using an electronic module.","In the method for protecting a logic or mathematical operator, or a similar functional circuit able to be used in the execution of a program in the electronic module including a microprocessor and a memory, the execution of said operator by the microprocessor is replaced by the execution of a sequence of replacement operations whose final result is identical to that of the function of said operator, said result being stored in the memory.","The electronic module with a protected operator and comprising at least one microprocessor and a memory storing a program to be executed comprising at least one logic or mathematical operator or similar functional circuit to be protected wherein it comprises means to replace execution of the operator with the aid of the microprocessor by the execution of a sequence of operations whose final result is identical to that of the function de the operator, said result being stored in the memory."]],"string":"[\n [\n \"Method for Protecting a Logic or Mathematical operator Installed in an Electronic Module with a Microprocessor, as well as the Associated Embarked Electronic Module and the System\",\n \"The method for protecting a logic or mathematical operator of the NOR operator type, able to be used for executing a program in a microprocessor electronic module wherein the execution of the NOR operator is replaced by the execution (CAL-XORSEC(1) of a sequence Si operations having for final result a result identical to that of the XOR function.\",\n \"The sequence of operations Si composed of elementary operations with AND, OR and NOT is selected at each XOR operator from a set of eight equivalent sequences (S1 to S8) after determination CAL-NDO) of an order number NDO=1 according to the parameters of the program and/or a random parameter R supplied by a pseudo-random number generator (14).\"\n ],\n [\n \"1.Method for protecting a logic or mathematical operator or a similar functional circuit able to be used for executing a program in an electronic module with a processor, wherein the execution of said operator is replaced by a sequence of replacement operations whose or final result is identical to that of the function of said operator.\",\n \"2.Method according to claim 1, wherein said sequence of replacement operations is selected on each call from said operator from a set of equivalent sequences.\",\n \"3.Method according to claim 2, wherein the order number in said set of the selected sequence is determined according to certain parameters of the program being executed and/or a random parameter obtained from a pseudo-random number generator.\",\n \"4.Method according to claim 2, wherein certain sequences include at least one non-operative instruction intended for introducing a delay time in the execution of the sequences in question, said non-operative instruction being selected from non-operative instructions in relation to the microprocessor or from instructions which are normally operative but rendered ineffective via their positions in the in the sequence of operations.\",\n \"5.Method according to claim 2, wherein at least one sequence of replacement operations is made up of elementary logic operators.\",\n \"6.Method according to claim 5, wherein at least one sequence of replacement operations is made up of AND, OR and NOT elementary logic operators.\",\n \"7.Electronic module including at least one microprocessor and one program to be executed and including at least one logic or mathematical operator or similar functional circuit to be protected, wherein the module includes means for replacing the execution of said operator by the execution of a sequence of operations whose final result is identical to that of the function of said operator.\",\n \"8.Electronic module according to claim 7, wherein it includes means for selecting said sequence of operations on each call of said operator from a set of equivalent sequences.\",\n \"9.Electronic module according to claim 8, wherein it includes data processing means so as to determine the order number in said set of the selected sequence according to certain parameters of the program being executed and/or a random parameter generated by a pseudo-random numbers generator.\",\n \"10.Installed system, wherein it includes an electronic module according to claim 7.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>The multiplication of applications in the current life of smart cards and other embarked systems and the generalization of their use in certain fields, such as those relating to bank cards, health cards or pay television, has made it necessary to introduce protection procedures, such as cryptographic procedures and/or data scrambling procedures, for example the data transiting on the internal bus of the microcontroller.\",\n \"These protective procedures concern in particular the authentication of the user, the authentication of the transaction and its validity, the retaining of the confidentiality of the data and the encryption/decryption of said data.\",\n \"Although the fraudulent use of smart cards is not a new phenomenon, the increase of the volume and value of smart card transactions has led to fraudulent persons using increasingly sophisticated means and methods.\",\n \"In particular, attacks by short radiations targeted on the chip have the result in modifying the data and/or the codes transiting from the ROM to the microprocessor on the internal bus which results in the non-execution or irregular execution of certain portions of the code, for example the execution of inoperable instructions instead of one or several protective operations.\",\n \"It has been shown that the precise marking of the position of a sensitive data operation (for example relating to encryption and/or decryption) in a program encoded in a ROM memory makes it easier to target the attack and significantly increase the nuisance power of the latter.\",\n \"So as to carry out this localization, fraudulent persons use the SPA method (Simple Power analysis) which consists of recording the current consumption at certain points of the microcontroller.\",\n \"The SPA method can be completed by the DPA (Differential Power Analysis) method based on the comparative analysis of the signals.\",\n \"From this point of view, it happens that the execution of certain programmed operations (such as the [OR exclusive] operation frequently used in encryption/decryption) reveals a sufficiently characteristic <<signature>> to allow identification of the operation in question and accordingly its localization in the program.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The object of the present invention is to protect certain vulnerable operations inside a program, especially logic or mathematical operators or similar operational circuits rendering them difficult, even more difficult, to identify.\",\n \"With this purpose in mind, the invention concerns a method for protecting a logic or mathematical operator or a similar operational circuit able to be used in the execution of a program in an electronic module with a microprocessor, wherein in said method, execution of said operator is replaced by the execution of a sequence of replacement operations whose final result is identical to the result of the function of said operator.\",\n \"Thus, the highly characteristic signature of a complex operator, such as the XOR operator, is replaced by the signal of a sequence of operations, often less complex but not necessarily, such as a sequence of elementary operations of less characterized adjacent or identical signatures, the identification of the operator being therefore rendered more difficult.\",\n \"According to one preferred implementation of the method of the invention, the sequence of replacement operations is selected on each call from the operator to be protected from a set of equivalent sequences.\",\n \"Thus, the difficulty of identification is further increased by the multiple changes of the sequence of operations replacing the operator to be protected during the execution of a given program.\",\n \"Advantageously, the set of sequences comprises at least four equivalent sequences and preferably eight equivalent sequences, which makes even more difficult identification of the sequence as the operator sought by fraudulent persons.\",\n \"According to a first variant of the invention, the order number in its entirety of the selected sequence is determined according to certain parameters of the current program being executed and/or a random parameter advantageously obtained from a generator of pseudo-random numbers.\",\n \"This scrambling mechanism for scrambling sequences proves to be extremely effective when the operator to be protected is repeated several times in a program, as for example the XOR operator in an encryption/decryption processing.\",\n \"According to a second variant of the invention (not exclusive of the first variant), the sequences of operations of a given set all have the same period of execution.\",\n \"Advantageously, certain sequences comprise at least one non-operative instruction intended to introduce a delay time in the execution of the sequences concerned.\",\n \"In particular, the non-operative sequence is selected from non-operative instructions in relation to the microprocessor or from normally operative instructions but rendered ineffective via their positions in the sequence of operations.\",\n \"This mechanism for making uniform the periods of execution of the sequences makes it more difficult to distinguish these sequences with respect to one another.\",\n \"According to one first application of the method of the invention, the operator to be protected is by way of example a non-restrictive logic operator, such as the XOR (or OR exclusive) logic operator.\",\n \"Advantageously, at least one sequence of replacement operations is composed from elementary logic operators.\",\n \"For example, at least one sequence of operations is composed from AND, OR and NOT elementary logic operators.\",\n \"As regards the processing on protecting the XOR operator, the invention also concerns the sets of sequences of replacement operations S 1 to S 8 and S′ 1 to S′ 8 as given below: S 1 =(x OR y) AND NOT (x AND y) S 2 =(x OR y) AND (NOT x OR NOT y) S 3 =NOT (NOT x AND NOT y) AND NOT (x AND y) S 4 =NOT (NOT x AND NOT y) AND (NOT x OR NOT y) S 5 =NOT(NOT(x OR y) OR (x AND y)) S 6 =NOT ((NOT x AND NOT y) OR (x AND y)) S 7 =NOT ((NOT x AND NOT y) OR NOT (NOT x OR NOT y)) S 8 =NOT (NOT (x OR y) OR NOT (NOT x OR NOT y)) and with sequences of replacement operations with the same period of execution: S′ 1 =(x OR NOP y OR y) AND NOP NOT (x AND NOP y AND y) S′ 2 =(x OR y NOP OR y) AND NOP (NOT x OR NOP NOT y) S′ 3 =NOT (NOT x AND NOP NOT y) AND NOT (x AND y AND y) S′ 4 =NOT (NOT x AND NOP NOT y) AND (NOT x OR NOT y) S′ 5 =NOT(NOT(x NOP OR y OR y) OR (x NOP AND y AND y)) S′ 6 =NOT ((NOT x AND NOT y NOP) OR (x AND y NOP AND y)) S′ 7 =NOT ((NOT x AND NOT y) OR NOT (NOT x OR NOT y)) S′ 8 =NOT (NOT (x OR y OR y) OR NOT (NOT x OR NOT y)) in which the instruction NOP corresponds to a non-operative instruction in relation to the microprocessor.\",\n \"According to other applications of the method of the invention, the operator to be protected is a mathematical operator, such as a one-digit adder, an adder, a subtractor or a multiplier, or a functional circuit similar to a logic or mathematical operator, such as a combinational circuit, especially a multiplexer and/or a demultiplexer, an encoder and/or decoder, a generator and/or parity detector or a comparator.\",\n \"In particular, according to a second application of the method of the invention, the operator is a mathematical operator of the “multiplication by two” obtained by shifting to the left of one bit with resetting of the least significant bit, the notation at C ANSI of this operator being written (x<<1).\",\n \"As regards processing protection of the operator (x<<1), the replacement sequences S′′ 1 to S′′ 4 following equivalents: S′′ 1 =(x ADD x) S′′ 2 =(x AND FOh) ADD x ADD (x AND OFh) S′′ 3 =(NOT ((NOT x) ADD (NOT x))) SUB 1 S′′ 4 =(y ADD x) SUB (y SUB x), in which the ADD operator is the standard addition operator on one octet, the SUB operator being the standard subtraction operator on one octet and the suffix <<h>> indicates a hexadecimal value.\",\n \"The invention also concerns an electronic module with a protected operator and comprising at least one microprocessor and a program to be executed comprising at least one logic or mathematical operator or a similar operational circuit to be protected, wherein the module comprises means for replacing the execution of the operator by the execution of a sequence of operations whose final result is identical to the result of the function of the operator.\",\n \"Advantageously, the electronic module of the invention comprises means for selecting the sequence of operations on each call of the operator from a set of equivalent sequences.\",\n \"According to one extremely advantageous variant, the module comprises computer processing means for determining the order number concerning the sequence selected according to certain parameters of the program being executed and/or a random parameter generated by a generator of pseudo-random numbers.\",\n \"The invention also concerns an electronic module with a protected operator and comprising at least one microprocessor and a program to be executed comprising at least one logic or mathematical operator or a similar circuit to be protected, wherein it comprises hardware and/or software means for implementing the method of the invention shown above.\",\n \"The invention also concerns an embarked system or a microcircuit card comprising an electronic module with a protected operator as previously defined in its various variants.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention concerns the protection of embarked systems comprising one or several electronic modules incorporating at least one microprocessor, a ROM type memory containing at least one program to be executed and input/output means for communicating with the outside world.\",\n \"Certain modules also comprise other accessory circuits, such as RAM and EEPROM memories for more elaborate applications and are also known under the name of microcontrollers.\",\n \"These modules are most often embodied in the form of a monolithic electronic microcircuit or chip.\",\n \"These modules can be used firstly as such once physically protected and mounted for example on a portable smart card object, microcircuit card or similar element able to used in various domains, especially those relating to bank and/or commercial cards, mobile radiotelephony, paying television, health and transport, and secondly in other embarked systems ranging from relatively simple systems, such as sensors and circuit breakers, to more complex systems, such as industrial controllers, multiswitches and other sub-units and electrically controlled devices up to complete industrial systems electronically controlled.\",\n \"BACKGROUND OF THE INVENTION The multiplication of applications in the current life of smart cards and other embarked systems and the generalization of their use in certain fields, such as those relating to bank cards, health cards or pay television, has made it necessary to introduce protection procedures, such as cryptographic procedures and/or data scrambling procedures, for example the data transiting on the internal bus of the microcontroller.\",\n \"These protective procedures concern in particular the authentication of the user, the authentication of the transaction and its validity, the retaining of the confidentiality of the data and the encryption/decryption of said data.\",\n \"Although the fraudulent use of smart cards is not a new phenomenon, the increase of the volume and value of smart card transactions has led to fraudulent persons using increasingly sophisticated means and methods.\",\n \"In particular, attacks by short radiations targeted on the chip have the result in modifying the data and/or the codes transiting from the ROM to the microprocessor on the internal bus which results in the non-execution or irregular execution of certain portions of the code, for example the execution of inoperable instructions instead of one or several protective operations.\",\n \"It has been shown that the precise marking of the position of a sensitive data operation (for example relating to encryption and/or decryption) in a program encoded in a ROM memory makes it easier to target the attack and significantly increase the nuisance power of the latter.\",\n \"So as to carry out this localization, fraudulent persons use the SPA method (Simple Power analysis) which consists of recording the current consumption at certain points of the microcontroller.\",\n \"The SPA method can be completed by the DPA (Differential Power Analysis) method based on the comparative analysis of the signals.\",\n \"From this point of view, it happens that the execution of certain programmed operations (such as the [OR exclusive] operation frequently used in encryption/decryption) reveals a sufficiently characteristic <<signature>> to allow identification of the operation in question and accordingly its localization in the program.\",\n \"SUMMARY OF THE INVENTION The object of the present invention is to protect certain vulnerable operations inside a program, especially logic or mathematical operators or similar operational circuits rendering them difficult, even more difficult, to identify.\",\n \"With this purpose in mind, the invention concerns a method for protecting a logic or mathematical operator or a similar operational circuit able to be used in the execution of a program in an electronic module with a microprocessor, wherein in said method, execution of said operator is replaced by the execution of a sequence of replacement operations whose final result is identical to the result of the function of said operator.\",\n \"Thus, the highly characteristic signature of a complex operator, such as the XOR operator, is replaced by the signal of a sequence of operations, often less complex but not necessarily, such as a sequence of elementary operations of less characterized adjacent or identical signatures, the identification of the operator being therefore rendered more difficult.\",\n \"According to one preferred implementation of the method of the invention, the sequence of replacement operations is selected on each call from the operator to be protected from a set of equivalent sequences.\",\n \"Thus, the difficulty of identification is further increased by the multiple changes of the sequence of operations replacing the operator to be protected during the execution of a given program.\",\n \"Advantageously, the set of sequences comprises at least four equivalent sequences and preferably eight equivalent sequences, which makes even more difficult identification of the sequence as the operator sought by fraudulent persons.\",\n \"According to a first variant of the invention, the order number in its entirety of the selected sequence is determined according to certain parameters of the current program being executed and/or a random parameter advantageously obtained from a generator of pseudo-random numbers.\",\n \"This scrambling mechanism for scrambling sequences proves to be extremely effective when the operator to be protected is repeated several times in a program, as for example the XOR operator in an encryption/decryption processing.\",\n \"According to a second variant of the invention (not exclusive of the first variant), the sequences of operations of a given set all have the same period of execution.\",\n \"Advantageously, certain sequences comprise at least one non-operative instruction intended to introduce a delay time in the execution of the sequences concerned.\",\n \"In particular, the non-operative sequence is selected from non-operative instructions in relation to the microprocessor or from normally operative instructions but rendered ineffective via their positions in the sequence of operations.\",\n \"This mechanism for making uniform the periods of execution of the sequences makes it more difficult to distinguish these sequences with respect to one another.\",\n \"According to one first application of the method of the invention, the operator to be protected is by way of example a non-restrictive logic operator, such as the XOR (or OR exclusive) logic operator.\",\n \"Advantageously, at least one sequence of replacement operations is composed from elementary logic operators.\",\n \"For example, at least one sequence of operations is composed from AND, OR and NOT elementary logic operators.\",\n \"As regards the processing on protecting the XOR operator, the invention also concerns the sets of sequences of replacement operations S1 to S8 and S′1 to S′8 as given below: S1=(x OR y) AND NOT (x AND y) S2=(x OR y) AND (NOT x OR NOT y) S3=NOT (NOT x AND NOT y) AND NOT (x AND y) S4=NOT (NOT x AND NOT y) AND (NOT x OR NOT y) S5=NOT(NOT(x OR y) OR (x AND y)) S6=NOT ((NOT x AND NOT y) OR (x AND y)) S7=NOT ((NOT x AND NOT y) OR NOT (NOT x OR NOT y)) S8=NOT (NOT (x OR y) OR NOT (NOT x OR NOT y)) and with sequences of replacement operations with the same period of execution: S′1=(x OR NOP y OR y) AND NOP NOT (x AND NOP y AND y) S′2=(x OR y NOP OR y) AND NOP (NOT x OR NOP NOT y) S′3=NOT (NOT x AND NOP NOT y) AND NOT (x AND y AND y) S′4=NOT (NOT x AND NOP NOT y) AND (NOT x OR NOT y) S′5=NOT(NOT(x NOP OR y OR y) OR (x NOP AND y AND y)) S′6=NOT ((NOT x AND NOT y NOP) OR (x AND y NOP AND y)) S′7=NOT ((NOT x AND NOT y) OR NOT (NOT x OR NOT y)) S′8=NOT (NOT (x OR y OR y) OR NOT (NOT x OR NOT y)) in which the instruction NOP corresponds to a non-operative instruction in relation to the microprocessor.\",\n \"According to other applications of the method of the invention, the operator to be protected is a mathematical operator, such as a one-digit adder, an adder, a subtractor or a multiplier, or a functional circuit similar to a logic or mathematical operator, such as a combinational circuit, especially a multiplexer and/or a demultiplexer, an encoder and/or decoder, a generator and/or parity detector or a comparator.\",\n \"In particular, according to a second application of the method of the invention, the operator is a mathematical operator of the “multiplication by two” obtained by shifting to the left of one bit with resetting of the least significant bit, the notation at C ANSI of this operator being written (x<<1).\",\n \"As regards processing protection of the operator (x<<1), the replacement sequences S′′1 to S′′4 following equivalents: S′′1=(x ADD x) S′′2=(x AND FOh) ADD x ADD (x AND OFh) S′′3=(NOT ((NOT x) ADD (NOT x))) SUB 1 S′′4=(y ADD x) SUB (y SUB x), in which the ADD operator is the standard addition operator on one octet, the SUB operator being the standard subtraction operator on one octet and the suffix <<h>> indicates a hexadecimal value.\",\n \"The invention also concerns an electronic module with a protected operator and comprising at least one microprocessor and a program to be executed comprising at least one logic or mathematical operator or a similar operational circuit to be protected, wherein the module comprises means for replacing the execution of the operator by the execution of a sequence of operations whose final result is identical to the result of the function of the operator.\",\n \"Advantageously, the electronic module of the invention comprises means for selecting the sequence of operations on each call of the operator from a set of equivalent sequences.\",\n \"According to one extremely advantageous variant, the module comprises computer processing means for determining the order number concerning the sequence selected according to certain parameters of the program being executed and/or a random parameter generated by a generator of pseudo-random numbers.\",\n \"The invention also concerns an electronic module with a protected operator and comprising at least one microprocessor and a program to be executed comprising at least one logic or mathematical operator or a similar circuit to be protected, wherein it comprises hardware and/or software means for implementing the method of the invention shown above.\",\n \"The invention also concerns an embarked system or a microcircuit card comprising an electronic module with a protected operator as previously defined in its various variants.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS Other aims, advantages and characteristics of the invention shall appear on a reading of the following description of the implementation of the method of the invention applied to the protection of the XOR operator and an embodiment of an electronic module with a microprocessor according to the invention and given by way of non-restrictive example with reference to the accompanying drawings on which: FIG.\",\n \"1 shows a diagrammatic representation of an embodiment of an electronic module with a microprocessor and a protected XOR operator according to the invention, and FIG.\",\n \"2 shows a diagrammatic representation of the equivalent execution of the XOR operator implementing the method of the invention in the module of FIG.\",\n \"1.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The monolithic electronic module 10 with a microprocessor shown on FIG.\",\n \"1 according to the present invention and described by way of non-restrictive example generally comprises one microprocessor or central unit CPU 11 connected bidirectionally by an internal bus 12 to a live RAM memory 14, a dead ROM memory 16, an EEPROM memory 18 and an I/O input/output interface 20.The module 10 also comprises a Timer 22 with automatic resetting (in an optional variant) and a generator of pseudo-random numbers GNPA 24 connected to the internal bus 12.Application programs are installed at ROM 16, such as applications of bank card transactions or medical card applications which for reasons of confidentiality and protection comprise encryption/decryption, operator authentication or transaction validation sub-program in which the XOR operator is frequently present, especially for carrying out comparisons octet by octet.\",\n \"As regards execution of the XOR operator, this operator most commonly used generally forms part of the set of arithmetic instructions with two operands OP1 et OP2) of the central unit CPU or microprocessor 11.In the embodiment described here, the means for implementing the method for protecting the XOR operator are mainly software items in the form of a protected XOR calculation routine (or XORSEC routine) diagrammatically shown on FIG.\",\n \"2.Thus, on each call from the XOR instruction OP1, OP2) the program is rerouted with the two operands OP1 and OP2 towards the XORSEC routine which shall be executed in place of the XOR instruction, it being understood that the execution of the XORSEC routine carries out a function equivalent to that of the original XOR instruction.\",\n \"According to the main characteristic of the invention, the execution of the XOR instruction is replaced in the XORSEC routine by the execution of a sequence of operations, namely of the type but not exclusively of operations with a degree of less complexity, such as elementary operations, whose final result is identical to the result of the function of the XOR operator (condition easily verified amongst others by identical outlet tables).\",\n \"By way of non-restrictive examples, a set of eight sequences S1 to S8 equivalent to the XOR instruction is given below: S1=(x OR y) AND NOT (x AND y) S2=(x OR y) AND (NOT x OR NOT y) S3=NOT (NOT x AND NOT y) AND NOT (x AND y) S4=NOT (NOT x AND NOT y) AND (NOT x OR NOT y) S5=NOT(NOT(x OR y) OR (x AND y)) S6=NOT ((NOT x AND NOT y) OR (x AND y)) S7=NOT ((NOT x AND NOT y) OR NOT (NOT x OR NOT y)) S8=NOT (NOT (x OR y) OR NOT (NOT x OR NOT y)) It shall be observed that all these sequences S1 to S8 are based on the use of at least two of three AND, NOT and OR logic elementary instructions and have the same truth table outlet as for the XOR instruction.\",\n \"By using the traditional presentation of truth tables with two inlets x, y and one outlet s, it is possible to write for the XOR, AND and OR operators and for the sequence S5 (selected by way of non-restrictive example) the following four truth tables: XOR AND OR x y s(XOR) x y s(AND) x y s(OR) 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 0 1 1 1 0 1 1 0 0 1 0 1 1 1 0 1 1 1 1 1 1 and for S5=NOT(NOT(x OR y) OR (x AND y)) with A=(x OR y), B=NOT A, C=(x AND y) D=(NOT(x OR y) OR (x AND y)=B OR C, and s(S5)=E=NOT D x y A B C D E 0 0 0 1 0 1 0 0 1 1 0 0 0 1 1 0 1 0 0 0 1 1 1 1 0 1 1 0 It is thus verified that s(S5)=E is identical to s(XOR).\",\n \"It shall be observed that the sequence automatically selected to replace the XOR operator is made up of five elementary operations whose signature shall be significantly different from the XOR operator.\",\n \"Thus, it is possible to embody the simplest variant for implementing the method of the invention.\",\n \"According to one optional, but extremely advantageous, characteristic of the invention used in the embodiment described here, the sequence of replacement operations, namely the set ES constituted by the eight sequences S1 to S8 given above.\",\n \"Thus, the difficulty of identification of the XOR operator is further increased by the multiple changes of the sequence of operations replacing the XOR operator during execution of the program, the sequences S1 to S8 able to be used having all different signatures.\",\n \"According to another optional characteristic of the invention, but also extremely advantageous, used in the embodiment described here, the order number NDO=i (i ranging from 1 to 8) in its entirety (S1 to S8) of the sequence S1 selected to be executed is determined according to certain parameters of the program currently being executed and/or of a random parameter.\",\n \"Advantageously, said random parameter is obtained from a pseudo-random numbers generator.\",\n \"This mechanism for scrambling sequences rendering random the sequence effectively selected to replace the XOR operator on each call proves to be extremely effective, especially in an encryption/decryption processing when the XOR operator is called several times in the program.\",\n \"As shown on FIG.\",\n \"2, the XORSEC routine comprises four main processing phases, namely in the order of execution: a start or initialization phase IN-XORSEC with in particular the storing of the program counter values, and the operands OP1 and OP2, a phase CAL-NDO for determining by calculation the order number NDO=i (i ranging from 1 to 8) of the sequence of operations Si to be executed to replace the instruction XOR with branching towards the corresponding sub-routine CAL-XORSEC(i) and present in the set CAL-XORSEC of eight sub-routines CAL-XORSEC(1) to CAL-XORSEC(8), a phase CAL-XORSEC(i) for executing the sub-routine CAL-XORSEC(i) by logic calculation with the operands OP1 and OP2 of the sequence of operations Si selected by the phase CAL_NDO, a phase OUT-XORSEC for return to the main program so as to resume its execution and transfer of the results of the CAL-XORSEC(i) equivalent to the calculation of an XOR between the two operands OP1 and OP2.It shall be noted that for the phase CAL-NDO, the random generator GNPA 24 supplies on demand one random octet R used as a calculation parameter alone or with other parameters extracted from the values of the operands OP1 and OP2, the final result of the calculation being one octet F(R).\",\n \"By using for example an operation of the type NOD=i=F(R) AND 07 h, the three less significant bits are extracted from this octet so as to obtain the binary value of NOD=i (from 000 to 111 namely 00h to 07h), order number of the sequence Si to be executed.\",\n \"It is to be noted that the value of the order number is a sensitive data element of the algorithm in question.\",\n \"Finally, the routine XORSEC exhibits a further improved variant as regards the difficulty of identification in which the series of replacement sequences is constituted by sequences with a given period of execution (and owing to this more difficult to distinguish).\",\n \"So as to achieve this, certain sequences comprise at least one non-operative instruction designed to introduce a delay time in the execution of the sequences concerned.\",\n \"In particular, the non-operative instruction is selected from non-operative instructions in relation to the microprocessor or from normally operative instructions but rendered ineffective via their positions in the sequence of operations.\",\n \"If it is assumed that the elementary operations AND, OR and NOT have approximately equal periods of execution (for example 4 cycle times of the clock of the central unit CPU 11), as for the non-operative <<blank>> operation NOP, the set ES of the sequences S1 to S8 is modified into a new set of sequences made uniform to 9 operations ES′ being written by way of non-restrictive example as follows (and in which the added operations appear in thick type): S′1=(x OR NOP y OR y) AND NOP NOT (x AND NOP y AND y) S′2=(x OR y NOP OR y) AND NOP (NOT x OR NOP NOT y) S′3=NOT (NOT x AND NOP NOT y) AND NOT (x AND y AND y) S′4=NOT (NOT x AND NOP NOT y) AND (NOT x OR NOT y) S′5=NOT(NOT(x NOP OR y OR y) OR (x NOP AND y AND y)) S′6=NOT ((NOT x AND NOT y NOP) OR (x AND y NOP AND y)) S′7=NOT ((NOT x AND NOT y) OR NOT (NOT x OR NOT y)) S′8=NOT (NOT (x OR y OR y) OR NOT (NOT x OR NOT y)) For example, for the sequence S′5: S′5=NOT(NOT(x NOP OR y OR y) OR (x NOP AND y AND y)) with x′=x NOP, A=(x′ OR y), A′=A OR y , B=NOT A′, C=(x′ AND y), C′=C AND y, D=B OR C′ and s(S′5)=E=NOT D It is possible to write the truth table of the sequence S′5 which also verifies s(S′5)=s(XOR) S′5 x y x′ A A′ B C C′ D E 0 0 0 0 0 1 0 0 1 0 0 1 0 1 1 0 0 0 0 1 1 0 1 1 1 0 0 0 0 1 1 1 1 1 1 0 1 1 1 0 It shall be observed that for sequences with nine elementary operations, the machine time remains quite reasonable.\",\n \"The invention is not limited to its application concerning the protection of logic operators, but is also applicable to protection of mathematical operators, such as one-digit adders, adders, subtractors or multipliers or functional circuits similar to logic or mathematical operators, such as combinatory circuits, especially multiplexers and/or demultiplexers, coders and/or decoders, generators and/or parity detectors or comparators.\",\n \"For example, according to another application of the method of the invention, the operator to be protected is the mathematical operator of the <<multiplication by two>> obtained by shifting left one bit with resetting of the low order bit.\",\n \"In notation C ANSI, this operator is also denoted (x<<1).\",\n \"Thus, on each call from the operator (x<<1), its execution shall be replaced by the execution of a sequence of operations selected from the following equivalent sequences: S′′1=(x ADD x) S′′2=(x AND FOh) ADD x ADD (x AND OFh) S′′3=(NOT ((NOT x) ADD (NOT x))) SUB 1 S′′4=(y ADD x) SUB (y SUB x), in which the operator ADD is the standard addition operator on one octet, the instruction SUB is the standard subtraction operator on one octet and the suffix <<h>> indicates a hexadecimal value.\",\n \"Generally speaking, the choice of the equivalent sequence S′′i and its implementation are embodied similarly, sometimes identically, to what has been described in details as previously for the XOR operator.\",\n \"It shall also be noted that, without departing from the context of the invention, the smart card accommodating the electronic module with a protected operator according to the invention can be replaced by any other embarked system.\",\n \"One embodiment of the invention is shown below concerning its implementation using an electronic module.\",\n \"In the method for protecting a logic or mathematical operator, or a similar functional circuit able to be used in the execution of a program in the electronic module including a microprocessor and a memory, the execution of said operator by the microprocessor is replaced by the execution of a sequence of replacement operations whose final result is identical to that of the function of said operator, said result being stored in the memory.\",\n \"The electronic module with a protected operator and comprising at least one microprocessor and a memory storing a program to be executed comprising at least one logic or mathematical operator or similar functional circuit to be protected wherein it comprises means to replace execution of the operator with the aid of the microprocessor by the execution of a sequence of operations whose final result is identical to that of the function de the operator, said result being stored in the memory.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471323"}}},{"rowIdx":588,"cells":{"text":{"kind":"list like","value":[["Diagnosis and treatment of blood disorders","Based on the discovery of the nucleotide and amino acid differences which distinguish the Gova and Govb allelic forms of the membrane glycoprotein CD109, and which comprise the biallelic Gov platelet alloantigen system, compositions and methods are provided for determining the Gov genotype and phenotype of individuals.","Also provided, on the basis of this discovery, are compositions and methods for treating disorders associated with Gov alloantigen incompatibility, such as the bleeding disorders post-transfusion purpura, post-transfusion platelet refractoriness, and neonatal alloimmune thrombocytopenia.","The two allelic forms of CD109 differ by a single amino acid.","The Gova allelic form has Tyr at amino acid position 703 in the CD109 sequence.","The Govb allelic form has Ser at the same position.","This amino acid difference is due to a single change, from A for the Gova allele to C for the Govb allele, in the CD109 gene."],["1.An oligonucleotide comprising a sequence which binds specifically to (i) a region of CD109 nucleic acid that includes a single nucleotide polymorphism that is distinctive of a Gova allele and/or (ii) a region of CD109 nucleic acid that includes a single nucleotide polymorphism that is distinctive of a Govb allele.","2.The oligonucleotide of claim 1, comprising 8 to 50 nucleotides.","3.The oligonucleotide of claim 1, wherein the nucleic acid specifically binds to one of (i) or (ii) under high stringency hybridization conditions.","4.The oligonucleotide of claim 2, wherein the stringent hybridization conditions comprise 0.1×SSC, 0.1% SDS at 65° C. 5.The oligonucleotide of claim 1, wherein the CD109 nucleic acid comprises genomic DNA, cDNA, or RNA corresponding to the Gova allele of the CD109 gene or locus, or comprises genomic DNA, cDNA, or RNA corresponding to the Govb allele of the CD109 gene or locus.","6.The oligonucleotide of claim 5, wherein the Gova allele comprises an A at a position corresponding to position 2108 of SEQ ID NO:1 and corresponding to position 954 of SEQ ID No:5.7.The oligonucleotide of claim 5, wherein the Govb allele comprises a C at a position corresponding to position 2108 of SEQ ID NO:3 and corresponding to position 954 of SEQ ID NO:5.8.The oligonucleotide of claim 1, comprising a sequence complementary to the Gova allele or to the Govb allele.","9.The oligonucleotide of claim 6, comprising a sequence selected from the group consisting of: (a) 8-50 nucleotides of SEQ ID NO:1; (b) a sequence that is complementary to a sequence specified in (a); and (c) a sequence having at least 70% sequence identity to a sequence in (a) or (b), wherein the sequence having identity is capable of hybridization to CD109 under high stringency hybridization conditions.","10.The oligonucleotide of claim 7, comprising a sequence selected from the group consisting of: (a) 8-50 nucleotides of SEQ ID NO:3; (b) a sequence that is complementary to a sequence specified in (a); and (c) a sequence having at least 70% sequence identity to a sequence in (a) or (b), wherein the sequence having identity is capable of hybridization to CD109 under high stringency hybridization conditions.","11.A oligonucleotide comprising all or part of any one of SEQ ID NO:6-SEQ ID NO:14 or a complement thereof.","12.The oligonucleotide of claim 11, comprising 8 to 50 nucleic acids.","13.The oligonucleotide of claim 1, wherein the nucleic acid is capable of use as a probe in a hybridization assay.","14.The oligonucleotide of claim 1, wherein the nucleic acid sequence is detectably labelled.","15.The oligonucleotide of claim 14, wherein the detectable label comprises: (a) a fluorogenic dye; and/or (b) a biotinylation modification; and/or (c) a radiolabel.","16.The oligonucleotide of claim 1, wherein the sequence comprises DNA, a DNA analog, RNA or an RNA analog.","17.The oligonucleotide of claim 1, wherein the oligonucleotide is attached to a substrate.","18.The oligonucleotide of claim 1, wherein the oligonucleotide is capable of use as a primer that will specifically bind proximate to, and/or cause elongation through, a CD109 sequence, including the single nucleotide polymorphism distinctive of the Gova or Govb alleles.","19.A Gov genotyping kit comprising a detection agent for detecting the presence of a Gov allele-specific target sequence in a CD109 nucleic acid derived from a subject.","20.The kit of claim 19, wherein the detection agent comprises a nucleic acid and/or a restriction enzyme.","21.The kit of claim 19, further comprising a container.","22.The kit of claim 21, wherein the container comprises a biological sample container for housing the detection agent.","23.The kit of claim 19, further comprising a plate having a plurality of wells and having bound thereto probes having a nucleic acid sequence which specifically binds to a CD109 sequence including a Gova or a Govb allele target sequence.","24.The kit of claim 20, wherein the restriction enzyme is selected from the group consisting of Bst2UI, BstNI, BstOI, EcoRII, MaeIII, MspR91, MvaI, ScrFI or an isoschizomer thereof.","25.The kit of claim 19, further comprising an amplification agent for amplifying the nucleic acid.","26.The kit of claim 25, wherein the amplification agent amplifies a region of CD109 platelet, T cell, or endothelial cell mRNA including the single nucleotide polymorphism distinctive of a Gova or Govb allele.","27.The kit of claim 25, wherein the amplification agent comprises a primer set including first and second primers, wherein the first primer is a nucleic acid that will specifically bind proximate to, and/or cause elongation through, CD109 sequence that includes the single nucleotide polymorphism distinctive of a Gova allele and the second primer is a nucleic acid that will specifically bind proximate to, and/or cause elongation through, CD109 sequence that includes the single nucleotide polymorphism distinctive of a Govb allele.","28.The kit of claim 19, wherein the nucleic acid is obtained by amplification with all or part of the nucleic acid of any one of SEQ ID NO:6- SEQ ID NO:14 or the complement thereof.","29.The kit of claim 19, further comprising all or part of a CD109 gene, a CD109-encoding mRNA, or a CD109 cDNA made from a CD109-encoding mRNA.","30.The kit of claim 19, comprising the oligonucleotide of any of claims 1 to 18.31.The kit of claim 19, for detecting that the subject has or is at risk of a disease, disorder or abnormal physical state.","32.The kit of claim 31, wherein the disease, disorder or abnormal physical state comprises a blood disease, disorder or abnormal physical state.","33.The kit of claim 32, wherein the blood disease, disorder or abnormal physical state comprises bleeding of the subject, or increased risk of bleeding, due to destruction of blood platelets.","34.The kit of claim 33, wherein the blood disease, disorder or abnormal physical state comprises post-transfusion purpura (“PTP”), post-transfusion platelet refractoriness (“PR”) or neonatal alloimmune thrombocytopenia (“NAIT”).","35.The kit of claim 33, wherein the nucleic acid is obtained from mRNA from human platelets, T cells, endothelial cells, or human genomic DNA.","36.A method of Gov alloantigen genotyping a subject comprising: (a) providing a CD109 nucleic acid sample derived from the subject; and (b) detecting a region of CD109 nucleic acid that includes a single nucleotide polymorphism distinctive of a Gova or a Govb allele.","37.The method of claim 36 comprising determining whether the subject is homozygous or heterozygous for the Gov alleles.","38.The method of claim 37, wherein the subject is a human and the Gov genotype is used to determine that the subject has, or is at risk of a disease, disorder or abnormal physical state.","39.The method of claim 38, wherein the disease, disorder or abnormal physical state comprises a blood disease, disorder or abnormal physical state.","40.The method of claim 39, wherein the blood disease, disorder or abnormal physical state comprises bleeding of the subject, or increased risk of bleeding, due to destruction of blood platelets.","41.The method of claim 40, wherein the blood disease, disorder or abnormal physical state comprises post-transfusion purpura (“PTP”), post-transfusion platelet refractoriness (“PR”) or neonatal alloimmune thrombocytopenia (“NAIT”).","42.The method of claim 41, wherein the nucleic acid is obtained by amplifying the nucleic acid from the subject.","43.The method of claim 42, wherein the nucleic acid is obtained by amplification with all or part of the oligonucleotide of any of claims 1 to 18.44.The method of claim 41, wherein the nucleic acid is obtained from mRNA from human platelets, T cells, endothelial cells, or human genomic DNA.","45.The method of claim 36, wherein the detection step comprises determining the nucleotide sequence of the CD109 nucleic acid.","46.The method of clam 36, wherein the detection step comprises contacting the nucleic acid with the oligonucleotide of any of claims 1 to 18 under high stringency conditions.","47.The method of claim 46, wherein the oligonucleotide will selectively hybridize to (i) a region of CD109 nucleic acid that includes a single polymorphism distinctive of a Gova allele or (ii) a region of CD109 nucleic acid that includes a single polymorphism distinctive of a Govb allele.","48.The method of claim 36, wherein the detecting step comprises: (a) performing a restriction endonuclease digestion of the nucleic acid, thereby providing a nucleic acid digest; and (b) contacting the digest with the oligonucleotide.","49.The method of claim 47, wherein the hybridization occurs either during or subsequent to PCR amplification and the analysis is by “Real-Time” PCR analysis, or fluorimetric analysis.","50.The method of claim 36, wherein the detection step comprises: (a) incubation of the amplified nucleic acid with a restriction endonuclease under conditions whereby the DNA will be cleaved if the nucleic acid comprises a recognition site for the enzyme; and (b) determining whether the nucleic acid contains a recognition site for the restriction enzyme characteristic of cDNA made from mRNA encoding a Gova or Govb allele of CD109.51.The method of claim 50, wherein the restriction enzyme is selected from the group consisting of Bst2UI, BstNI, BstOI, EcoRII, MaeIII, MspR91, MvaI, ScrFI or an isoschizomer thereof.","52.The method of claim 50, wherein the determination step includes size analysis of the nucleic acid.","53.The method of claim 50, wherein the amplified nucleic acid is analyzed by electrophoretic mobility and the mobility of the amplified nucleic acid is compared to the characteristic mobility of amplified nucleic acid fragments corresponding to the Gova or Govb alleles of CD109.54.A method of amplifying CD109 mRNA comprising amplifying the mRNA by PCR using an oligonucleotide of claim 1.55.A Gova specific antibody.","56.The antibody of claim 55, that recognizes specifically a Gova allele-specific CD109 epitope corresponding to the polypeptide encoded by a CD109 nucleic acid containing an A at the position corresponding to position 2108 of SEQ ID NO: and position 954 of SEQ ID NO:5., and containing the amino acid Tyrosine at the position corresponding to position 703 of the CD109 protein encoded by SEQ ID NO:1.57.A Govb specific antibody.","58.The antibody of claim 57, that recognizes specifically a Govb allele-specific CD109 epitope corresponding to the polypeptide encoded by a CD109 nucleic acid containing a C at the position corresponding to position 2108 of SEQ ID NO:3 and position 954 of SEQ ID NO:5., and containing the amino acid Serine at the position corresponding to position 703 of the CD109 protein encoded by SEQ ID NO:3.59.The antibody of claim 55, further comprising a monoclonal antibody or a polyclonal antibody.","60.The antibody of claim 55, further comprising a detectable label.","61.An immunogenic composition comprising a Gov specific antibody of claim 55.62.A method of Gov alloantigen phenotyping a subject, comprising: (a) providing a CD109 polypeptide sample derived from the subject; and (b) detecting the presence of a Gova or a Govb antigen in the CD109 polypeptide.","63.The method of claim 62, wherein the CD109 is membrane bound CD109 or isolated CD109.64.The method of claim 62, wherein the detection step comprises contacting the polypeptide sample with the antibody of claim 55.65.A diagnostic kit for Gov alloantigen phenotyping a subject, comprising a Gova antibody and/or a Govb antibody of claim 55.66.The kit of claim 65, further comprising a container.","67.An isolated polypeptide containing Gova allele-specific amino acid sequence and which is specifically reactive with a Gova antibody.","68.An isolated polypeptide containing Govb allele-specific amino acid sequence and which is specifically reactive with a Govb antibody.","69.The isolated polypeptide of claim 67, comprising between 4 and 100 amino acids.","70.The isolated polypeptide of claim 67, comprising a full-length CD109 polypeptide, or a fragment of a CD109 polypeptide.","71.An isolated CD109 polypeptide fragment, comprising a Gova or a Govb antigen.","72.The polypeptide fragment of claim 71, comprising all of, or a fragment of, the protein encoded by SEQ ID NO:1., and in which the amino acid corresponding to position 703 of the protein encoded by SEQ ID NO:1 is a Tyrosine.","73.The polypeptide fragment of claim 72, comprising all of, or a fragment of, the protein encoded by SEQ ID NO:3., and in which the amino acid corresponding to position 703 of the protein encoded by SEQ ID NO:3 is a Serine.","74.The polypeptide fragment of claim 72, comprising between 4 and 100 amino acids.","75.The polypeptide fragment of claim 74, comprising between 7 and 50 amino acids.","76.The polypeptide fragment of claim 67, in which the polypeptide is purified from native CD109, is synthetic, or is prepared by recombinant means.","77.The polypeptide fragment of claim 67, in which the polypeptide is bound to a substrate.","78.A fusion compound comprising the polypeptide of any of claim 67 connected to an immunogenic carrier.","79.The fusion compound of claim 78, wherein the immunogenic carrier comprises a proteinaceous carrier.","80.The fusion compound of claim 79, wherein the immunogenic carrier comprises a detectable label.","81.A Gova or Govb specific antibody recognizing the fusion compound of claim 80.82.An immunogenic composition comprising the polypeptide, polypeptide fragment or fusion compound of claim 67.83.A method of producing a Gova or Govb specific antibody, comprising contacting an animal with the immunogenic composition of claim 82, so that the animal produces antibodies against the immunogenic composition.","84.The method of claim 83, wherein the animal is a bird or a mammal.","85.A method of screening an antibody producing culture to determine whether the culture produces Gova or Govb specific antibody, comprising: (a) contacting a polypeptide of the invention with the culture; and (b) detecting Gova or Govb specific antibody.","86.The method of claim 85, wherein the polypeptide comprises a detectable label.","87.The method of claim 86, wherein the polypeptide is attached to a substrate.","88.A method of purifying a Gov allele-specific antibody from a sample, comprising: (a) contacting a Gov allele-specific antibody with a polypeptide of the invention comprising a Gova or Govb antigen, so that an antibody:polypeptide complex is formed; (b) separating the complex from the sample; and (c) next separating the antibody from the polypeptide.","89.The method of claim 88, wherein the polypeptide is bound to a substrate.","90.The method of claim 89, wherein the polypeptide comprises a detectable label.","91.A method of purifying a Gov polypeptide from a sample, comprising: (a) contacting a Gov allele-specific antibody with a polypeptide of the invention containing a Gova or Govb-specific epitope, so that an antibody: polypeptide complex is formed; (b) separating the complex from the sample; and (c) next separating the antibody from the polypeptide.","92.The method of claim 91, wherein the antibody is bound to a substrate.","93.The method of claim 92, wherein the antibody comprises a detectable label.","94.A method of screening a subject sample to determine whether the sample contains Gova or Govb-specific antibodies, comprising: (a) contacting a polypeptide of the invention with the sample; and (b) detecting the presence or absence of Gova or Govb specific antibody.","95.The method of claim 94, wherein the polypeptide comprises a detectable label.","96.The method of claim 95, wherein the polypeptide is attached to a substrate.","97.The method of claim 96, wherein the subject comprises a mother of a fetus or a newborn infant, or the fetus or newborn infant itself, and the presence of Gova or Govb-specific antibody indicates that the fetus or infant has, or is at risk of NAIT.","98.The method of claim 97, wherein the presence of Gova or Govb specific antibody indicates that the subject has, or is at risk of a blood disease, disorder or abnormal physical state.","99.The method of claim 98, wherein the blood disease, disorder or abnormal physical state comprises bleeding of the subject, or increased risk of bleeding, due to destruction of blood platelets.","100.The method of claim 99, wherein the blood disease, disorder or abnormal physical state comprises post-transfusion purpura (“PTP”), post-transfusion platelet refractoriness (“PR”) or neonatal alloimmune thrombocytopenia (NAIT).","101.The method of claim 100, wherein the sample comprises human serum or plasma.","102.A diagnostic kit for detection of Gova or Govb specific antibody, comprising a polypeptide of claim 67.103.The kit of claim 102, further comprising a container.","104.A method of determining Gov antibody specificity, comprising: (a) contacting an antibody with a first polypeptide comprising a Gova antigen and a second polypeptide comprising a Govb antigen; and (b) determining whether the antibody binds to either or both of the first and second polypeptide.","105.A method of blocking Gova antibody binding to an antigen, comprising: contacting the antibody with a polypeptide of the invention comprising a Gova antigen so that an antibody:polypeptide complex is formed.","106.The method of claim 105, wherein the polypeptide comprises a detectable label.","107.The method of claim 106, wherein the polypeptide is bound to a substrate.","108.A method of blocking Govb antibody binding to an antigen, comprising: contacting the antibody with a polypeptide of the invention comprising a Govb antigen so that an antibody:polypeptide complex is formed.","109.The method of claim 108, wherein the polypeptide comprises a detectable label.","110.The method of claim 109, wherein the polypeptide is bound to a substrate.","111.A pharmaceutical composition comprising the polypeptide of claim 67.112.A method of immunizing a subject so that the subject will produce anti-idiotypic antibodies, comprising administering to the subject the immunogenic composition of claim 82.113.A method of blocking Gova or Govb specific antibodies from binding to CD109 in a subject, comprising: administering to the subject polypeptides of the invention capable of binding to Gova and/or Govb specific antibodies.","114.The method of claim 113, wherein the polypeptide comprises a detectable label.","115.The method of claim 114, wherein the binding of the polypeptide to the Gova or Govb specific antibody prevents alloimmune cell destruction by the antibody.","116.The method of claim 115, wherein the binding of the polypeptide to the Gova or Govb-specific antibody depletes the antibody.","117.The method of claim 116, wherein the subject has or is at risk of a blood disease, disorder or abnormal physical state.","118.The method of claim 117, wherein the blood disease, disorder or abnormal physical state comprises bleeding of the subject, or increased risk of bleeding, due to alloimmune destruction of blood platelets.","119.The method of claim 118, wherein the blood disease, disorder or abnormal physical state comprises post-transfusion purpura (“PTP”), post-transfusion platelet refractoriness (“PR”) or neonatal alloimmune thrombocytopenia (“NAIT”)."],["<SOH> BACKGROUND OF THE INVENTION <EOH>Among the disorders, which the invention concerns, are those involving abnormal and excessive bleeding due to destruction of blood platelets (“platelets”).","These disorders include, but are not restricted to, post-transfusion purpura (“PTP”) and post-transfusion platelet refractoriness (“PTPR”), which are suffered by some persons who receive blood, platelets, leukocyte concentrates, or plasma from other persons by transfusion or the like.","The disorders also include one that is suffered by fetuses and newborns and is known as “neonatal alloimmune thrombocytopenia” (“NATP”).","This disorder can cause death of fetuses and serious birth defects or death of newborns.","NATP is estimated to affect about 1 in 1000 newborns.","In NATP, fetal platelets, which enter the mother's blood stream, induce production in the mother of antibodies directed against fetal platelets.","These maternal antibodies then pass with the mother's blood into the fetus and mediate destruction of platelets in the fetus.","A mother, whose fetus or newborn suffers from NATP, is at increased risk of suffering PTP or PTPR.","When platelets from a first human (a “donor”) are introduced into the blood system of a second human (a “recipient”) by transfusion, through the placenta (in the case of fetal blood entering the mother), or the like, the recipient may mount an immune response against the platelets from the donor.","Such an immune response is referred to as an “alloimmune” response, because it involves antibodies reacting-against antigens of a different individual of the same species.","The alloimmune response to platelets is due to an immune response of the recipient against “alloantigens” (antigens of the same species as that mounting the immune response) on platelets from the donor.","These alloantigens are found on membrane glycoproteins that occur in the cell membranes, which define the outer surfaces of platelets (“platelet membranes”).","In this invention, the glycoprotein is anchored to the membrane in an atypical manner through an anchor consisting of glycosylphosphatidylinositol (GPI), which anchors an extracellular domain or segment of the glycoprotein exposed to the outside of the platelet.","It is thought that alloantibodies, which are generated in an alloimmune response against platelet alloantigens, interact with the extracellular domains of the alloantigens.","The platelet alloantigens that a person has are determined by the person's genetics.","A donor, because of his or her genetics, may have a platelet alloantigen, which a recipient, who receives blood, platelets, leukocytes or plasma from the donor, does not have, because of the recipient's genetics.","In such a situation, the immune system of the recipient may recognize the donor's alloantigen as “non-self,” and raise an immune response against, the platelet alloantigen, which the donor has but the recipient does not.","Membrane glycoprotein alloantigens have been characterised for both human red blood cells and human platelets.","It is noteworthy, however, that they also occur on other cell types, such as leukocytes and endothelial cells, where they may also occasion various disorders through alloimmune responses.","Recognised classes of red blood cell and platelet alloantigens have been described, over the past 30 years, based on observations of antibody reactions occurring when blood recipients have been exposed to blood from donors.","A recent review of human platelet alloantigen systems is provided by Ouwehand, W., and Navarrete, C., in Molecular Haematology , Provan, D. and Gribben, J. eds.","Blackwell (1999).","Several biallelic platelet alloantigen “systems” have been characterised.","In each of these systems, there are two alloantigens, each of which is provided by one of two alleles of the gene comprising the system.","Because each gene occurs twice in the normal human genome, a person can be homozygous for one or the other of the two alloantigens, or heterozygous for the two alloantigens, comprising a biallelic system.","The alloantigens described to date occur on glycoprotein molecules which may exist in various forms (transmembrane, GPI-linked and soluble, for example).","In such a case, the alloantigens are found on each of the variant forms of the glycoprotein.","For all of the biallelic platelet alloantigen systems that have been characterised at the level of protein and gene sequences, it has been found in all cases, except for one, that the difference between the two alleles is based on a single nucleotide polymorphism in the relevant gene.","One biallelic system of human platelet alloantigens is the Gov a /Gov b biallelic system associated with CD109, a membrane glycoprotein which occurs on platelets and various other cell types, including leukocytes and endothelial cells.","Each Gov allele corresponds to one CD109 glycoprotein (Sutherland, D. R. et al, 1991; Smith et al., 1995; Berry, J. et al., 2000), consistent with the known tissue distribution of CD109.The frequencies for the Gov alleles are 0.4 for Gov a and 0.6 for Gov b in the Caucasian population.","Thus, in this population, 40.7% are heterozygous for the Gov alleles, and will not mount an alloimmune response due to Gov incompatibility (not possessing the Gov alloantigen found on platelets received from another).","In contrast, 19.8% of Caucasians are homozygous for the Gov a allele and thus may mount an immune response due to Gov alloantigen incompatibility against platelets received from anyone in the 80.5% of the Caucasian population that is not homozygous for the Gov a allele, while 39.8% are homozygous for the Gov b allele and thus may mount an immune response due to Gov alloantigen incompatibility against platelets received from anyone in the 60.2% of the Caucasian population that is not homozygous for the Gov b allele.","As indicated above, alloimmunization based on Gov incompatibility (the introduction into the blood stream of donor platelets bearing a Gov alloantigen not carried by the recipient) can result in bleeding disorders due to platelet destruction, including NATP, PTPR, and PTP.","The location of the Gov antigens within the CD109 molecule, and the nature of the CD109 polymorphism which underlies the Gov a /Gov b alloantigen (both at the protein and at the gene level), have not heretofore been known.","Furthermore, it has not heretofore been possible to generate non-human antibody (polyclonal or monoclonal), as from a rat, mouse, goat, chicken, or the like, with specificity for the Gov a alloantigen but not the Gov b alloantigen (or vice-versa) sufficient for use in an immunoassay, for typing for Gov phenotype using platelets or CD109 molecules.","Previously developed technology, involving gene-specific amplification of platelet RNA-derived cDNA, followed by the determination of the nucleotide sequence of the amplified DNA, has been applied successfully to the elucidation of the molecular basis of other biallelic platelet alloantigen systems (Newman et al., J. Clin.","Invest.","82,739-744 (1988); Newman et al., J. Clin.","Invest.","83, 1778-1781 (1989)(P1A or HPA-1 system); Lyman et al., Blood 75, 2343-2348 (1990)(Bak or HPA-3system); Kuijpers et al., J. Clin.","Invest.","89, 381-384 (1992)(HPA-2 or Ko system); Wang et al., J. Clin.","Invest.","90, 2038-2043 (1992)(Pen system).","With one exception, it has been found in each case that a single amino acid difference at a single position differentiates the amino acid sequences of the two alleles, and that this difference arises from a single allele-specific nucleotide substitution in the coding region of the mRNA and gene.","There remains a need to elucidate the molecular basis of the biallelic Gov platelet alloantigen system."],["<SOH> SUMMARY OF THE INVENTION <EOH>The Gov a/Gov b CD109 Single Nucleotide Polymorphism We have now discovered that a single amino acid difference in the CD109 glycoprotein distinguishes the Gov a and Gov b allelic forms.","The two alleles differ at amino acid position 703 of the full-length 1445 amino acid CD109 molecule, with the Gov a allele [SEQ ID NO:2] containing a Tyr at this position, while the Gov b allele [SEQ ID NO:4] contains Ser.","Further, we have discovered that this difference in amino acid sequence between the allelic forms of CD109 is due to a single nucleotide polymorphism at position 2108 of the coding portion of full-length mRNA encoding CD109, or of the corresponding coding strand of the cDNA corresponding to this mRNA.","Specifically, the Gov a allele [SEQ ID NO:1] contains adenine at position 2108, the second nucleotide of the codon encoding the amino acid at position 703 of the full-length CD109 protein, while the Gov b allele contains cytosine at position 2108, as shown in SEQ ID NO:3 The Gov a /Gov b single nucleotide polymorphism of CD109, lies at position 2108 in SEQ ID NO:1.SEQ ID NO:1 is the cDNA sequence encoding the full-length 1445 amino acid CD109 precursor encoding the Gov a alleleln the Gov b allele form [SEQ ID NO:3], C occurs at position 2108, rather than A.","The ATG at the 5′-end of the sequence in SEQ ID NO:1 corresponds to the translation start of the full-length precursor form (including leader peptide) of CD109.The triplet corresponding to the N-terminal amino acid of the mature CD109 protein is at positions 64-66 in SEQ ID NO:1.The Gov a /Gov b single nucleotide polymorphism of CD109, lies at position 954 in SEQ ID NO:5.SEQ ID NO:5 is the genomic DNA sequence of human CD109 exon 19 and the contiguous introns, introns 18 and 19.The Gov a /Gov b single nucleotide polymorphism of CD109 is found within CD109 exon 19, and specifically is located at position 3 of CD109 exon 19.The sequence presented in SEQ ID NO:5 contains A at position 954, and thus corresponds to the Gov a allele.","The corresponding Gov b sequence contains C at position 954 of SEQ ID NO:5 (nucleotide position 3 of exon 19).","In view of this discovery, it will be readily apparent to the skilled what the present invention provides: Gov allele-specific oligonucleotides and polynucleotides: Based on the discovery, the present invention provides oligonucleotides and polynucleotides (seems repetitive), including (but not limited to) probes which can be used to determine whether a person is homozygous for one or the other of the Gov alleles, or heterozygous for these alleles, thereby to determine that person's Gov genotype, and by extension, their Gov phenotype (i.e., the Gov alloantigen(s) which their cells express).","Further, the invention provides methods of using such oligonucleotides, and test kits to facilitate their use, in such Gov genotype and phenotype determinations.","These oligonucleotides of the invention can be used to determine whether, in the CD109 gene, or in the mRNA encoding CD109, the internal nucleotide (nucleotide 2108) of the codon (in CD109 gene or in the mRNA encoding CD109) which corresponds to the amino acid at position 703 in the sequence of full-length CD109 is adenine or cytosine.","Such probes will typically be cDNA but may be genomic DNA, mRNA or RNA, and may be labelled for detection.","The oligonucleotides of the invention can be used as probes to detect nucleic acid molecules according to techniques known in the art (for example, see U.S. Pat.","Nos.","5,792,851 and 5,851,788).","For example, an oligonucleotide of the invention may be converted to a probe by being end-labelled using digoxigenin-11-deoxyuridine triphosphate.","Such probes may be detected immunologically using alkaline-phosphate-conjugated polyclonal sheep antidigoxigenin F(ab) fragments and nitro blue tetrazolium with 5-bromo-4-chloro-3-indoyl phosphate as chromogenic substrate.","Gov allele-specific antibodies: Still further, based on the discovery, which underlies the invention, of the molecular basis for the Gov a /Gov b alloantigen system, the invention provides non-human polyclonal and monoclonal antibodies, which can be used to distinguish one Gov allelic form of CD109 from the other, whether the CD109 is part of a complex embedded in or isolated from a membrane or is isolated.","These antibodies of the invention, which are preferably provided in an aqueous buffer solution, and the immunoassays of the invention which employ such antibodies, are useful for determining whether a person has one or both of the Gov alloantigens and for Gov phenotyping.","Methods of using the antibodies of the invention in the immunoassays of the invention, and in such determinations, are also encompassed by the invention.","The invention also provides test kits to facilitate carrying out such immunoassays and determinations.","Gov allele-specific peptides and polypeptides: Again, based on the discovery that underlies the invention, of the molecular basis for the Gov a /Gov b alloantigen system, the invention provides peptides or polypeptides, which are useful for various purposes.","These peptides or polypeptides are typically between 4 and 100, and more typically between 7 and 50, amino acids in length, and have amino acid sequences identical or having sequence identity to those of segments of the CD109 sequences, that include the amino acid at position 703 of full-length mature CD109.This amino acid (position 703) corresponds the triplet at positions 2107-2109 in the CD109 cDNA sequence presented in SEQ ID NO:1, or in the corresponding sequence for the CD109 cDNA that encodes the Gov b allelic form [SEQ ID NO:3].","These peptides or polypeptides may be synthetic, may be purified from native CD109 or may be prepared by recombinant means.","For guidance, one may consult the following U.S. Pat.","Nos.","5,840,537, 5,850,025, 5,858,719, 5,710,018, 5,792,851, 5,851,788, 5,759,788, 5,840,530, 5,789,202, 5,871,983, 5,821,096, 5,876,991, 5,422,108, 5,612,191, 5,804,693, 5,847,258, 5,880,328, 5,767,369, 5,756,684, 5,750,652, 5,824,864, 5,763,211, 5,767,375, 5,750,848, 5,859,337, 5,563,246, 5,346,815, and WO9713843.Many of these patents also provide guidance with respect to experimental assays, probes and antibodies, methods, transformation of host cells, which are described below.","These patents, like all other patents, publications (such as articles and database publications) in this application, are incorporated by reference in their entirety.","Gov allele-specific peptides and polypeptides as antigens and immunogens, and Gov allele-specific polyclonal and monoclonal antibodies: These peptides or polypeptides are useful as antigens (usually coupled to a larger, immunogenic carrier [proteinaceous or otherwise], as known in the art) for making the polyclonal or monoclonal antibodies of the invention.","The peptides or polypeptides are also useful in screening monoclonal antibody-producing cultures (hybridoma cultures/ E. coli cultures or so-called V gene phage antibodies) to identify those that produce monoclonal antibodies of the invention.","The invention also encompasses immunogenic compositions which comprise a peptide, polypeptide or fusion compound of the invention and which are immunogenic in a bird, including, without limitation, a chicken, or a mammal, such as, a mouse, rat, goat, rabbit, guinea pig, sheep or human.","The compositions may include an immunogenicity-imparting “carrier” which may be but is not necessarily a protein as known in the art, that is immunogenic in a bird or mammal, coupled to at least one peptide or polypeptide of the invention, which has an amino acid sequence that is the same as that of a segment of the sequence for CD109, that includes the amino acid at position 703 of the full length CD109 molecule.","The present invention also provides methods of using the peptides, polypeptides and immunogenic compositions of the invention for making antibodies of the invention, and methods of using the peptides and polypeptides of the invention in screening monoclonal antibody-producing hybridoma cultures or bacterial clones for those that produce monoclonal antibodies or fragments thereof of the invention.","Therapeutic and diagnostic application of Gov allele-specific peptides, polypeptides, and antibodies: These peptides or polypeptides, as well as antibodies, which are specific for the Gov a [SEQ ID NO:2] or Gov b [SEQ ID NO:4], but not both, allelic forms of CD109 in the platelet membrane, and which can be produced by a mammal (including an human) immunized with the peptides or polypeptides, which themselves happen to be immunogenic, or the immunogenic compositions of the invention, are also useful both therapeutically and diagnostically.","The invention also provides the methods of using the peptides and polypeptides of the invention, and antibodies made using the peptides that are immunogenic and the immunogenic compositions of the invention, in therapeutic and diagnostic applications.","The Gov allele-specific peptides or polypeptides can also be used diagnostically to detect the presence of Gov a or Gov b specific antibodies in human plasma or serum samples, using methods that are readily apparent to those skilled in the art.","Such analyses would be useful in the investigation of cases of acquired alloimmune thrombocytopenia, including PTP, PTPR, and NATP.","In the latter case, this approach could also be used to detect the presence of Gov allele-specific antibodies in the mother of the affected fetus or newborn.","The presence of Gov allele-specific antibodies can also be detected using platelets of known Gov phenotype.","However, this approach has numerous technical disadvantages that are eliminated by the use of Gov allele-specific peptides or polypeptides for Gov allele-specific antibody detection.","Administration to a person, who is suffering from, or at risk for, for example, PTP or PTPR, or a mother at risk for passing NATP-causing alloantibodies to her fetus, of one of the peptides or polypeptides, that would be bound by the anti-Gov alloantibodies in such a person, would inhibit the binding of the alloantibodies to the person's (or the fetus's platelets and thereby inhibit the platelet destruction and abnormal bleeding associated with the disorders.","Alternatively, administration to such a person of antibodies (particularly human antibodies), which are produced using a peptide or polypeptide of the invention, which is immunogenic by itself, or an immunogenic composition of the invention, and which are specific for the Gov allelic form of the CD109 on the person's platelets which is associated with the PTP or PTPR, from which the person is suffering or may suffer, would induce the production of anti-idiotypic antibodies, which, in turn, would inhibit the platelet-destructive effects of the anti-Gov alloantibodies, which are generated by the person's own immune system and which are causing or threatening to cause the PTP, PTPR or NATP.","These therapeutic applications of peptides and polypeptides of the invention would be especially useful in treating NATP in a newborn, because the alloantibody giving rise to NATP in the newborn is not continuously produced by the immune system of the newborn, but rather is acquired passively, and therefore in limited, non-replenished quantity, by the newborn from its mother.","Thus, in accordance with one aspect of the present invention, an oligonucleotideprobe is provided that hybridizes to a portion of the CD109 gene, or a portion of CD109-encoding mRNA or cDNA prepared from such mRNA, which portion includes a nucleotide corresponding to the internal nucleotide of the codon for the amino acid at position 703 of the full-length CD109 molecule, and that is capable of distinguishing one Gov allele from the other through the ability to hybridize under stringent conditions to the portion in question only when the nucleotide in question is A (or dA), when the probe is to detect the Gov a allele, or C (or dC), when the probe is to detect the Gov b allele.","The nucleotide in question is at position 2108 of the coding region of the CD109 cDNA sequence and lies at position 2108 in SEQ ID NO:1.The cDNA sequence has A at this position, and so is the sequence corresponding to the Gov a allele.","The nucleotide in question lies at position 954 of the sequence presented as SEQ ID NO:5 and contains an A in this position, and thus also corresponds to Gov a allele.","The Gov allele-specific oligonucleotide hybridization probes of the invention may comprise genomic DNA, cDNA, or RNA, although preferably it is DNA.","Such oligonucleotide probes can be synthesised by automated synthesis and will preferably contain about 10-30 bases, although as understood in the oligonucleotide probe hybridization assay art, as few as 8 and as many as about 50 nucleotides may be useful, depending on the position within the probe where the potential mismatch with the target is located, the extent to which a label on the probe might interfere with hybridization, and the physical conditions (e.g., temperature, pH, ionic strength) under which the hybridization of probe with target is carried out.","In accordance with another aspect of the present invention, a test kit for Gov alloantigen typing is provided comprising: (a) means for amplifying nucleic acid that comprises at least a portion of a CD109 gene, a CD109-encoding mRNA, or a CD109 cDNA made from such RNA, wherein the portion includes a nucleotide (nucleotide 2108 in SEQ ID NO:1, or nucleotide 954 in SEQ ID NO:5) corresponding to the internal nucleotide of the codon encoding amino acid 703 of the full length CD109 protein.","(b) an oligonucleotide probe of the invention, that distinguishes one Gov allele from the other.","The “means for amplifying” will, as the skilled will readily understand, depend on the amplification method to be used.","Thus, for example, these means might include suitable primers, a suitable DNA polymerase, and the four 2′-deoxyribonucleoside triphosphates (dA, dC, dG, dT), if amplification is to be by the PCR method.","To cite another example, if the amplification is to be by a method relying on transcription, such as the 3SR method, the means will include two primers, at least one of which, when made double-stranded, will provide a promoter, an RNA polymerase capable of transcribing from that promoter, a reverse transcriptase to function in primer-initiated, DNA-directed and RNA-directed, DNA polymerization and possibly also in RNAse H degradation of RNA to free DNA strands from RNA/RNA hybrids, the four ribonucleoside triphosphates (A, C, G and U), and the four 2′-deoxyribonucleoside triphosphates.","In another example, if the amplification is by the ligase chain reaction, the means will include two oligonucleotides (DNAs) and a suitable DNA ligase that will join the two if a target, to which both can hybridize adjacent to one another in ligatable orientation, is present.","The oligonucleotide probes of the invention will preferably be labelled.","The label may be any of the various labels available in the art for such probes, including, but not limited to 32 P; 35 S; biotin (to which a signal generating moiety, bound to or complexed with avidin can be complexed); a fluorescent moiety; an enzyme such as alkaline phosphatase (which is capable of catalysing a chromogenic reaction); digoxigenin, as described above; or the like.","As indicated in the examples, RFLP analysis can be employed, using BstNI (or isoschizomers thereof), in analysing cDNA or genomic DNA (with or without amplification) to determine Gov genotype.","As indicated further in the examples, electrophoretic SSCP analysis may be used to determine Gov genotype.","And as indicated in the examples, the hybridization studies outlined above may use fluorescent probes, and may be directly coupled to the DNA amplification step, as in “Real-Time PCR” or related methods.","There has also been provided, in accordance with another aspect of the present invention, a method of typing for Gov allele-specific target sequence in a CD 109 nucleic acid derived from a subject, comprising the steps of, (a) obtaining, by a target nucleic acid amplification process applied to mRNA from human platelets, endothelial cells, or T cells, an assayable quantity of amplified nucleic acid with a sequence that is that of a subsequence (or the complement of a subsequence) of the mRNA that encodes a CD109 said subsequence including the nucleotide at the position in the mRNA corresponding to position 2108 in SEQ ID NO:1 or to nucleotide 954 in SEQ ID NO:5; and (b) analyzing (e.g., in a nucleic acid probe hybridization assay employing an oligonucleotide probe or probes according to the invention) the amplified nucleic acid obtained in step (a) to determine the base or bases at the position in the amplified nucleic acid that corresponds to position 2108 in SEQ ID NO:1 or to nucleotide 954 in SEQ ID NO:5.It is noteworthy that, if the product of the amplification is double-stranded DNA, analysis for Gov genotype can be carried out by a RFLP (restriction fragment length polymorphism) analysis comprising exposing the amplified DNA to the restriction endonuclease BstNI (or isoschizomer thereof) under conditions whereby the DNA will be cleaved if it includes a site for cleavage by that enzyme.","Such DNA, prepared from mRNA encoding the Gov b alloantigen, containing a C rather than an A at the position corresponding to nucleotide 2108 in SEQ ID NO:1 (or to nucleotide 954 in SEQ ID NO:5), includes a recognition site for that endonuclease, while such DNA prepared from mRNA encoding the Gov a alloantigen, does not If the analysis, by whatever method, of the amplified nucleic acid reveals that there is only an A (or dA) at the position corresponding to position 12108, the platelets (and blood from which they came) have only the Gov a alloantigen, and the individual from whom the platelets came, is homozygous for Gov a .","Alternatively, if the analysis of the amplified nucleic acid reveals that there is only a C (or dC) at the position corresponding to position 2108, the platelets (and blood from which they came) have only the Gov b alloantigen and the individual, from whom the platelets came, is homozygous for the Gov b allele.","Finally, if the analysis indicates that there is either an A (or dA) or a C (or dC) at that position, the platelets (and blood from which they came) have both Gov alloantigens, and the individual from whom the platelets came, is heterozygous for Gov alloantigen.","In one application of the typing methods of the invention, the methods are applied to two individuals to determine whether blood or platelets from one would provoke an alloimmune response, and possibly PTP or PTPR, in the other.","The typing method can be applied with a man and a woman, who are contemplating conceiving or have conceived a child together, to determine the risk that the child would be at risk for NATP and the risk that the woman would be at increased risk for PTP or PTPR.","If the woman were heterozygous for the Gov alloantigens there would be, due to Gov alloantigen incompatibility, no risk of NATP and no increased risk for the woman of PTP or PTPR.","If, however, the woman were homozygous for one of the Gov alloantigens, there would be, due to Gov alloantigen incompatibility, risk of NATP in a child and increased risk of PTP or PTPR for the woman, unless the man is homozygous for the same Gov alloantigen as is the woman.","In accordance with yet another aspect of the present invention, a method of typing an individual for Gov alloantigen is provided that comprises analyzing the genomic DNA of the individual to determine the Gov alloantigen(s) of the individual.","Applications of this method are substantially the same as those of the method of the invention for typing for Gov alloantigen that begins with platelet, endothelial cell, or T cell mRNA.","This method of the invention, entailing analysis of genomic DNA, can be carried out in substantially the same way as outlined above for analysis of mRNA, namely first amplifying the genomic DNA and then analyzing to product of the amplification to ascertain whether there is only dA, only dC, or both dA and dC, at the position in the coding region of the genomic DNA corresponding to position 2108 in SEQ ID NO:1, or to nucleotide 954 in SEQ ID NO:5.In accordance with a further aspect of the present invention, a test kit for Gov alloantigen typing is provided comprising a non-human antibody (or antibodies) that distinguishes the two allelic forms of CD109.The antibody (or antibodies) of the kit may be polyclonal, or preferably monoclonal, and in addition to its (their) specificity for either but not both Gov alloantigens (on the surface of platelets or separated therefrom) or the CD109 subunit of one but not both of such alloantigens, typically will recognise a polypeptide molecule encoded by a nucleotide sequence encoding at least amino acid 703 of a CD109 polypeptide (the amino acid at the position corresponding to nucleotides 2107-2109 in SEQ ID NO:1, or to nucleotides 953-955 in SEQ ID NO:5).","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["FIELD OF THE INVENTION The present invention provides novel compositions and methods for use in diagnosing the occurrence of certain serious disorders, especially certain bleeding disorders, and novel compositions and methods for use in treating such a disorder, in a person in which the disorder has occurred, and novel compositions and methods for use in avoiding such a disorder, in an individual who is susceptible thereto.","BACKGROUND OF THE INVENTION Among the disorders, which the invention concerns, are those involving abnormal and excessive bleeding due to destruction of blood platelets (“platelets”).","These disorders include, but are not restricted to, post-transfusion purpura (“PTP”) and post-transfusion platelet refractoriness (“PTPR”), which are suffered by some persons who receive blood, platelets, leukocyte concentrates, or plasma from other persons by transfusion or the like.","The disorders also include one that is suffered by fetuses and newborns and is known as “neonatal alloimmune thrombocytopenia” (“NATP”).","This disorder can cause death of fetuses and serious birth defects or death of newborns.","NATP is estimated to affect about 1 in 1000 newborns.","In NATP, fetal platelets, which enter the mother's blood stream, induce production in the mother of antibodies directed against fetal platelets.","These maternal antibodies then pass with the mother's blood into the fetus and mediate destruction of platelets in the fetus.","A mother, whose fetus or newborn suffers from NATP, is at increased risk of suffering PTP or PTPR.","When platelets from a first human (a “donor”) are introduced into the blood system of a second human (a “recipient”) by transfusion, through the placenta (in the case of fetal blood entering the mother), or the like, the recipient may mount an immune response against the platelets from the donor.","Such an immune response is referred to as an “alloimmune” response, because it involves antibodies reacting-against antigens of a different individual of the same species.","The alloimmune response to platelets is due to an immune response of the recipient against “alloantigens” (antigens of the same species as that mounting the immune response) on platelets from the donor.","These alloantigens are found on membrane glycoproteins that occur in the cell membranes, which define the outer surfaces of platelets (“platelet membranes”).","In this invention, the glycoprotein is anchored to the membrane in an atypical manner through an anchor consisting of glycosylphosphatidylinositol (GPI), which anchors an extracellular domain or segment of the glycoprotein exposed to the outside of the platelet.","It is thought that alloantibodies, which are generated in an alloimmune response against platelet alloantigens, interact with the extracellular domains of the alloantigens.","The platelet alloantigens that a person has are determined by the person's genetics.","A donor, because of his or her genetics, may have a platelet alloantigen, which a recipient, who receives blood, platelets, leukocytes or plasma from the donor, does not have, because of the recipient's genetics.","In such a situation, the immune system of the recipient may recognize the donor's alloantigen as “non-self,” and raise an immune response against, the platelet alloantigen, which the donor has but the recipient does not.","Membrane glycoprotein alloantigens have been characterised for both human red blood cells and human platelets.","It is noteworthy, however, that they also occur on other cell types, such as leukocytes and endothelial cells, where they may also occasion various disorders through alloimmune responses.","Recognised classes of red blood cell and platelet alloantigens have been described, over the past 30 years, based on observations of antibody reactions occurring when blood recipients have been exposed to blood from donors.","A recent review of human platelet alloantigen systems is provided by Ouwehand, W., and Navarrete, C., in Molecular Haematology, Provan, D. and Gribben, J. eds.","Blackwell (1999).","Several biallelic platelet alloantigen “systems” have been characterised.","In each of these systems, there are two alloantigens, each of which is provided by one of two alleles of the gene comprising the system.","Because each gene occurs twice in the normal human genome, a person can be homozygous for one or the other of the two alloantigens, or heterozygous for the two alloantigens, comprising a biallelic system.","The alloantigens described to date occur on glycoprotein molecules which may exist in various forms (transmembrane, GPI-linked and soluble, for example).","In such a case, the alloantigens are found on each of the variant forms of the glycoprotein.","For all of the biallelic platelet alloantigen systems that have been characterised at the level of protein and gene sequences, it has been found in all cases, except for one, that the difference between the two alleles is based on a single nucleotide polymorphism in the relevant gene.","One biallelic system of human platelet alloantigens is the Gova/Govb biallelic system associated with CD109, a membrane glycoprotein which occurs on platelets and various other cell types, including leukocytes and endothelial cells.","Each Gov allele corresponds to one CD109 glycoprotein (Sutherland, D. R. et al, 1991; Smith et al., 1995; Berry, J. et al., 2000), consistent with the known tissue distribution of CD109.The frequencies for the Gov alleles are 0.4 for Gova and 0.6 for Govb in the Caucasian population.","Thus, in this population, 40.7% are heterozygous for the Gov alleles, and will not mount an alloimmune response due to Gov incompatibility (not possessing the Gov alloantigen found on platelets received from another).","In contrast, 19.8% of Caucasians are homozygous for the Gova allele and thus may mount an immune response due to Gov alloantigen incompatibility against platelets received from anyone in the 80.5% of the Caucasian population that is not homozygous for the Gova allele, while 39.8% are homozygous for the Govb allele and thus may mount an immune response due to Gov alloantigen incompatibility against platelets received from anyone in the 60.2% of the Caucasian population that is not homozygous for the Govb allele.","As indicated above, alloimmunization based on Gov incompatibility (the introduction into the blood stream of donor platelets bearing a Gov alloantigen not carried by the recipient) can result in bleeding disorders due to platelet destruction, including NATP, PTPR, and PTP.","The location of the Gov antigens within the CD109 molecule, and the nature of the CD109 polymorphism which underlies the Gova/Govb alloantigen (both at the protein and at the gene level), have not heretofore been known.","Furthermore, it has not heretofore been possible to generate non-human antibody (polyclonal or monoclonal), as from a rat, mouse, goat, chicken, or the like, with specificity for the Gova alloantigen but not the Govb alloantigen (or vice-versa) sufficient for use in an immunoassay, for typing for Gov phenotype using platelets or CD109 molecules.","Previously developed technology, involving gene-specific amplification of platelet RNA-derived cDNA, followed by the determination of the nucleotide sequence of the amplified DNA, has been applied successfully to the elucidation of the molecular basis of other biallelic platelet alloantigen systems (Newman et al., J. Clin.","Invest.","82,739-744 (1988); Newman et al., J. Clin.","Invest.","83, 1778-1781 (1989)(P1A or HPA-1 system); Lyman et al., Blood 75, 2343-2348 (1990)(Bak or HPA-3system); Kuijpers et al., J. Clin.","Invest.","89, 381-384 (1992)(HPA-2 or Ko system); Wang et al., J. Clin.","Invest.","90, 2038-2043 (1992)(Pen system).","With one exception, it has been found in each case that a single amino acid difference at a single position differentiates the amino acid sequences of the two alleles, and that this difference arises from a single allele-specific nucleotide substitution in the coding region of the mRNA and gene.","There remains a need to elucidate the molecular basis of the biallelic Gov platelet alloantigen system.","SUMMARY OF THE INVENTION The Gov a/Gov b CD109 Single Nucleotide Polymorphism We have now discovered that a single amino acid difference in the CD109 glycoprotein distinguishes the Gova and Govb allelic forms.","The two alleles differ at amino acid position 703 of the full-length 1445 amino acid CD109 molecule, with the Gova allele [SEQ ID NO:2] containing a Tyr at this position, while the Govb allele [SEQ ID NO:4] contains Ser.","Further, we have discovered that this difference in amino acid sequence between the allelic forms of CD109 is due to a single nucleotide polymorphism at position 2108 of the coding portion of full-length mRNA encoding CD109, or of the corresponding coding strand of the cDNA corresponding to this mRNA.","Specifically, the Gova allele [SEQ ID NO:1] contains adenine at position 2108, the second nucleotide of the codon encoding the amino acid at position 703 of the full-length CD109 protein, while the Govb allele contains cytosine at position 2108, as shown in SEQ ID NO:3 The Gova/Govb single nucleotide polymorphism of CD109, lies at position 2108 in SEQ ID NO:1.SEQ ID NO:1 is the cDNA sequence encoding the full-length 1445 amino acid CD109 precursor encoding the Gova alleleln the Govb allele form [SEQ ID NO:3], C occurs at position 2108, rather than A.","The ATG at the 5′-end of the sequence in SEQ ID NO:1 corresponds to the translation start of the full-length precursor form (including leader peptide) of CD109.The triplet corresponding to the N-terminal amino acid of the mature CD109 protein is at positions 64-66 in SEQ ID NO:1.The Gova/Govb single nucleotide polymorphism of CD109, lies at position 954 in SEQ ID NO:5.SEQ ID NO:5 is the genomic DNA sequence of human CD109 exon 19 and the contiguous introns, introns 18 and 19.The Gova/Govb single nucleotide polymorphism of CD109 is found within CD109 exon 19, and specifically is located at position 3 of CD109 exon 19.The sequence presented in SEQ ID NO:5 contains A at position 954, and thus corresponds to the Gova allele.","The corresponding Govb sequence contains C at position 954 of SEQ ID NO:5 (nucleotide position 3 of exon 19).","In view of this discovery, it will be readily apparent to the skilled what the present invention provides: Gov allele-specific oligonucleotides and polynucleotides: Based on the discovery, the present invention provides oligonucleotides and polynucleotides (seems repetitive), including (but not limited to) probes which can be used to determine whether a person is homozygous for one or the other of the Gov alleles, or heterozygous for these alleles, thereby to determine that person's Gov genotype, and by extension, their Gov phenotype (i.e., the Gov alloantigen(s) which their cells express).","Further, the invention provides methods of using such oligonucleotides, and test kits to facilitate their use, in such Gov genotype and phenotype determinations.","These oligonucleotides of the invention can be used to determine whether, in the CD109 gene, or in the mRNA encoding CD109, the internal nucleotide (nucleotide 2108) of the codon (in CD109 gene or in the mRNA encoding CD109) which corresponds to the amino acid at position 703 in the sequence of full-length CD109 is adenine or cytosine.","Such probes will typically be cDNA but may be genomic DNA, mRNA or RNA, and may be labelled for detection.","The oligonucleotides of the invention can be used as probes to detect nucleic acid molecules according to techniques known in the art (for example, see U.S. Pat.","Nos.","5,792,851 and 5,851,788).","For example, an oligonucleotide of the invention may be converted to a probe by being end-labelled using digoxigenin-11-deoxyuridine triphosphate.","Such probes may be detected immunologically using alkaline-phosphate-conjugated polyclonal sheep antidigoxigenin F(ab) fragments and nitro blue tetrazolium with 5-bromo-4-chloro-3-indoyl phosphate as chromogenic substrate.","Gov allele-specific antibodies: Still further, based on the discovery, which underlies the invention, of the molecular basis for the Gova/Govb alloantigen system, the invention provides non-human polyclonal and monoclonal antibodies, which can be used to distinguish one Gov allelic form of CD109 from the other, whether the CD109 is part of a complex embedded in or isolated from a membrane or is isolated.","These antibodies of the invention, which are preferably provided in an aqueous buffer solution, and the immunoassays of the invention which employ such antibodies, are useful for determining whether a person has one or both of the Gov alloantigens and for Gov phenotyping.","Methods of using the antibodies of the invention in the immunoassays of the invention, and in such determinations, are also encompassed by the invention.","The invention also provides test kits to facilitate carrying out such immunoassays and determinations.","Gov allele-specific peptides and polypeptides: Again, based on the discovery that underlies the invention, of the molecular basis for the Gova/Govb alloantigen system, the invention provides peptides or polypeptides, which are useful for various purposes.","These peptides or polypeptides are typically between 4 and 100, and more typically between 7 and 50, amino acids in length, and have amino acid sequences identical or having sequence identity to those of segments of the CD109 sequences, that include the amino acid at position 703 of full-length mature CD109.This amino acid (position 703) corresponds the triplet at positions 2107-2109 in the CD109 cDNA sequence presented in SEQ ID NO:1, or in the corresponding sequence for the CD109 cDNA that encodes the Govb allelic form [SEQ ID NO:3].","These peptides or polypeptides may be synthetic, may be purified from native CD109 or may be prepared by recombinant means.","For guidance, one may consult the following U.S. Pat.","Nos.","5,840,537, 5,850,025, 5,858,719, 5,710,018, 5,792,851, 5,851,788, 5,759,788, 5,840,530, 5,789,202, 5,871,983, 5,821,096, 5,876,991, 5,422,108, 5,612,191, 5,804,693, 5,847,258, 5,880,328, 5,767,369, 5,756,684, 5,750,652, 5,824,864, 5,763,211, 5,767,375, 5,750,848, 5,859,337, 5,563,246, 5,346,815, and WO9713843.Many of these patents also provide guidance with respect to experimental assays, probes and antibodies, methods, transformation of host cells, which are described below.","These patents, like all other patents, publications (such as articles and database publications) in this application, are incorporated by reference in their entirety.","Gov allele-specific peptides and polypeptides as antigens and immunogens, and Gov allele-specific polyclonal and monoclonal antibodies: These peptides or polypeptides are useful as antigens (usually coupled to a larger, immunogenic carrier [proteinaceous or otherwise], as known in the art) for making the polyclonal or monoclonal antibodies of the invention.","The peptides or polypeptides are also useful in screening monoclonal antibody-producing cultures (hybridoma cultures/E.","coli cultures or so-called V gene phage antibodies) to identify those that produce monoclonal antibodies of the invention.","The invention also encompasses immunogenic compositions which comprise a peptide, polypeptide or fusion compound of the invention and which are immunogenic in a bird, including, without limitation, a chicken, or a mammal, such as, a mouse, rat, goat, rabbit, guinea pig, sheep or human.","The compositions may include an immunogenicity-imparting “carrier” which may be but is not necessarily a protein as known in the art, that is immunogenic in a bird or mammal, coupled to at least one peptide or polypeptide of the invention, which has an amino acid sequence that is the same as that of a segment of the sequence for CD109, that includes the amino acid at position 703 of the full length CD109 molecule.","The present invention also provides methods of using the peptides, polypeptides and immunogenic compositions of the invention for making antibodies of the invention, and methods of using the peptides and polypeptides of the invention in screening monoclonal antibody-producing hybridoma cultures or bacterial clones for those that produce monoclonal antibodies or fragments thereof of the invention.","Therapeutic and diagnostic application of Gov allele-specific peptides, polypeptides, and antibodies: These peptides or polypeptides, as well as antibodies, which are specific for the Gova [SEQ ID NO:2] or Govb [SEQ ID NO:4], but not both, allelic forms of CD109 in the platelet membrane, and which can be produced by a mammal (including an human) immunized with the peptides or polypeptides, which themselves happen to be immunogenic, or the immunogenic compositions of the invention, are also useful both therapeutically and diagnostically.","The invention also provides the methods of using the peptides and polypeptides of the invention, and antibodies made using the peptides that are immunogenic and the immunogenic compositions of the invention, in therapeutic and diagnostic applications.","The Gov allele-specific peptides or polypeptides can also be used diagnostically to detect the presence of Gova or Govb specific antibodies in human plasma or serum samples, using methods that are readily apparent to those skilled in the art.","Such analyses would be useful in the investigation of cases of acquired alloimmune thrombocytopenia, including PTP, PTPR, and NATP.","In the latter case, this approach could also be used to detect the presence of Gov allele-specific antibodies in the mother of the affected fetus or newborn.","The presence of Gov allele-specific antibodies can also be detected using platelets of known Gov phenotype.","However, this approach has numerous technical disadvantages that are eliminated by the use of Gov allele-specific peptides or polypeptides for Gov allele-specific antibody detection.","Administration to a person, who is suffering from, or at risk for, for example, PTP or PTPR, or a mother at risk for passing NATP-causing alloantibodies to her fetus, of one of the peptides or polypeptides, that would be bound by the anti-Gov alloantibodies in such a person, would inhibit the binding of the alloantibodies to the person's (or the fetus's platelets and thereby inhibit the platelet destruction and abnormal bleeding associated with the disorders.","Alternatively, administration to such a person of antibodies (particularly human antibodies), which are produced using a peptide or polypeptide of the invention, which is immunogenic by itself, or an immunogenic composition of the invention, and which are specific for the Gov allelic form of the CD109 on the person's platelets which is associated with the PTP or PTPR, from which the person is suffering or may suffer, would induce the production of anti-idiotypic antibodies, which, in turn, would inhibit the platelet-destructive effects of the anti-Gov alloantibodies, which are generated by the person's own immune system and which are causing or threatening to cause the PTP, PTPR or NATP.","These therapeutic applications of peptides and polypeptides of the invention would be especially useful in treating NATP in a newborn, because the alloantibody giving rise to NATP in the newborn is not continuously produced by the immune system of the newborn, but rather is acquired passively, and therefore in limited, non-replenished quantity, by the newborn from its mother.","Thus, in accordance with one aspect of the present invention, an oligonucleotideprobe is provided that hybridizes to a portion of the CD109 gene, or a portion of CD109-encoding mRNA or cDNA prepared from such mRNA, which portion includes a nucleotide corresponding to the internal nucleotide of the codon for the amino acid at position 703 of the full-length CD109 molecule, and that is capable of distinguishing one Gov allele from the other through the ability to hybridize under stringent conditions to the portion in question only when the nucleotide in question is A (or dA), when the probe is to detect the Gova allele, or C (or dC), when the probe is to detect the Govb allele.","The nucleotide in question is at position 2108 of the coding region of the CD109 cDNA sequence and lies at position 2108 in SEQ ID NO:1.The cDNA sequence has A at this position, and so is the sequence corresponding to the Gova allele.","The nucleotide in question lies at position 954 of the sequence presented as SEQ ID NO:5 and contains an A in this position, and thus also corresponds to Gova allele.","The Gov allele-specific oligonucleotide hybridization probes of the invention may comprise genomic DNA, cDNA, or RNA, although preferably it is DNA.","Such oligonucleotide probes can be synthesised by automated synthesis and will preferably contain about 10-30 bases, although as understood in the oligonucleotide probe hybridization assay art, as few as 8 and as many as about 50 nucleotides may be useful, depending on the position within the probe where the potential mismatch with the target is located, the extent to which a label on the probe might interfere with hybridization, and the physical conditions (e.g., temperature, pH, ionic strength) under which the hybridization of probe with target is carried out.","In accordance with another aspect of the present invention, a test kit for Gov alloantigen typing is provided comprising: (a) means for amplifying nucleic acid that comprises at least a portion of a CD109 gene, a CD109-encoding mRNA, or a CD109 cDNA made from such RNA, wherein the portion includes a nucleotide (nucleotide 2108 in SEQ ID NO:1, or nucleotide 954 in SEQ ID NO:5) corresponding to the internal nucleotide of the codon encoding amino acid 703 of the full length CD109 protein.","(b) an oligonucleotide probe of the invention, that distinguishes one Gov allele from the other.","The “means for amplifying” will, as the skilled will readily understand, depend on the amplification method to be used.","Thus, for example, these means might include suitable primers, a suitable DNA polymerase, and the four 2′-deoxyribonucleoside triphosphates (dA, dC, dG, dT), if amplification is to be by the PCR method.","To cite another example, if the amplification is to be by a method relying on transcription, such as the 3SR method, the means will include two primers, at least one of which, when made double-stranded, will provide a promoter, an RNA polymerase capable of transcribing from that promoter, a reverse transcriptase to function in primer-initiated, DNA-directed and RNA-directed, DNA polymerization and possibly also in RNAse H degradation of RNA to free DNA strands from RNA/RNA hybrids, the four ribonucleoside triphosphates (A, C, G and U), and the four 2′-deoxyribonucleoside triphosphates.","In another example, if the amplification is by the ligase chain reaction, the means will include two oligonucleotides (DNAs) and a suitable DNA ligase that will join the two if a target, to which both can hybridize adjacent to one another in ligatable orientation, is present.","The oligonucleotide probes of the invention will preferably be labelled.","The label may be any of the various labels available in the art for such probes, including, but not limited to 32P; 35S; biotin (to which a signal generating moiety, bound to or complexed with avidin can be complexed); a fluorescent moiety; an enzyme such as alkaline phosphatase (which is capable of catalysing a chromogenic reaction); digoxigenin, as described above; or the like.","As indicated in the examples, RFLP analysis can be employed, using BstNI (or isoschizomers thereof), in analysing cDNA or genomic DNA (with or without amplification) to determine Gov genotype.","As indicated further in the examples, electrophoretic SSCP analysis may be used to determine Gov genotype.","And as indicated in the examples, the hybridization studies outlined above may use fluorescent probes, and may be directly coupled to the DNA amplification step, as in “Real-Time PCR” or related methods.","There has also been provided, in accordance with another aspect of the present invention, a method of typing for Gov allele-specific target sequence in a CD 109 nucleic acid derived from a subject, comprising the steps of, (a) obtaining, by a target nucleic acid amplification process applied to mRNA from human platelets, endothelial cells, or T cells, an assayable quantity of amplified nucleic acid with a sequence that is that of a subsequence (or the complement of a subsequence) of the mRNA that encodes a CD109 said subsequence including the nucleotide at the position in the mRNA corresponding to position 2108 in SEQ ID NO:1 or to nucleotide 954 in SEQ ID NO:5; and (b) analyzing (e.g., in a nucleic acid probe hybridization assay employing an oligonucleotide probe or probes according to the invention) the amplified nucleic acid obtained in step (a) to determine the base or bases at the position in the amplified nucleic acid that corresponds to position 2108 in SEQ ID NO:1 or to nucleotide 954 in SEQ ID NO:5.It is noteworthy that, if the product of the amplification is double-stranded DNA, analysis for Gov genotype can be carried out by a RFLP (restriction fragment length polymorphism) analysis comprising exposing the amplified DNA to the restriction endonuclease BstNI (or isoschizomer thereof) under conditions whereby the DNA will be cleaved if it includes a site for cleavage by that enzyme.","Such DNA, prepared from mRNA encoding the Govb alloantigen, containing a C rather than an A at the position corresponding to nucleotide 2108 in SEQ ID NO:1 (or to nucleotide 954 in SEQ ID NO:5), includes a recognition site for that endonuclease, while such DNA prepared from mRNA encoding the Gova alloantigen, does not If the analysis, by whatever method, of the amplified nucleic acid reveals that there is only an A (or dA) at the position corresponding to position 12108, the platelets (and blood from which they came) have only the Gova alloantigen, and the individual from whom the platelets came, is homozygous for Gova.","Alternatively, if the analysis of the amplified nucleic acid reveals that there is only a C (or dC) at the position corresponding to position 2108, the platelets (and blood from which they came) have only the Govb alloantigen and the individual, from whom the platelets came, is homozygous for the Govb allele.","Finally, if the analysis indicates that there is either an A (or dA) or a C (or dC) at that position, the platelets (and blood from which they came) have both Gov alloantigens, and the individual from whom the platelets came, is heterozygous for Gov alloantigen.","In one application of the typing methods of the invention, the methods are applied to two individuals to determine whether blood or platelets from one would provoke an alloimmune response, and possibly PTP or PTPR, in the other.","The typing method can be applied with a man and a woman, who are contemplating conceiving or have conceived a child together, to determine the risk that the child would be at risk for NATP and the risk that the woman would be at increased risk for PTP or PTPR.","If the woman were heterozygous for the Gov alloantigens there would be, due to Gov alloantigen incompatibility, no risk of NATP and no increased risk for the woman of PTP or PTPR.","If, however, the woman were homozygous for one of the Gov alloantigens, there would be, due to Gov alloantigen incompatibility, risk of NATP in a child and increased risk of PTP or PTPR for the woman, unless the man is homozygous for the same Gov alloantigen as is the woman.","In accordance with yet another aspect of the present invention, a method of typing an individual for Gov alloantigen is provided that comprises analyzing the genomic DNA of the individual to determine the Gov alloantigen(s) of the individual.","Applications of this method are substantially the same as those of the method of the invention for typing for Gov alloantigen that begins with platelet, endothelial cell, or T cell mRNA.","This method of the invention, entailing analysis of genomic DNA, can be carried out in substantially the same way as outlined above for analysis of mRNA, namely first amplifying the genomic DNA and then analyzing to product of the amplification to ascertain whether there is only dA, only dC, or both dA and dC, at the position in the coding region of the genomic DNA corresponding to position 2108 in SEQ ID NO:1, or to nucleotide 954 in SEQ ID NO:5.In accordance with a further aspect of the present invention, a test kit for Gov alloantigen typing is provided comprising a non-human antibody (or antibodies) that distinguishes the two allelic forms of CD109.The antibody (or antibodies) of the kit may be polyclonal, or preferably monoclonal, and in addition to its (their) specificity for either but not both Gov alloantigens (on the surface of platelets or separated therefrom) or the CD109 subunit of one but not both of such alloantigens, typically will recognise a polypeptide molecule encoded by a nucleotide sequence encoding at least amino acid 703 of a CD109 polypeptide (the amino acid at the position corresponding to nucleotides 2107-2109 in SEQ ID NO:1, or to nucleotides 953-955 in SEQ ID NO:5).","DETAILED DESCRIPTION OF THE INVENTION Definitions The following definitions are provided in order to aid those skilled in the art in understanding the detailed description of the present invention.","The term “alloantigens” refers to antigens of an individual that are responsible for eliciting an alloimmune response.","The phrase “alloimmune response” refers to an immune response, which occurs when antibodies from one individual react against antigens of a different individual of the same species.","The phrase “anti-idiotypic antibodies” refers to antibodies which can bind endogenous or foreign idiotypic antibodies and which can be used to treat or prevent pathological conditions associated with an immune response to a foreign alloantigen.","The phrase “Gova/Govb biallelic system” refers to a system of human platelet alloantigens in which an individual can be homozygous for either Gova or Govb allelic forms of CD 109, or an individual can be Gova/Govb heterozygous for CD109.“GPI” refers to glycosylphosphatidylinositol.","The term “NATP” refers to neonatal alloimmune thrombocytopenia.","“Nucleic acid” includes DNA and RNA, whether single or double stranded.","The term is also intended to include a strand that is a mixture of nucleic acids and nucleic acid analogs and/or nucleotide analogs, or that is made entirely of nucleic acid analogs and/or nucleotide analogs.","“Nucleic acid analogue” refers to modified nucleic acids or species unrelated to nucleic acids that are capable of providing selective binding to nucleic acids or other nucleic acid analogues.","As used herein, the term “nucleotide analogues” includes nucleic acids where the internucleotide phosphodiester bond of DNA or RNA is modified to enhance biostability of the oligomer and “tune” the selectivity/specificity for target molecules (Ulhmann, et al., 1990, Angew.","Chem.","Int.","Ed.","Eng., 90: 543; Goodchild, 1990, J. Bioconjugate Chem., I: 165; Englisch et al., 1991, Angew, Chem.","Int.","Ed.","Eng., 30: 613).","Such modifications may include and are not limited to phosphorothioates, phosphorodithioates, phosphotriesters, phosphoramidates or methylphosphonates.","The 2′-O-methyl, allyl and 2′-deoxy-2′-fluoro RNA analogs, when incorporated into an oligomer show increased biostability and stabilization of the RNA/DNA duplex (Lesnik et al., 1993, Biochemistry, 32: 7832).","As used herein, the term “nucleic acid analogues” also include alpha anomers (α-DNA), L-DNA (mirror image DNA), 2′-5′ linked RNA, branched DNA/RNA or chimeras of natural DNA or RNA and the above-modified nucleic acids.","For the puposes of the present invention, any nucleic acid containing a “nucleotide analogue” shall be considered as a nucleic acid analogue.","Backbone replaced nucleic acid analogues can also be adapted to for use as immobilised selective moieties of the present invention.","For purposes of the present invention, the peptide nucleic acids (PNAs) (Nielsen et al, 1993, Anti-Cancer Drug Design, 8: 53; Engels et al., 1992, Angew, Chem.","Int.","Ed.","Eng., 31: 1008) and carbamate-bridged morpholino-type oligonucleotide analogs (Burger, D. R., 1993, J.","Clinical Immunoassay, 16: 224; Uhlmann, et al., 1993, Methods in Molecular Biology, 20,.","“Protocols for Oligonucleotides and Analogs,” ed.","Sudhir Agarwal, Humana Press, NJ, U.S.A., pp.","335-389) are also embraced by the term “nucleic acid analogues”.","Both exhibit sequence-specific binding to DNA with the resulting duplexes being more thermally stable than the natural DNA/DNA duplex.","Other backbone-replaced nucleic acids are well known to those skilled in the art and may also be used in the present invention (See e.g., Uhlmann et al 1993, Methods in Molecular Biology, 20, “Protocols for Oligonucleotides and Analogs,” ed.","Sudhir Agrawal, Humana Press, NJ, U.S.A., pp.","335).","The term “PTP” refers to post-transfusion purpura.","The term “PTPR” refers to post-transfusion platelet refractorines.","“SNP” refers to single nucleotide polymorphism.","The standard, one-letter codes “A,” “C,” “G, ” and “T” are used herein for the nucleotides adenylate, cytidylate, guanylate, and thymidylate, respectively.","The skilled will understand that, in DNAs, the nucleotides are 2′-deoxyribonucleotide-5′-phosphates (or, at the 5′-end, possibly triphosphates) while, in RNAs, the nucleotides are ribonucleotide-5′-phosphates (or, at the 5′-end, possibly triphosphates) and uridylate (U) occurs in place of T. “N” means any one of the four nucleotides.","On occasion herein, dA, dC, dG and dT might be used for the respective 2′-deoxyribonucleotides.","Unless otherwise specified or required by the context, “nucleic acid” means DNA or RNA and “nucleotide” means ribonucleotide or 2′-deoxyribonucleotide.","Reference herein to a “full-length” CD109 molecule or protein means the 1445-amino acid-long polypeptide, for which the amino acid sequence, deduced from a cDNA sequence, is provided in SEQ ID NO:1 and in SEQ ID NO:3 and which is denoted as the full-length translated product (i.e., including the amino-terminal leader peptide, and excluding carboxyl-terminal processing associated with GPI anchor addition).","The Gova alloantigen bearing form of CD109 may be referred to herein as 703Tyr CD109.The Govb alloantigen bearing form of CD109 may be referred to herein as 703Ser CD109.It has been determined that a single nucleotide of the CD109 gene is responsible for the Gov polymorphism in CD109.Extensive serological studies initially demonstrated that the polymorphism underlying the Gov system resides solely on the CD109 molecule [Sutherland, D. R. (1991); Smith et al.","(1995)].","Further, extensive deglycosylation of CD109 does not affect the binding the anti-Gova and anti-Govb antibodies to molecules of the appropriate phenotype, or to cells bearing the appropriate CD109 variant, indicating that carbohydrate residues are not involved in the formation of Gov antigenic epitopes.","Further work has indicated that the Gov allele-specific antibody binding can however, be abrogated by denaturation of CD109 with the detergent SDS [Smith et al.","(1995)].","Taken together, these observations indicate that the Gov alleles of CD109 are protein epitopes that are likely defined by the primary amino acid sequence of CD109.Following the isolation of a CD109 cDNA the nature of the two Gov alleles was characterised further using platelet RNA-derived cDNA in the polymerase chain reaction (“PCR”).","Platelet mRNA transcripts were obtained from serologically defined Gova/a, Gova/b and Govb/b individuals.","The RNA was then converted to cDNA, and the entire CD109 cDNA coding region was then amplified as a series of overlapping PCR products.","The Gova [SEQ ID NO:1] and Govb [SEQ ID NO:3] alleles differ by an A to C substitution at position 2108 of the coding region of the CD109 cDNA.","This single nucleotide polymorphism also results in a BstNI restriction site in the Govb allele that is not present in its Gova counterpart.","On the basis of this BstNI site, Gova can by distinguished from Govb by restriction fragment length polymorphism (RFLP) analysis.","This single nucleotide polymorphism can also be detected by SSCP analysis, and by allele-specific hybridization studies, including “Real-Time” PCR analyses.","As a result of this A2108C single nucleotide polymorphism, the Gova allele [SEQ ID NO:2] of CD109 contains a Tyr at position 703 of the full-length protein, while the Govb allele [SEQ ID NO:4] contains a Ser in this position.","The polymorphism does not alter the ability of Gova and Govb homozygous platelets to adhere to collagen types I, III and V. Additionally, the binding of anti-Gova and ant-Govb antibodies to platelets of the appropriate phenotype did not interfere with platelet adhesion to any of the above collagen types.","Thus, while the Tyr703Ser results in the formation of the Gov alloantigen epitopes, it does not appear to impair platelet function.","Identification and characterisation of the Gov alloantigen system permits pre- and post-natal diagnosis of the Gov phenotype of an individual, providing a warning for the possibility of NATP, PTP and PTPR.","Allelic Gov typing of CD109 equates with the Gov status of the CD109 protein of an individual.","The Gov system led to diagnostic and therapeutic strategies to avoid or control diseases that result from Gov incompatibility.","The present invention can be applied to these tasks and goals in a variety of ways, illustrative examples of which are discussed below.","For example, an oligonucleotide probe can be synthesized, in accordance with the present invention, that will hybridize to a cDNA segment, derived from CD109 mRNA, that contains the nucleotide G at polymorphic nucleotide 2108 (nucleotide=guanylate).","Alternatively, an oligonucleotide probe can be synthesized that will hybridize with a CD109 cDNA segment containing the base adenine at nucleotide 2108.(nucleotide=adenylate).","These allele-specific probes can be appropriately labelled and added to the generated cDNA segments under annealing conditions, such that only one of the allele-specific probes hybridizes and can be detected, thereby identifying the specific Gova or Govb allele.","In accordance with conventional procedures, the design of an oligonucleotide probe according to the present invention preferably involves adjusting probe length to accommodate hybridization conditions (temperature, ionic strength, exposure time) while assuring allele-specificity.","A length of ten to thirty nucleotides is typical.","Diagnostic kits can also be used, in accordance with the present invention, for the determination and diagnosis of alloantigen phenotypes via the procedures described herein.","Such a kit can include, among others, antibodies or antibody fragments to an antigenic determinant expressed by either of the above-described Gova- and Govb-encoding sequences.","These antibodies would react with the blood sample of an individual so as to indicate whether that individual has a Gova or Govb phenotype.","Alternatively, all the reagents required for the detection of nucleotide(s) that distinguish the Gov alloantigens, by means described herein, can be provided in a single kit that uses isolated genomic DNA, platelet (or other cellular) mRNA or total RNA, or corresponding cDNA from an individual.","A kit containing a labelled probe that distinguishes, for example, nucleotide 2108 of CD109 can be utilised for Gov alloantigen genotyping and phenotyping.","A further beneficial use of the nucleotide sequences that distinguish the Gova allele from the Govb allele is to obtain or synthesize the respective expression product, in the form of a peptide or polypeptide, encoded by these nucleotide sequences.","These polypeptides can be used to generate antibodies for diagnostic and therapeutic uses, for example, with regard to pathological conditions such as PTP, PTPR or NATP.","These polypeptides can also be used diagnostically to detect the presence of Gova or Govb specific antibodies in patient plasma or serum, or used therapeutically (see below; assays may be adopted, for example, from U.S. Pat.","No.","5,851,788).","A polypeptide within the present invention which can be used for the purpose of generating such antibodies preferably comprises an amino-acid sequence that corresponds to (i.e., is coincident with or functionally equivalent to) a fragment of the CD109 molecule that includes amino acid 703.When amino acid 703 is Tyrosine, the polypeptide can be used, as described above, to produce antibodies that specifically bind the Gova form of CD109; in contrast, when it is Serine, antibodies can be obtained that specifically recognise the Govb form.","The class of polypeptides thus defined, in accordance with the present invention, is not intended to include the native CD109 molecule, but does encompass fragments of the molecule, as well as synthetic polypeptides meeting the aforementioned definition.","Although the length of a polypeptide within this class is not critical, the requirement for immunogenicity may require that the polypeptide be attached to an immunogenicity-imparting carrier.","Such carriers include a particulate carrier such as a liposome or a soluble macromolecule (protein or polysaccharide) with a molecular weight in the range of about 10,000 to 1,000,000 Daltons Additionally, it may be desirable to administer the polypeptide with an adjuvant, such as complete Freund's adjuvant For artificial polypeptides, as distinguished from CD109 fragments, maximum length is determined largely by the limits of techniques available for peptide synthesis, which are currently about fifty amino acids.","Thus, a synthetic polypeptide of the present invention is preferably between four to about fifty amino acids in length.","In the context of the present invention, the term “antibody” encompasses monoclonal and polyclonal antibodies produced by any available means.","Such antibodies can belong to any antibody class (IgG, IgM, IgA, etc.)","and may be chimeric.","Examples of the preparation and uses of polyclonal antibodies are disclosed in U.S. Pat.","Nos.","5,512,282, 4,828,985,5,225,331 and 5,124,147 which are incorporated by reference in their entirety The term “antibody” also encompasses antibody fragments, such as Fab and F(ab′)2 fragments, of anti-Gova or anti-Govb antibodies, conjugates of such fragments, and so-called “antigen binding proteins” (single-chain antibodies) which are based on anti-Gova or anti-Govb antibodies, in accordance, for example, with U.S. Pat.","No.","4,704,692, the contents of which are hereby incorporated by reference.","Alternatively, monoclonal antibodies or fragments thereof within the present invention can be produced using conventional procedures via the expression of isolated DNA that encodes variable regions of such a monoclonal antibody in host cells such as E. coli (see, e.g., Ward et al., Nature, 341:544-546 (1989)) or transfected murine myeloma cells (see Gillies et al., Biotechnol.","7:799-804 (1989); Nakatani et al., Biotechnol.","7:805-810 (1989)).","For additional examples of methods of the preparation and uses of monoclonal antibodies, see U.S. Pat.","Nos.","5,688,681, 5,688,657, 5,683,693, 5,667,781, 5,665,356, 5,591,628, 5,510,241, 5,503,987, 5,501,988, 5,500,345 and 5,496,705 that are incorporated by reference in their entirety.","While human alloantisera currently used for serological typing are specifically excluded from this definition, the use of CD109 or Gov allele-specific peptides to detect anti-Gov antibodies in human plasma or serum, or to determine the specificity of such alloantibodies, are specifically included.","Similarly, the use of such CD109 peptides or Gov allele-specific peptides to purify CD109 antibodies, or allele-specific CD109 antibodies from human serum is specifically included.","Similarly, the use in vitro of such CD109 peptides or Gov allele-specific peptides to deplete allele-specific antibody activity from human serum samples, or to block CD109 antibody binding, or allele-specific antibody binding, is specifically included.","Diagnostic applications of these antibodies are exemplified, according to the present invention, by the use of a kit containing an anti-Gova or an anti-Govb antibody, which undergoes a reaction with a sample of an individual's blood to determine a Gova or Govb platelet phenotype.","Such a reaction involves the binding of anti-Gova antibody to Gova antigen or the binding of anti-Govb antibody to Govb antigen.","The observation of antibody-antigen complex in a blood sample would indicate a positive result.","A kit of this type could be used to diagnose, or to help prevent the occurrence of pathological conditions like PTP, PTPR, or NATP.","A polypeptide of the present invention that is recognised specifically by anti-Gova or ant-Govb antibodies can also be used therapeutically.","Thus, antibodies raised against such a polypeptide can be employed in the generation, via conventional methods, of anti-idiotypic antibodies, that is, antibodies that bind an anti-Gova or anti-Govb antibody.","See, e.g., U.S. Pat.","No.","4,699,880, the contents of which are hereby incorporated by reference.","Such anti-idiotypic antibodies would bind endogenous or foreign anti-Gov antibodies in the blood of an individual, which would treat or prevent pathological conditions associated with an immune response to a “foreign” Gov alloantigen.","Alternatively, a polypeptide within the present invention can be administered to an individual, with a physiologically-compatible carrier, to achieve the same qualitative effect, namely, the selective reduction or elimination of circulating anti-Gov antibodies from a patient suffering or at risk from an immune response, or the abrogation by competitive binding to administered peptide, of the binding of Gov-specific antibodies to the platelets of such an individual The present invention is further described below by reference to the following, illustrative examples.","EXAMPLE 1 PCR Amplification and Analysis of PCR Products Platelet total RNA was isolated from EDTA anticoagulated blood of Govaa and Govbb individuals in the manner described in Lymann et al., Blood 75:234348 (1990).","First, platelet mRNA in 10 μl aliquots was heated to 70° C. for 10 minutes and quickly cooled on ice before reverse transcription.","The first strand cDNA was then synthesized using 10 μM oligo dT, 40 units RNAsin (Promega), 2 mM of each dNTP (dN triphosphate) (Pharmacia), 500 units of cloned MMLV reverse transcriptase and 5× enzyme buffer (Gibco) in a total volume of 50 μl.","The cDNA synthesis was carried out at 42° C. for 45 minutes and was stopped by chilling to 0° C. Overlapping sets of oligonucleotide primers (Table 2) based on the sequence of CD109 were then used to amplify by PCR the entire coding region of platelet CD109 in 8 overlapping segments that spanned the entire CD109 open reading frame.","TABLE 2 Anneal- ing Temper- Frag- Size ature ment Sense Primer Antisense Primer (bp) (° C.) 1 K1-80 (−24) K1-650 544 568 59 5′ GTAGCCCAGGCAGACGCC 3′ 5′ GTGACAACCACTGTTGGATCAA 3′ 2 K1-1 445 K1-1120 1014 570 50 5′ CGCATTGTTACACTCTTCTC 3′ 5′ TACATTTCTTGAAATACCTG 3′ 3 K1-1022 910 K1-REV-1 1747 838 50 5′ GATTCTTCAAATGGACTTT 3′ 5′ GGCTGTGTCACAGAGATC 3′ 4 KI-1400 1291 GSP3 2165 875 55 5′ TGAATTCCCAATCCTGGAGGA 3′ 5′ GCCACCCAAGAAGTGATAGA 3′ 5 K1-M43 1898 6R4N 2998 1101 56 5′ TTCAGGAATGTGGACTCTGG 3′ 5′ CGGCTTCAAGGAAACATCT 3′ 6 K1-3080 2948 1-SN 3859 912 56 5′ CTGGGAGCACTTGGTTGTCA 3′ 5′ CAGCAACATCTAAATCAAAGGC 3′ 7 K1-3570 3462 7U3N 4337 876 50 5′ ACAATTTCAGACTTCTGAGG 3′ 5′ CACAGCCAAAGTTCCATA 3′ 8 K1-3920 3812 K1-4600 4489 678 55 5′ GACGAAGATCTATCCAAAATC 3′ 5′ GCTAGGACCTGTTGTACACC 3′ Table 2 lists the position of the 5′ end of each oligonucleotide with respect to the CD109 cDNA sequence, which includes both 3′ and 5′ untranslated regions, is noted in parentheses.","The CD109 ORF encompasses nucleotides 1-4335 of the published CD109 cDNA, and corresponds exactly to the CD109 cDNA sequence presented in SEQ ID NO:1.The size of each PCR product, and the annealing temperature used for the corresponding primer pair, is listed.","PCR reactions (50:I) containing 1×PCR buffer (Gibco Life Technologies), 1.5 mM MgCl2, 200:M of each dNTP, 1:M of each primer, 1.25 units Taq polymerase (Gibco Life Technologies), and 3:I cDNA underwent 40 cycles of 94° C. (45 seconds), primer-specific annealing temperature (Table 2; 45 seconds), and 72° C. (45-60 seconds), using a Perkin Elmer 2400 thermocycler.","PCR products (30:I) were subsequently size-separated electrophoretically on a 1.2% agarose/TAE gel containing 1:g/ml ethidium bromide.","Bands were subsequently excised and purified (50:I) using the QIAquick (Qiagen) kit for direct sequencing and subcloning.","Sequencing reactions (3-5:1 purified product per reaction) were carried out using the Thermosequenase Cy5.5 dye terminator sequencing kit (Amersham Pharmacia Biotech) and the same primers that had been used for initial PCR amplification (Table 2), or selected internal CD109-specific primers as appropriate, and were subsequently analysed using the Open Gene automated DNA sequencing system (Visible Genetics).","In parallel, PCR products were cloned into PmeI-digested pMABI, a pBS SK(−) (Stratagene) derivative containing a PmeI restriction site within the polylinker.","Resultant plasmid clones were analysed by alkaline lysis/restriction digestion, and as appropriate (and following an additional overnight 13% PEG/ 1.6 M NaCl precipitation), by DNA sequence analysis as above.","By combining direct PCR sequencing and the analysis of subcloned fragments, it was ensured that the DNA sequence of each PCR-derived cDNA fragment was obtained independently at least twice, with each fragment being sequenced in both directions in its entirety.","This analysis revealed that the CD109 cDNA sequences of Govaa and Govbb individuals differed by a single nucleotide at position 2108 of the sequence shown in SEQ ID NO:1.Gova/a individuals have an A at position 2108, whereas Govb/b individuals have a C at the same position.","This change results in a Tyr-Ser amino acid polymorphism at residue 703 of the full-length CD109 polypeptide chain.","This single nucleotide polymorphism also results in a BstNI restriction site in the Govb allele that is not present in the Gova allele.","Analysis of the other regions of the CD109 cDNA in their entirety revealed no other nucleotide differences that segregated with Gov phenotype (i.e., that could be used to distinguish the Gova allele from the Govb allele).","To facilitate subsequent genomic DNA analyses of the Gova/b alleles, the intron/exon junctions of the exon bearing the putative Gov-specific nucleotide substitution identified above, as well as the DNA sequence of the flanking introns, were determined.","CD109 cDNA-specific oligonucleotides binding in the vicinity of this substitution were used for the direct sequencing of p4L10, a pCYPAC—1-derived PAC clone bearing the human CD109 locus using the Open Gene system (Visible Genetics) as above.","The nucleotide sequence of the Gov polymorphism-containing exon, as well as of the flanking introns, is presented in SEQ ID NO:5.The Gov polymorphism lies at nucleotide position 954 in SEQ ID NO:5.Subsequent work has mapped the intron-exon structure of the entire human CD109 locus, and has determined that the Gov single nucleotide polymorphism of CD109 lies in exon 19 of the CD109 gene.","EXAMPLE 2 RFLP Analysis of PCR Amplified Genomic DNA The A-C Gov CD109 polymorphism corresponds to the internal nucleotide of the first complete codon of exon 19 of the CD109 gene.","As this exon comprises only 118 nucleotides, and the Gov polymorphism lies almost at the extreme 5′ end of this exon, we determined the nucleotide sequence of both introns flanking this exon to facilitate subsequent genomic DNA analyses of the Gova/b alleles.","The DNA sequence of CD109 exon 19 and its flanking introns (CD109 introns 18 and 19) is presented as SEQ ID NO:5.To confirm that the A to C polymorphism at position 2108 of the CD109 open reading frame (nucleotide 2108, SEQ ID NO: 1; nucleotide 954, SEQ ID NO:5) segregates with the Gov phenotype, RFLP analysis was carried out on PCR amplified genomic CD109 DNA using the BstNI restriction endonuclease, which recognises the DNA sequence 5′CCAGG 3′ found in the Govb cDNA (nucleotides position 2108-2112 in SEQ ID NO:3; the corresponding Gova sequence, 5′ ACAGG 3′, is nucleotides 2108-2112 in SEQ ID NO:1).","This enzyme does not cleave at 5′ ACAGG 3′ (found in Gova; nucleotides 2108-2112 in SEQ ID NO: 1).","A 448 bp genomic fragment was PCR-amplified from Govaa, Govab, and Govbb individuals using the pair of oligonucleotides SEQ ID NO:9 and SEQ ID NO:10.These oligonucleotides flank exon 19.The former binds within intron 18 (nucleotides 875-892 SEQ ID NO:5), while the latter binds within intron 19 to the sequence complementary to nucleotides 1305-1322 of SEQ ID NO:5).","The resultant 448 bp PCR product, when digested with BstNI, yielded the restriction fragments predicted on the basis that the A to C polymorphism at position 2108 (SEQ ID NO: 1) segregates with the Gov phenotype.","EXAMPLE 3 Hybridization Analysis of PCR Amplified Genomic DNA To further confirm that the A to C polymorphism at position 2108 of the CD109 open reading frame (nucleotide 2108, SEQ ID NO:1; nucleotide 954, SEQ ID NO:5) segregates with the Gov phenotype, we also performed an alternative analysis involving the selective hybridization of Gov allele-specific DNA probes to PCR amplified genomic CD109 DNA.","Two primers flanking the polymorphic A-C site at position 2108 (SEQ ID NO:1; position 954, SEQ ID NO:5) were designed to amplify by PCR a 105 bp genomic DNA fragment containing the polymorphic site from genomic DNA isolated from Govaa, Govab, and Govbb individuals.","The first primer (SEQ ID NO:11) binds within intron 18 to nucleotides 902-928 of SEQ ID NO:5.The second primer (SEQ ID NO:12) binds within exon 19 to the sequence complementary to nucleotides 977-1106 of SEQ ID NO:5.Two additional nucleotide probes were designed—one specific for the target sequence of the Gova allele of the CD109 gene, and the other for the Govb allele of the CD109 gene.","The first probe (SEQ ID NO:13) overlaps the CD109 intron 18/exon 19 junction, binds to the Gova allele at nucleotides 935-974 of SEQ ID NO:5, and was tagged with the fluorescent dye 6-FAM.","The second probe (SEQ ID NO:14), also overlapping the CD109 intron 18/exon 19 junction, binds to the Govb allele at the position corresponding to nucleotides 935-971 of SEQ ID NO:5, and was tagged with the fluorescent dye VIC.","Genomic DNA was isolated from Gov phenotyped human peripheral blood leukocytes, and PCR/hybridization analysis was carried out using Taqman real-time PCR technology (Perkin Elmer).","Genomic DNA was amplified using primers SEQ ID NO:11 and SEQ ID NO:12, with each reaction additionally containing 100 nM FAM-labelled Gova probe and 200 nM VIC-labelled Govb probe.","Allelic discrimination, based on allele-specific fluorescence, was then determined using a post-PCR plate reader (Perkin Elmer).","In all cases, PCR/fluorescence-based Gov genotyping correlated with the Gov phenotype, indicating that the A to C polymorphism at position 2108 (SEQ ID NO: 1) does indeed segregate with the Gov phenotype.","EXAMPLE 4 SSP Analysis of PCR Amplified Genomic DNA To further confirm that the A to C polymorphism at position 2108 of the CD109 open reading frame (nucleotide 2108, SEQ ID NO:1; nucleotide 954, SEQ ID NO:5) segregates with the Gov phenotype, we also performed an alternative analysis involving SSCP analysis of PCR amplified genomic CD109 DNA.","Two Gov allele-specific antisense oligonucleotides—SEQ ID NO:6 and SEQ ID NO:7—differing by a single 3′ nucleotide (and binding to sequence complementary to nucleotides 954-976 of SEQ ID NO:5, and of the Govb counterpart of SEQ ID NO:5, respectively), were combined with a common sense primer—SEQ ID NO:8 binds within intron 18 and which corresponds to nucleotides 752-773 of SEQ ID NO:5, to amplify a 225 bp genomic DNA fragment containing the Gov polymorphic site from genomic DNA isolated from Govaa, Govab, and Govbb individuals.","In all cases, complete concordance between PCR-SSP analysis and Gov phenotyping was observed.","Sequences: SEQ ID NO: 1 consists of the entire 4335 nucleotide CD109 cDNA open reading frame encoding the Gova allele.","The Gova allele comprises an A at nucleotide position 2108.SEQ ID NO:2 consists of the entire 1445 aa protein sequence produced from CD109 Gova cDNA.","The Gova allele comprises a Tyr at amino acid 703.SEQ ID NO: 3 consists of the entire 4335 nucleotide CD109 cDNA open reading frame encoding the Govb allele.","The Govb allele comprises a C at nucleotide position 2108.SEQ ID NO: 4 consists of the entire 1445 aa protein sequence produced from the CD109 Govb cDNA.","The Govb allele comprises a Ser at amino acid 703.SEQ ID NO: 5 consists of the CD109 genomic DNA comprising CD109 exon 19 and the flanking introns, introns 18 and 19.The 118 nucleotide exon 19, comprising nucleotides 952-1069 of SEQ ID NO:5, corresponds to nucleotides 2106-2223 of SEQ ID NO: 1.The A to C Gov polymorphism of CD109 (corresponding to nucleotide 2108 of SEQ ID NO: 1) therefore corresponds to nucleotide 954 of SEQ ID NO:5.In the Gova allele, nucleotide 954 is A, while in the Govb allele nucleotide 954 is C. Thus, SEQ ID NO:5 corresponds to the Gova allele of CD109.Within SEQ ID NO:5, nucleotides 1-951 correspond to CD109 intron 18, while nucleotides 1070-2608 correspond to intron 19.We note that nucleotides 2108-2112 of SEQ ID NO: 1, and the corresponding nucleotides 954-958 of SEQ ID NO:5, which consist of the sequence 5′ ACAGG 3′ (and which contains the Gova allele-specific polymorphic nucleotide at its 5′ end), is not cleavable by the restriction endonuclease BstNI.","However, in the corresponding Govb allele, the corresponding sequence—5′ CCAGG 3′—is cleavable by BstNI, and that the two Gov alleles can be discriminated on this basis.","We note also that a group of restriction endonucleases—Bst2UI, BstNI, BstOI, EcoRII, MaeIII, MspR91, MvaI, or ScrFI (or one of their isoschizomers)—is capable of differentiating between the Gova and Govb alleles on this basis.","SEQ ID NO:6- SEQ ID NO:14 comprise oligonucleotides for the PCR amplification of Gov polymorphism containing CD109 sequence from RNA, cDNA derived from RNA, or from genomic DNA, and for the Gov typing analyses of such amplified DNA fragments.","SEQ ID NO:6.SEQ ID NO: 3, an antisense oligonucleotide specific for the Gova allele, binds to exon 19 sequence complementary to nucleotides 954-976 of SEQ ID NO:5.SEQ ID NO:6 and SEQ ID NO: 7 (see below) differ by a single allele-specific 3′ nucleotide SEQ ID NO:7.SEQ ID NO:7, an antisense oligonucleotide specific for the Govb allele, binds to exon 19 sequence complementary to nucleotides 954-976 of the Govb counterpart of SEQ ID NO:5.SEQ ID NO:6 (see above) and SEQ ID NO:7 differ by a single allele-specific 3′ nucleotide.","SEQ ID NO:8.SEQ ID NO:8 binds within intron 18, and corresponds to nucleotides 752-773 of SEQ ID NO:5.SEQ ID NO:9.SEQ ID NO:9 binds within intron 18 (nucleotides 875-892 SEQ ID NO:5).","SEQ ID NO:10.SEQ ID NO:10 binds within intron 19 to the sequence complementary to nucleotides 1305-1322 of SEQ ID NO:5.SEQ ID NO:11 SEQ ID NO:1 binds within intron 18 to nucleotides 902-928 of SEQ ID NO:5.SEQ ID NO:12.SEQ ID NO:12, binds within exon 19 to the sequence complementary to nucleotides 977-1006 of SEQ ID NO:5.SEQ ID NO:13.SEQ ID NO:13, specific for the Gova allele, overlaps the CD109 intron 18/exon 19 junction, and binds to the Gova allele at nucleotides 935-974 of SEQ ID NO:5.SEQ ID NO:14.SEQ ID NO:14, specific for the Govb allele, overlaps the CD109 intron 18/exon 19 junction, and binds to the Govb allele at the position corresponding to nucleotides 935-971 of SEQ ID NO:5."]],"string":"[\n [\n \"Diagnosis and treatment of blood disorders\",\n \"Based on the discovery of the nucleotide and amino acid differences which distinguish the Gova and Govb allelic forms of the membrane glycoprotein CD109, and which comprise the biallelic Gov platelet alloantigen system, compositions and methods are provided for determining the Gov genotype and phenotype of individuals.\",\n \"Also provided, on the basis of this discovery, are compositions and methods for treating disorders associated with Gov alloantigen incompatibility, such as the bleeding disorders post-transfusion purpura, post-transfusion platelet refractoriness, and neonatal alloimmune thrombocytopenia.\",\n \"The two allelic forms of CD109 differ by a single amino acid.\",\n \"The Gova allelic form has Tyr at amino acid position 703 in the CD109 sequence.\",\n \"The Govb allelic form has Ser at the same position.\",\n \"This amino acid difference is due to a single change, from A for the Gova allele to C for the Govb allele, in the CD109 gene.\"\n ],\n [\n \"1.An oligonucleotide comprising a sequence which binds specifically to (i) a region of CD109 nucleic acid that includes a single nucleotide polymorphism that is distinctive of a Gova allele and/or (ii) a region of CD109 nucleic acid that includes a single nucleotide polymorphism that is distinctive of a Govb allele.\",\n \"2.The oligonucleotide of claim 1, comprising 8 to 50 nucleotides.\",\n \"3.The oligonucleotide of claim 1, wherein the nucleic acid specifically binds to one of (i) or (ii) under high stringency hybridization conditions.\",\n \"4.The oligonucleotide of claim 2, wherein the stringent hybridization conditions comprise 0.1×SSC, 0.1% SDS at 65° C. 5.The oligonucleotide of claim 1, wherein the CD109 nucleic acid comprises genomic DNA, cDNA, or RNA corresponding to the Gova allele of the CD109 gene or locus, or comprises genomic DNA, cDNA, or RNA corresponding to the Govb allele of the CD109 gene or locus.\",\n \"6.The oligonucleotide of claim 5, wherein the Gova allele comprises an A at a position corresponding to position 2108 of SEQ ID NO:1 and corresponding to position 954 of SEQ ID No:5.7.The oligonucleotide of claim 5, wherein the Govb allele comprises a C at a position corresponding to position 2108 of SEQ ID NO:3 and corresponding to position 954 of SEQ ID NO:5.8.The oligonucleotide of claim 1, comprising a sequence complementary to the Gova allele or to the Govb allele.\",\n \"9.The oligonucleotide of claim 6, comprising a sequence selected from the group consisting of: (a) 8-50 nucleotides of SEQ ID NO:1; (b) a sequence that is complementary to a sequence specified in (a); and (c) a sequence having at least 70% sequence identity to a sequence in (a) or (b), wherein the sequence having identity is capable of hybridization to CD109 under high stringency hybridization conditions.\",\n \"10.The oligonucleotide of claim 7, comprising a sequence selected from the group consisting of: (a) 8-50 nucleotides of SEQ ID NO:3; (b) a sequence that is complementary to a sequence specified in (a); and (c) a sequence having at least 70% sequence identity to a sequence in (a) or (b), wherein the sequence having identity is capable of hybridization to CD109 under high stringency hybridization conditions.\",\n \"11.A oligonucleotide comprising all or part of any one of SEQ ID NO:6-SEQ ID NO:14 or a complement thereof.\",\n \"12.The oligonucleotide of claim 11, comprising 8 to 50 nucleic acids.\",\n \"13.The oligonucleotide of claim 1, wherein the nucleic acid is capable of use as a probe in a hybridization assay.\",\n \"14.The oligonucleotide of claim 1, wherein the nucleic acid sequence is detectably labelled.\",\n \"15.The oligonucleotide of claim 14, wherein the detectable label comprises: (a) a fluorogenic dye; and/or (b) a biotinylation modification; and/or (c) a radiolabel.\",\n \"16.The oligonucleotide of claim 1, wherein the sequence comprises DNA, a DNA analog, RNA or an RNA analog.\",\n \"17.The oligonucleotide of claim 1, wherein the oligonucleotide is attached to a substrate.\",\n \"18.The oligonucleotide of claim 1, wherein the oligonucleotide is capable of use as a primer that will specifically bind proximate to, and/or cause elongation through, a CD109 sequence, including the single nucleotide polymorphism distinctive of the Gova or Govb alleles.\",\n \"19.A Gov genotyping kit comprising a detection agent for detecting the presence of a Gov allele-specific target sequence in a CD109 nucleic acid derived from a subject.\",\n \"20.The kit of claim 19, wherein the detection agent comprises a nucleic acid and/or a restriction enzyme.\",\n \"21.The kit of claim 19, further comprising a container.\",\n \"22.The kit of claim 21, wherein the container comprises a biological sample container for housing the detection agent.\",\n \"23.The kit of claim 19, further comprising a plate having a plurality of wells and having bound thereto probes having a nucleic acid sequence which specifically binds to a CD109 sequence including a Gova or a Govb allele target sequence.\",\n \"24.The kit of claim 20, wherein the restriction enzyme is selected from the group consisting of Bst2UI, BstNI, BstOI, EcoRII, MaeIII, MspR91, MvaI, ScrFI or an isoschizomer thereof.\",\n \"25.The kit of claim 19, further comprising an amplification agent for amplifying the nucleic acid.\",\n \"26.The kit of claim 25, wherein the amplification agent amplifies a region of CD109 platelet, T cell, or endothelial cell mRNA including the single nucleotide polymorphism distinctive of a Gova or Govb allele.\",\n \"27.The kit of claim 25, wherein the amplification agent comprises a primer set including first and second primers, wherein the first primer is a nucleic acid that will specifically bind proximate to, and/or cause elongation through, CD109 sequence that includes the single nucleotide polymorphism distinctive of a Gova allele and the second primer is a nucleic acid that will specifically bind proximate to, and/or cause elongation through, CD109 sequence that includes the single nucleotide polymorphism distinctive of a Govb allele.\",\n \"28.The kit of claim 19, wherein the nucleic acid is obtained by amplification with all or part of the nucleic acid of any one of SEQ ID NO:6- SEQ ID NO:14 or the complement thereof.\",\n \"29.The kit of claim 19, further comprising all or part of a CD109 gene, a CD109-encoding mRNA, or a CD109 cDNA made from a CD109-encoding mRNA.\",\n \"30.The kit of claim 19, comprising the oligonucleotide of any of claims 1 to 18.31.The kit of claim 19, for detecting that the subject has or is at risk of a disease, disorder or abnormal physical state.\",\n \"32.The kit of claim 31, wherein the disease, disorder or abnormal physical state comprises a blood disease, disorder or abnormal physical state.\",\n \"33.The kit of claim 32, wherein the blood disease, disorder or abnormal physical state comprises bleeding of the subject, or increased risk of bleeding, due to destruction of blood platelets.\",\n \"34.The kit of claim 33, wherein the blood disease, disorder or abnormal physical state comprises post-transfusion purpura (“PTP”), post-transfusion platelet refractoriness (“PR”) or neonatal alloimmune thrombocytopenia (“NAIT”).\",\n \"35.The kit of claim 33, wherein the nucleic acid is obtained from mRNA from human platelets, T cells, endothelial cells, or human genomic DNA.\",\n \"36.A method of Gov alloantigen genotyping a subject comprising: (a) providing a CD109 nucleic acid sample derived from the subject; and (b) detecting a region of CD109 nucleic acid that includes a single nucleotide polymorphism distinctive of a Gova or a Govb allele.\",\n \"37.The method of claim 36 comprising determining whether the subject is homozygous or heterozygous for the Gov alleles.\",\n \"38.The method of claim 37, wherein the subject is a human and the Gov genotype is used to determine that the subject has, or is at risk of a disease, disorder or abnormal physical state.\",\n \"39.The method of claim 38, wherein the disease, disorder or abnormal physical state comprises a blood disease, disorder or abnormal physical state.\",\n \"40.The method of claim 39, wherein the blood disease, disorder or abnormal physical state comprises bleeding of the subject, or increased risk of bleeding, due to destruction of blood platelets.\",\n \"41.The method of claim 40, wherein the blood disease, disorder or abnormal physical state comprises post-transfusion purpura (“PTP”), post-transfusion platelet refractoriness (“PR”) or neonatal alloimmune thrombocytopenia (“NAIT”).\",\n \"42.The method of claim 41, wherein the nucleic acid is obtained by amplifying the nucleic acid from the subject.\",\n \"43.The method of claim 42, wherein the nucleic acid is obtained by amplification with all or part of the oligonucleotide of any of claims 1 to 18.44.The method of claim 41, wherein the nucleic acid is obtained from mRNA from human platelets, T cells, endothelial cells, or human genomic DNA.\",\n \"45.The method of claim 36, wherein the detection step comprises determining the nucleotide sequence of the CD109 nucleic acid.\",\n \"46.The method of clam 36, wherein the detection step comprises contacting the nucleic acid with the oligonucleotide of any of claims 1 to 18 under high stringency conditions.\",\n \"47.The method of claim 46, wherein the oligonucleotide will selectively hybridize to (i) a region of CD109 nucleic acid that includes a single polymorphism distinctive of a Gova allele or (ii) a region of CD109 nucleic acid that includes a single polymorphism distinctive of a Govb allele.\",\n \"48.The method of claim 36, wherein the detecting step comprises: (a) performing a restriction endonuclease digestion of the nucleic acid, thereby providing a nucleic acid digest; and (b) contacting the digest with the oligonucleotide.\",\n \"49.The method of claim 47, wherein the hybridization occurs either during or subsequent to PCR amplification and the analysis is by “Real-Time” PCR analysis, or fluorimetric analysis.\",\n \"50.The method of claim 36, wherein the detection step comprises: (a) incubation of the amplified nucleic acid with a restriction endonuclease under conditions whereby the DNA will be cleaved if the nucleic acid comprises a recognition site for the enzyme; and (b) determining whether the nucleic acid contains a recognition site for the restriction enzyme characteristic of cDNA made from mRNA encoding a Gova or Govb allele of CD109.51.The method of claim 50, wherein the restriction enzyme is selected from the group consisting of Bst2UI, BstNI, BstOI, EcoRII, MaeIII, MspR91, MvaI, ScrFI or an isoschizomer thereof.\",\n \"52.The method of claim 50, wherein the determination step includes size analysis of the nucleic acid.\",\n \"53.The method of claim 50, wherein the amplified nucleic acid is analyzed by electrophoretic mobility and the mobility of the amplified nucleic acid is compared to the characteristic mobility of amplified nucleic acid fragments corresponding to the Gova or Govb alleles of CD109.54.A method of amplifying CD109 mRNA comprising amplifying the mRNA by PCR using an oligonucleotide of claim 1.55.A Gova specific antibody.\",\n \"56.The antibody of claim 55, that recognizes specifically a Gova allele-specific CD109 epitope corresponding to the polypeptide encoded by a CD109 nucleic acid containing an A at the position corresponding to position 2108 of SEQ ID NO: and position 954 of SEQ ID NO:5., and containing the amino acid Tyrosine at the position corresponding to position 703 of the CD109 protein encoded by SEQ ID NO:1.57.A Govb specific antibody.\",\n \"58.The antibody of claim 57, that recognizes specifically a Govb allele-specific CD109 epitope corresponding to the polypeptide encoded by a CD109 nucleic acid containing a C at the position corresponding to position 2108 of SEQ ID NO:3 and position 954 of SEQ ID NO:5., and containing the amino acid Serine at the position corresponding to position 703 of the CD109 protein encoded by SEQ ID NO:3.59.The antibody of claim 55, further comprising a monoclonal antibody or a polyclonal antibody.\",\n \"60.The antibody of claim 55, further comprising a detectable label.\",\n \"61.An immunogenic composition comprising a Gov specific antibody of claim 55.62.A method of Gov alloantigen phenotyping a subject, comprising: (a) providing a CD109 polypeptide sample derived from the subject; and (b) detecting the presence of a Gova or a Govb antigen in the CD109 polypeptide.\",\n \"63.The method of claim 62, wherein the CD109 is membrane bound CD109 or isolated CD109.64.The method of claim 62, wherein the detection step comprises contacting the polypeptide sample with the antibody of claim 55.65.A diagnostic kit for Gov alloantigen phenotyping a subject, comprising a Gova antibody and/or a Govb antibody of claim 55.66.The kit of claim 65, further comprising a container.\",\n \"67.An isolated polypeptide containing Gova allele-specific amino acid sequence and which is specifically reactive with a Gova antibody.\",\n \"68.An isolated polypeptide containing Govb allele-specific amino acid sequence and which is specifically reactive with a Govb antibody.\",\n \"69.The isolated polypeptide of claim 67, comprising between 4 and 100 amino acids.\",\n \"70.The isolated polypeptide of claim 67, comprising a full-length CD109 polypeptide, or a fragment of a CD109 polypeptide.\",\n \"71.An isolated CD109 polypeptide fragment, comprising a Gova or a Govb antigen.\",\n \"72.The polypeptide fragment of claim 71, comprising all of, or a fragment of, the protein encoded by SEQ ID NO:1., and in which the amino acid corresponding to position 703 of the protein encoded by SEQ ID NO:1 is a Tyrosine.\",\n \"73.The polypeptide fragment of claim 72, comprising all of, or a fragment of, the protein encoded by SEQ ID NO:3., and in which the amino acid corresponding to position 703 of the protein encoded by SEQ ID NO:3 is a Serine.\",\n \"74.The polypeptide fragment of claim 72, comprising between 4 and 100 amino acids.\",\n \"75.The polypeptide fragment of claim 74, comprising between 7 and 50 amino acids.\",\n \"76.The polypeptide fragment of claim 67, in which the polypeptide is purified from native CD109, is synthetic, or is prepared by recombinant means.\",\n \"77.The polypeptide fragment of claim 67, in which the polypeptide is bound to a substrate.\",\n \"78.A fusion compound comprising the polypeptide of any of claim 67 connected to an immunogenic carrier.\",\n \"79.The fusion compound of claim 78, wherein the immunogenic carrier comprises a proteinaceous carrier.\",\n \"80.The fusion compound of claim 79, wherein the immunogenic carrier comprises a detectable label.\",\n \"81.A Gova or Govb specific antibody recognizing the fusion compound of claim 80.82.An immunogenic composition comprising the polypeptide, polypeptide fragment or fusion compound of claim 67.83.A method of producing a Gova or Govb specific antibody, comprising contacting an animal with the immunogenic composition of claim 82, so that the animal produces antibodies against the immunogenic composition.\",\n \"84.The method of claim 83, wherein the animal is a bird or a mammal.\",\n \"85.A method of screening an antibody producing culture to determine whether the culture produces Gova or Govb specific antibody, comprising: (a) contacting a polypeptide of the invention with the culture; and (b) detecting Gova or Govb specific antibody.\",\n \"86.The method of claim 85, wherein the polypeptide comprises a detectable label.\",\n \"87.The method of claim 86, wherein the polypeptide is attached to a substrate.\",\n \"88.A method of purifying a Gov allele-specific antibody from a sample, comprising: (a) contacting a Gov allele-specific antibody with a polypeptide of the invention comprising a Gova or Govb antigen, so that an antibody:polypeptide complex is formed; (b) separating the complex from the sample; and (c) next separating the antibody from the polypeptide.\",\n \"89.The method of claim 88, wherein the polypeptide is bound to a substrate.\",\n \"90.The method of claim 89, wherein the polypeptide comprises a detectable label.\",\n \"91.A method of purifying a Gov polypeptide from a sample, comprising: (a) contacting a Gov allele-specific antibody with a polypeptide of the invention containing a Gova or Govb-specific epitope, so that an antibody: polypeptide complex is formed; (b) separating the complex from the sample; and (c) next separating the antibody from the polypeptide.\",\n \"92.The method of claim 91, wherein the antibody is bound to a substrate.\",\n \"93.The method of claim 92, wherein the antibody comprises a detectable label.\",\n \"94.A method of screening a subject sample to determine whether the sample contains Gova or Govb-specific antibodies, comprising: (a) contacting a polypeptide of the invention with the sample; and (b) detecting the presence or absence of Gova or Govb specific antibody.\",\n \"95.The method of claim 94, wherein the polypeptide comprises a detectable label.\",\n \"96.The method of claim 95, wherein the polypeptide is attached to a substrate.\",\n \"97.The method of claim 96, wherein the subject comprises a mother of a fetus or a newborn infant, or the fetus or newborn infant itself, and the presence of Gova or Govb-specific antibody indicates that the fetus or infant has, or is at risk of NAIT.\",\n \"98.The method of claim 97, wherein the presence of Gova or Govb specific antibody indicates that the subject has, or is at risk of a blood disease, disorder or abnormal physical state.\",\n \"99.The method of claim 98, wherein the blood disease, disorder or abnormal physical state comprises bleeding of the subject, or increased risk of bleeding, due to destruction of blood platelets.\",\n \"100.The method of claim 99, wherein the blood disease, disorder or abnormal physical state comprises post-transfusion purpura (“PTP”), post-transfusion platelet refractoriness (“PR”) or neonatal alloimmune thrombocytopenia (NAIT).\",\n \"101.The method of claim 100, wherein the sample comprises human serum or plasma.\",\n \"102.A diagnostic kit for detection of Gova or Govb specific antibody, comprising a polypeptide of claim 67.103.The kit of claim 102, further comprising a container.\",\n \"104.A method of determining Gov antibody specificity, comprising: (a) contacting an antibody with a first polypeptide comprising a Gova antigen and a second polypeptide comprising a Govb antigen; and (b) determining whether the antibody binds to either or both of the first and second polypeptide.\",\n \"105.A method of blocking Gova antibody binding to an antigen, comprising: contacting the antibody with a polypeptide of the invention comprising a Gova antigen so that an antibody:polypeptide complex is formed.\",\n \"106.The method of claim 105, wherein the polypeptide comprises a detectable label.\",\n \"107.The method of claim 106, wherein the polypeptide is bound to a substrate.\",\n \"108.A method of blocking Govb antibody binding to an antigen, comprising: contacting the antibody with a polypeptide of the invention comprising a Govb antigen so that an antibody:polypeptide complex is formed.\",\n \"109.The method of claim 108, wherein the polypeptide comprises a detectable label.\",\n \"110.The method of claim 109, wherein the polypeptide is bound to a substrate.\",\n \"111.A pharmaceutical composition comprising the polypeptide of claim 67.112.A method of immunizing a subject so that the subject will produce anti-idiotypic antibodies, comprising administering to the subject the immunogenic composition of claim 82.113.A method of blocking Gova or Govb specific antibodies from binding to CD109 in a subject, comprising: administering to the subject polypeptides of the invention capable of binding to Gova and/or Govb specific antibodies.\",\n \"114.The method of claim 113, wherein the polypeptide comprises a detectable label.\",\n \"115.The method of claim 114, wherein the binding of the polypeptide to the Gova or Govb specific antibody prevents alloimmune cell destruction by the antibody.\",\n \"116.The method of claim 115, wherein the binding of the polypeptide to the Gova or Govb-specific antibody depletes the antibody.\",\n \"117.The method of claim 116, wherein the subject has or is at risk of a blood disease, disorder or abnormal physical state.\",\n \"118.The method of claim 117, wherein the blood disease, disorder or abnormal physical state comprises bleeding of the subject, or increased risk of bleeding, due to alloimmune destruction of blood platelets.\",\n \"119.The method of claim 118, wherein the blood disease, disorder or abnormal physical state comprises post-transfusion purpura (“PTP”), post-transfusion platelet refractoriness (“PR”) or neonatal alloimmune thrombocytopenia (“NAIT”).\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>Among the disorders, which the invention concerns, are those involving abnormal and excessive bleeding due to destruction of blood platelets (“platelets”).\",\n \"These disorders include, but are not restricted to, post-transfusion purpura (“PTP”) and post-transfusion platelet refractoriness (“PTPR”), which are suffered by some persons who receive blood, platelets, leukocyte concentrates, or plasma from other persons by transfusion or the like.\",\n \"The disorders also include one that is suffered by fetuses and newborns and is known as “neonatal alloimmune thrombocytopenia” (“NATP”).\",\n \"This disorder can cause death of fetuses and serious birth defects or death of newborns.\",\n \"NATP is estimated to affect about 1 in 1000 newborns.\",\n \"In NATP, fetal platelets, which enter the mother's blood stream, induce production in the mother of antibodies directed against fetal platelets.\",\n \"These maternal antibodies then pass with the mother's blood into the fetus and mediate destruction of platelets in the fetus.\",\n \"A mother, whose fetus or newborn suffers from NATP, is at increased risk of suffering PTP or PTPR.\",\n \"When platelets from a first human (a “donor”) are introduced into the blood system of a second human (a “recipient”) by transfusion, through the placenta (in the case of fetal blood entering the mother), or the like, the recipient may mount an immune response against the platelets from the donor.\",\n \"Such an immune response is referred to as an “alloimmune” response, because it involves antibodies reacting-against antigens of a different individual of the same species.\",\n \"The alloimmune response to platelets is due to an immune response of the recipient against “alloantigens” (antigens of the same species as that mounting the immune response) on platelets from the donor.\",\n \"These alloantigens are found on membrane glycoproteins that occur in the cell membranes, which define the outer surfaces of platelets (“platelet membranes”).\",\n \"In this invention, the glycoprotein is anchored to the membrane in an atypical manner through an anchor consisting of glycosylphosphatidylinositol (GPI), which anchors an extracellular domain or segment of the glycoprotein exposed to the outside of the platelet.\",\n \"It is thought that alloantibodies, which are generated in an alloimmune response against platelet alloantigens, interact with the extracellular domains of the alloantigens.\",\n \"The platelet alloantigens that a person has are determined by the person's genetics.\",\n \"A donor, because of his or her genetics, may have a platelet alloantigen, which a recipient, who receives blood, platelets, leukocytes or plasma from the donor, does not have, because of the recipient's genetics.\",\n \"In such a situation, the immune system of the recipient may recognize the donor's alloantigen as “non-self,” and raise an immune response against, the platelet alloantigen, which the donor has but the recipient does not.\",\n \"Membrane glycoprotein alloantigens have been characterised for both human red blood cells and human platelets.\",\n \"It is noteworthy, however, that they also occur on other cell types, such as leukocytes and endothelial cells, where they may also occasion various disorders through alloimmune responses.\",\n \"Recognised classes of red blood cell and platelet alloantigens have been described, over the past 30 years, based on observations of antibody reactions occurring when blood recipients have been exposed to blood from donors.\",\n \"A recent review of human platelet alloantigen systems is provided by Ouwehand, W., and Navarrete, C., in Molecular Haematology , Provan, D. and Gribben, J. eds.\",\n \"Blackwell (1999).\",\n \"Several biallelic platelet alloantigen “systems” have been characterised.\",\n \"In each of these systems, there are two alloantigens, each of which is provided by one of two alleles of the gene comprising the system.\",\n \"Because each gene occurs twice in the normal human genome, a person can be homozygous for one or the other of the two alloantigens, or heterozygous for the two alloantigens, comprising a biallelic system.\",\n \"The alloantigens described to date occur on glycoprotein molecules which may exist in various forms (transmembrane, GPI-linked and soluble, for example).\",\n \"In such a case, the alloantigens are found on each of the variant forms of the glycoprotein.\",\n \"For all of the biallelic platelet alloantigen systems that have been characterised at the level of protein and gene sequences, it has been found in all cases, except for one, that the difference between the two alleles is based on a single nucleotide polymorphism in the relevant gene.\",\n \"One biallelic system of human platelet alloantigens is the Gov a /Gov b biallelic system associated with CD109, a membrane glycoprotein which occurs on platelets and various other cell types, including leukocytes and endothelial cells.\",\n \"Each Gov allele corresponds to one CD109 glycoprotein (Sutherland, D. R. et al, 1991; Smith et al., 1995; Berry, J. et al., 2000), consistent with the known tissue distribution of CD109.The frequencies for the Gov alleles are 0.4 for Gov a and 0.6 for Gov b in the Caucasian population.\",\n \"Thus, in this population, 40.7% are heterozygous for the Gov alleles, and will not mount an alloimmune response due to Gov incompatibility (not possessing the Gov alloantigen found on platelets received from another).\",\n \"In contrast, 19.8% of Caucasians are homozygous for the Gov a allele and thus may mount an immune response due to Gov alloantigen incompatibility against platelets received from anyone in the 80.5% of the Caucasian population that is not homozygous for the Gov a allele, while 39.8% are homozygous for the Gov b allele and thus may mount an immune response due to Gov alloantigen incompatibility against platelets received from anyone in the 60.2% of the Caucasian population that is not homozygous for the Gov b allele.\",\n \"As indicated above, alloimmunization based on Gov incompatibility (the introduction into the blood stream of donor platelets bearing a Gov alloantigen not carried by the recipient) can result in bleeding disorders due to platelet destruction, including NATP, PTPR, and PTP.\",\n \"The location of the Gov antigens within the CD109 molecule, and the nature of the CD109 polymorphism which underlies the Gov a /Gov b alloantigen (both at the protein and at the gene level), have not heretofore been known.\",\n \"Furthermore, it has not heretofore been possible to generate non-human antibody (polyclonal or monoclonal), as from a rat, mouse, goat, chicken, or the like, with specificity for the Gov a alloantigen but not the Gov b alloantigen (or vice-versa) sufficient for use in an immunoassay, for typing for Gov phenotype using platelets or CD109 molecules.\",\n \"Previously developed technology, involving gene-specific amplification of platelet RNA-derived cDNA, followed by the determination of the nucleotide sequence of the amplified DNA, has been applied successfully to the elucidation of the molecular basis of other biallelic platelet alloantigen systems (Newman et al., J. Clin.\",\n \"Invest.\",\n \"82,739-744 (1988); Newman et al., J. Clin.\",\n \"Invest.\",\n \"83, 1778-1781 (1989)(P1A or HPA-1 system); Lyman et al., Blood 75, 2343-2348 (1990)(Bak or HPA-3system); Kuijpers et al., J. Clin.\",\n \"Invest.\",\n \"89, 381-384 (1992)(HPA-2 or Ko system); Wang et al., J. Clin.\",\n \"Invest.\",\n \"90, 2038-2043 (1992)(Pen system).\",\n \"With one exception, it has been found in each case that a single amino acid difference at a single position differentiates the amino acid sequences of the two alleles, and that this difference arises from a single allele-specific nucleotide substitution in the coding region of the mRNA and gene.\",\n \"There remains a need to elucidate the molecular basis of the biallelic Gov platelet alloantigen system.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The Gov a/Gov b CD109 Single Nucleotide Polymorphism We have now discovered that a single amino acid difference in the CD109 glycoprotein distinguishes the Gov a and Gov b allelic forms.\",\n \"The two alleles differ at amino acid position 703 of the full-length 1445 amino acid CD109 molecule, with the Gov a allele [SEQ ID NO:2] containing a Tyr at this position, while the Gov b allele [SEQ ID NO:4] contains Ser.\",\n \"Further, we have discovered that this difference in amino acid sequence between the allelic forms of CD109 is due to a single nucleotide polymorphism at position 2108 of the coding portion of full-length mRNA encoding CD109, or of the corresponding coding strand of the cDNA corresponding to this mRNA.\",\n \"Specifically, the Gov a allele [SEQ ID NO:1] contains adenine at position 2108, the second nucleotide of the codon encoding the amino acid at position 703 of the full-length CD109 protein, while the Gov b allele contains cytosine at position 2108, as shown in SEQ ID NO:3 The Gov a /Gov b single nucleotide polymorphism of CD109, lies at position 2108 in SEQ ID NO:1.SEQ ID NO:1 is the cDNA sequence encoding the full-length 1445 amino acid CD109 precursor encoding the Gov a alleleln the Gov b allele form [SEQ ID NO:3], C occurs at position 2108, rather than A.\",\n \"The ATG at the 5′-end of the sequence in SEQ ID NO:1 corresponds to the translation start of the full-length precursor form (including leader peptide) of CD109.The triplet corresponding to the N-terminal amino acid of the mature CD109 protein is at positions 64-66 in SEQ ID NO:1.The Gov a /Gov b single nucleotide polymorphism of CD109, lies at position 954 in SEQ ID NO:5.SEQ ID NO:5 is the genomic DNA sequence of human CD109 exon 19 and the contiguous introns, introns 18 and 19.The Gov a /Gov b single nucleotide polymorphism of CD109 is found within CD109 exon 19, and specifically is located at position 3 of CD109 exon 19.The sequence presented in SEQ ID NO:5 contains A at position 954, and thus corresponds to the Gov a allele.\",\n \"The corresponding Gov b sequence contains C at position 954 of SEQ ID NO:5 (nucleotide position 3 of exon 19).\",\n \"In view of this discovery, it will be readily apparent to the skilled what the present invention provides: Gov allele-specific oligonucleotides and polynucleotides: Based on the discovery, the present invention provides oligonucleotides and polynucleotides (seems repetitive), including (but not limited to) probes which can be used to determine whether a person is homozygous for one or the other of the Gov alleles, or heterozygous for these alleles, thereby to determine that person's Gov genotype, and by extension, their Gov phenotype (i.e., the Gov alloantigen(s) which their cells express).\",\n \"Further, the invention provides methods of using such oligonucleotides, and test kits to facilitate their use, in such Gov genotype and phenotype determinations.\",\n \"These oligonucleotides of the invention can be used to determine whether, in the CD109 gene, or in the mRNA encoding CD109, the internal nucleotide (nucleotide 2108) of the codon (in CD109 gene or in the mRNA encoding CD109) which corresponds to the amino acid at position 703 in the sequence of full-length CD109 is adenine or cytosine.\",\n \"Such probes will typically be cDNA but may be genomic DNA, mRNA or RNA, and may be labelled for detection.\",\n \"The oligonucleotides of the invention can be used as probes to detect nucleic acid molecules according to techniques known in the art (for example, see U.S. Pat.\",\n \"Nos.\",\n \"5,792,851 and 5,851,788).\",\n \"For example, an oligonucleotide of the invention may be converted to a probe by being end-labelled using digoxigenin-11-deoxyuridine triphosphate.\",\n \"Such probes may be detected immunologically using alkaline-phosphate-conjugated polyclonal sheep antidigoxigenin F(ab) fragments and nitro blue tetrazolium with 5-bromo-4-chloro-3-indoyl phosphate as chromogenic substrate.\",\n \"Gov allele-specific antibodies: Still further, based on the discovery, which underlies the invention, of the molecular basis for the Gov a /Gov b alloantigen system, the invention provides non-human polyclonal and monoclonal antibodies, which can be used to distinguish one Gov allelic form of CD109 from the other, whether the CD109 is part of a complex embedded in or isolated from a membrane or is isolated.\",\n \"These antibodies of the invention, which are preferably provided in an aqueous buffer solution, and the immunoassays of the invention which employ such antibodies, are useful for determining whether a person has one or both of the Gov alloantigens and for Gov phenotyping.\",\n \"Methods of using the antibodies of the invention in the immunoassays of the invention, and in such determinations, are also encompassed by the invention.\",\n \"The invention also provides test kits to facilitate carrying out such immunoassays and determinations.\",\n \"Gov allele-specific peptides and polypeptides: Again, based on the discovery that underlies the invention, of the molecular basis for the Gov a /Gov b alloantigen system, the invention provides peptides or polypeptides, which are useful for various purposes.\",\n \"These peptides or polypeptides are typically between 4 and 100, and more typically between 7 and 50, amino acids in length, and have amino acid sequences identical or having sequence identity to those of segments of the CD109 sequences, that include the amino acid at position 703 of full-length mature CD109.This amino acid (position 703) corresponds the triplet at positions 2107-2109 in the CD109 cDNA sequence presented in SEQ ID NO:1, or in the corresponding sequence for the CD109 cDNA that encodes the Gov b allelic form [SEQ ID NO:3].\",\n \"These peptides or polypeptides may be synthetic, may be purified from native CD109 or may be prepared by recombinant means.\",\n \"For guidance, one may consult the following U.S. Pat.\",\n \"Nos.\",\n \"5,840,537, 5,850,025, 5,858,719, 5,710,018, 5,792,851, 5,851,788, 5,759,788, 5,840,530, 5,789,202, 5,871,983, 5,821,096, 5,876,991, 5,422,108, 5,612,191, 5,804,693, 5,847,258, 5,880,328, 5,767,369, 5,756,684, 5,750,652, 5,824,864, 5,763,211, 5,767,375, 5,750,848, 5,859,337, 5,563,246, 5,346,815, and WO9713843.Many of these patents also provide guidance with respect to experimental assays, probes and antibodies, methods, transformation of host cells, which are described below.\",\n \"These patents, like all other patents, publications (such as articles and database publications) in this application, are incorporated by reference in their entirety.\",\n \"Gov allele-specific peptides and polypeptides as antigens and immunogens, and Gov allele-specific polyclonal and monoclonal antibodies: These peptides or polypeptides are useful as antigens (usually coupled to a larger, immunogenic carrier [proteinaceous or otherwise], as known in the art) for making the polyclonal or monoclonal antibodies of the invention.\",\n \"The peptides or polypeptides are also useful in screening monoclonal antibody-producing cultures (hybridoma cultures/ E. coli cultures or so-called V gene phage antibodies) to identify those that produce monoclonal antibodies of the invention.\",\n \"The invention also encompasses immunogenic compositions which comprise a peptide, polypeptide or fusion compound of the invention and which are immunogenic in a bird, including, without limitation, a chicken, or a mammal, such as, a mouse, rat, goat, rabbit, guinea pig, sheep or human.\",\n \"The compositions may include an immunogenicity-imparting “carrier” which may be but is not necessarily a protein as known in the art, that is immunogenic in a bird or mammal, coupled to at least one peptide or polypeptide of the invention, which has an amino acid sequence that is the same as that of a segment of the sequence for CD109, that includes the amino acid at position 703 of the full length CD109 molecule.\",\n \"The present invention also provides methods of using the peptides, polypeptides and immunogenic compositions of the invention for making antibodies of the invention, and methods of using the peptides and polypeptides of the invention in screening monoclonal antibody-producing hybridoma cultures or bacterial clones for those that produce monoclonal antibodies or fragments thereof of the invention.\",\n \"Therapeutic and diagnostic application of Gov allele-specific peptides, polypeptides, and antibodies: These peptides or polypeptides, as well as antibodies, which are specific for the Gov a [SEQ ID NO:2] or Gov b [SEQ ID NO:4], but not both, allelic forms of CD109 in the platelet membrane, and which can be produced by a mammal (including an human) immunized with the peptides or polypeptides, which themselves happen to be immunogenic, or the immunogenic compositions of the invention, are also useful both therapeutically and diagnostically.\",\n \"The invention also provides the methods of using the peptides and polypeptides of the invention, and antibodies made using the peptides that are immunogenic and the immunogenic compositions of the invention, in therapeutic and diagnostic applications.\",\n \"The Gov allele-specific peptides or polypeptides can also be used diagnostically to detect the presence of Gov a or Gov b specific antibodies in human plasma or serum samples, using methods that are readily apparent to those skilled in the art.\",\n \"Such analyses would be useful in the investigation of cases of acquired alloimmune thrombocytopenia, including PTP, PTPR, and NATP.\",\n \"In the latter case, this approach could also be used to detect the presence of Gov allele-specific antibodies in the mother of the affected fetus or newborn.\",\n \"The presence of Gov allele-specific antibodies can also be detected using platelets of known Gov phenotype.\",\n \"However, this approach has numerous technical disadvantages that are eliminated by the use of Gov allele-specific peptides or polypeptides for Gov allele-specific antibody detection.\",\n \"Administration to a person, who is suffering from, or at risk for, for example, PTP or PTPR, or a mother at risk for passing NATP-causing alloantibodies to her fetus, of one of the peptides or polypeptides, that would be bound by the anti-Gov alloantibodies in such a person, would inhibit the binding of the alloantibodies to the person's (or the fetus's platelets and thereby inhibit the platelet destruction and abnormal bleeding associated with the disorders.\",\n \"Alternatively, administration to such a person of antibodies (particularly human antibodies), which are produced using a peptide or polypeptide of the invention, which is immunogenic by itself, or an immunogenic composition of the invention, and which are specific for the Gov allelic form of the CD109 on the person's platelets which is associated with the PTP or PTPR, from which the person is suffering or may suffer, would induce the production of anti-idiotypic antibodies, which, in turn, would inhibit the platelet-destructive effects of the anti-Gov alloantibodies, which are generated by the person's own immune system and which are causing or threatening to cause the PTP, PTPR or NATP.\",\n \"These therapeutic applications of peptides and polypeptides of the invention would be especially useful in treating NATP in a newborn, because the alloantibody giving rise to NATP in the newborn is not continuously produced by the immune system of the newborn, but rather is acquired passively, and therefore in limited, non-replenished quantity, by the newborn from its mother.\",\n \"Thus, in accordance with one aspect of the present invention, an oligonucleotideprobe is provided that hybridizes to a portion of the CD109 gene, or a portion of CD109-encoding mRNA or cDNA prepared from such mRNA, which portion includes a nucleotide corresponding to the internal nucleotide of the codon for the amino acid at position 703 of the full-length CD109 molecule, and that is capable of distinguishing one Gov allele from the other through the ability to hybridize under stringent conditions to the portion in question only when the nucleotide in question is A (or dA), when the probe is to detect the Gov a allele, or C (or dC), when the probe is to detect the Gov b allele.\",\n \"The nucleotide in question is at position 2108 of the coding region of the CD109 cDNA sequence and lies at position 2108 in SEQ ID NO:1.The cDNA sequence has A at this position, and so is the sequence corresponding to the Gov a allele.\",\n \"The nucleotide in question lies at position 954 of the sequence presented as SEQ ID NO:5 and contains an A in this position, and thus also corresponds to Gov a allele.\",\n \"The Gov allele-specific oligonucleotide hybridization probes of the invention may comprise genomic DNA, cDNA, or RNA, although preferably it is DNA.\",\n \"Such oligonucleotide probes can be synthesised by automated synthesis and will preferably contain about 10-30 bases, although as understood in the oligonucleotide probe hybridization assay art, as few as 8 and as many as about 50 nucleotides may be useful, depending on the position within the probe where the potential mismatch with the target is located, the extent to which a label on the probe might interfere with hybridization, and the physical conditions (e.g., temperature, pH, ionic strength) under which the hybridization of probe with target is carried out.\",\n \"In accordance with another aspect of the present invention, a test kit for Gov alloantigen typing is provided comprising: (a) means for amplifying nucleic acid that comprises at least a portion of a CD109 gene, a CD109-encoding mRNA, or a CD109 cDNA made from such RNA, wherein the portion includes a nucleotide (nucleotide 2108 in SEQ ID NO:1, or nucleotide 954 in SEQ ID NO:5) corresponding to the internal nucleotide of the codon encoding amino acid 703 of the full length CD109 protein.\",\n \"(b) an oligonucleotide probe of the invention, that distinguishes one Gov allele from the other.\",\n \"The “means for amplifying” will, as the skilled will readily understand, depend on the amplification method to be used.\",\n \"Thus, for example, these means might include suitable primers, a suitable DNA polymerase, and the four 2′-deoxyribonucleoside triphosphates (dA, dC, dG, dT), if amplification is to be by the PCR method.\",\n \"To cite another example, if the amplification is to be by a method relying on transcription, such as the 3SR method, the means will include two primers, at least one of which, when made double-stranded, will provide a promoter, an RNA polymerase capable of transcribing from that promoter, a reverse transcriptase to function in primer-initiated, DNA-directed and RNA-directed, DNA polymerization and possibly also in RNAse H degradation of RNA to free DNA strands from RNA/RNA hybrids, the four ribonucleoside triphosphates (A, C, G and U), and the four 2′-deoxyribonucleoside triphosphates.\",\n \"In another example, if the amplification is by the ligase chain reaction, the means will include two oligonucleotides (DNAs) and a suitable DNA ligase that will join the two if a target, to which both can hybridize adjacent to one another in ligatable orientation, is present.\",\n \"The oligonucleotide probes of the invention will preferably be labelled.\",\n \"The label may be any of the various labels available in the art for such probes, including, but not limited to 32 P; 35 S; biotin (to which a signal generating moiety, bound to or complexed with avidin can be complexed); a fluorescent moiety; an enzyme such as alkaline phosphatase (which is capable of catalysing a chromogenic reaction); digoxigenin, as described above; or the like.\",\n \"As indicated in the examples, RFLP analysis can be employed, using BstNI (or isoschizomers thereof), in analysing cDNA or genomic DNA (with or without amplification) to determine Gov genotype.\",\n \"As indicated further in the examples, electrophoretic SSCP analysis may be used to determine Gov genotype.\",\n \"And as indicated in the examples, the hybridization studies outlined above may use fluorescent probes, and may be directly coupled to the DNA amplification step, as in “Real-Time PCR” or related methods.\",\n \"There has also been provided, in accordance with another aspect of the present invention, a method of typing for Gov allele-specific target sequence in a CD 109 nucleic acid derived from a subject, comprising the steps of, (a) obtaining, by a target nucleic acid amplification process applied to mRNA from human platelets, endothelial cells, or T cells, an assayable quantity of amplified nucleic acid with a sequence that is that of a subsequence (or the complement of a subsequence) of the mRNA that encodes a CD109 said subsequence including the nucleotide at the position in the mRNA corresponding to position 2108 in SEQ ID NO:1 or to nucleotide 954 in SEQ ID NO:5; and (b) analyzing (e.g., in a nucleic acid probe hybridization assay employing an oligonucleotide probe or probes according to the invention) the amplified nucleic acid obtained in step (a) to determine the base or bases at the position in the amplified nucleic acid that corresponds to position 2108 in SEQ ID NO:1 or to nucleotide 954 in SEQ ID NO:5.It is noteworthy that, if the product of the amplification is double-stranded DNA, analysis for Gov genotype can be carried out by a RFLP (restriction fragment length polymorphism) analysis comprising exposing the amplified DNA to the restriction endonuclease BstNI (or isoschizomer thereof) under conditions whereby the DNA will be cleaved if it includes a site for cleavage by that enzyme.\",\n \"Such DNA, prepared from mRNA encoding the Gov b alloantigen, containing a C rather than an A at the position corresponding to nucleotide 2108 in SEQ ID NO:1 (or to nucleotide 954 in SEQ ID NO:5), includes a recognition site for that endonuclease, while such DNA prepared from mRNA encoding the Gov a alloantigen, does not If the analysis, by whatever method, of the amplified nucleic acid reveals that there is only an A (or dA) at the position corresponding to position 12108, the platelets (and blood from which they came) have only the Gov a alloantigen, and the individual from whom the platelets came, is homozygous for Gov a .\",\n \"Alternatively, if the analysis of the amplified nucleic acid reveals that there is only a C (or dC) at the position corresponding to position 2108, the platelets (and blood from which they came) have only the Gov b alloantigen and the individual, from whom the platelets came, is homozygous for the Gov b allele.\",\n \"Finally, if the analysis indicates that there is either an A (or dA) or a C (or dC) at that position, the platelets (and blood from which they came) have both Gov alloantigens, and the individual from whom the platelets came, is heterozygous for Gov alloantigen.\",\n \"In one application of the typing methods of the invention, the methods are applied to two individuals to determine whether blood or platelets from one would provoke an alloimmune response, and possibly PTP or PTPR, in the other.\",\n \"The typing method can be applied with a man and a woman, who are contemplating conceiving or have conceived a child together, to determine the risk that the child would be at risk for NATP and the risk that the woman would be at increased risk for PTP or PTPR.\",\n \"If the woman were heterozygous for the Gov alloantigens there would be, due to Gov alloantigen incompatibility, no risk of NATP and no increased risk for the woman of PTP or PTPR.\",\n \"If, however, the woman were homozygous for one of the Gov alloantigens, there would be, due to Gov alloantigen incompatibility, risk of NATP in a child and increased risk of PTP or PTPR for the woman, unless the man is homozygous for the same Gov alloantigen as is the woman.\",\n \"In accordance with yet another aspect of the present invention, a method of typing an individual for Gov alloantigen is provided that comprises analyzing the genomic DNA of the individual to determine the Gov alloantigen(s) of the individual.\",\n \"Applications of this method are substantially the same as those of the method of the invention for typing for Gov alloantigen that begins with platelet, endothelial cell, or T cell mRNA.\",\n \"This method of the invention, entailing analysis of genomic DNA, can be carried out in substantially the same way as outlined above for analysis of mRNA, namely first amplifying the genomic DNA and then analyzing to product of the amplification to ascertain whether there is only dA, only dC, or both dA and dC, at the position in the coding region of the genomic DNA corresponding to position 2108 in SEQ ID NO:1, or to nucleotide 954 in SEQ ID NO:5.In accordance with a further aspect of the present invention, a test kit for Gov alloantigen typing is provided comprising a non-human antibody (or antibodies) that distinguishes the two allelic forms of CD109.The antibody (or antibodies) of the kit may be polyclonal, or preferably monoclonal, and in addition to its (their) specificity for either but not both Gov alloantigens (on the surface of platelets or separated therefrom) or the CD109 subunit of one but not both of such alloantigens, typically will recognise a polypeptide molecule encoded by a nucleotide sequence encoding at least amino acid 703 of a CD109 polypeptide (the amino acid at the position corresponding to nucleotides 2107-2109 in SEQ ID NO:1, or to nucleotides 953-955 in SEQ ID NO:5).\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention provides novel compositions and methods for use in diagnosing the occurrence of certain serious disorders, especially certain bleeding disorders, and novel compositions and methods for use in treating such a disorder, in a person in which the disorder has occurred, and novel compositions and methods for use in avoiding such a disorder, in an individual who is susceptible thereto.\",\n \"BACKGROUND OF THE INVENTION Among the disorders, which the invention concerns, are those involving abnormal and excessive bleeding due to destruction of blood platelets (“platelets”).\",\n \"These disorders include, but are not restricted to, post-transfusion purpura (“PTP”) and post-transfusion platelet refractoriness (“PTPR”), which are suffered by some persons who receive blood, platelets, leukocyte concentrates, or plasma from other persons by transfusion or the like.\",\n \"The disorders also include one that is suffered by fetuses and newborns and is known as “neonatal alloimmune thrombocytopenia” (“NATP”).\",\n \"This disorder can cause death of fetuses and serious birth defects or death of newborns.\",\n \"NATP is estimated to affect about 1 in 1000 newborns.\",\n \"In NATP, fetal platelets, which enter the mother's blood stream, induce production in the mother of antibodies directed against fetal platelets.\",\n \"These maternal antibodies then pass with the mother's blood into the fetus and mediate destruction of platelets in the fetus.\",\n \"A mother, whose fetus or newborn suffers from NATP, is at increased risk of suffering PTP or PTPR.\",\n \"When platelets from a first human (a “donor”) are introduced into the blood system of a second human (a “recipient”) by transfusion, through the placenta (in the case of fetal blood entering the mother), or the like, the recipient may mount an immune response against the platelets from the donor.\",\n \"Such an immune response is referred to as an “alloimmune” response, because it involves antibodies reacting-against antigens of a different individual of the same species.\",\n \"The alloimmune response to platelets is due to an immune response of the recipient against “alloantigens” (antigens of the same species as that mounting the immune response) on platelets from the donor.\",\n \"These alloantigens are found on membrane glycoproteins that occur in the cell membranes, which define the outer surfaces of platelets (“platelet membranes”).\",\n \"In this invention, the glycoprotein is anchored to the membrane in an atypical manner through an anchor consisting of glycosylphosphatidylinositol (GPI), which anchors an extracellular domain or segment of the glycoprotein exposed to the outside of the platelet.\",\n \"It is thought that alloantibodies, which are generated in an alloimmune response against platelet alloantigens, interact with the extracellular domains of the alloantigens.\",\n \"The platelet alloantigens that a person has are determined by the person's genetics.\",\n \"A donor, because of his or her genetics, may have a platelet alloantigen, which a recipient, who receives blood, platelets, leukocytes or plasma from the donor, does not have, because of the recipient's genetics.\",\n \"In such a situation, the immune system of the recipient may recognize the donor's alloantigen as “non-self,” and raise an immune response against, the platelet alloantigen, which the donor has but the recipient does not.\",\n \"Membrane glycoprotein alloantigens have been characterised for both human red blood cells and human platelets.\",\n \"It is noteworthy, however, that they also occur on other cell types, such as leukocytes and endothelial cells, where they may also occasion various disorders through alloimmune responses.\",\n \"Recognised classes of red blood cell and platelet alloantigens have been described, over the past 30 years, based on observations of antibody reactions occurring when blood recipients have been exposed to blood from donors.\",\n \"A recent review of human platelet alloantigen systems is provided by Ouwehand, W., and Navarrete, C., in Molecular Haematology, Provan, D. and Gribben, J. eds.\",\n \"Blackwell (1999).\",\n \"Several biallelic platelet alloantigen “systems” have been characterised.\",\n \"In each of these systems, there are two alloantigens, each of which is provided by one of two alleles of the gene comprising the system.\",\n \"Because each gene occurs twice in the normal human genome, a person can be homozygous for one or the other of the two alloantigens, or heterozygous for the two alloantigens, comprising a biallelic system.\",\n \"The alloantigens described to date occur on glycoprotein molecules which may exist in various forms (transmembrane, GPI-linked and soluble, for example).\",\n \"In such a case, the alloantigens are found on each of the variant forms of the glycoprotein.\",\n \"For all of the biallelic platelet alloantigen systems that have been characterised at the level of protein and gene sequences, it has been found in all cases, except for one, that the difference between the two alleles is based on a single nucleotide polymorphism in the relevant gene.\",\n \"One biallelic system of human platelet alloantigens is the Gova/Govb biallelic system associated with CD109, a membrane glycoprotein which occurs on platelets and various other cell types, including leukocytes and endothelial cells.\",\n \"Each Gov allele corresponds to one CD109 glycoprotein (Sutherland, D. R. et al, 1991; Smith et al., 1995; Berry, J. et al., 2000), consistent with the known tissue distribution of CD109.The frequencies for the Gov alleles are 0.4 for Gova and 0.6 for Govb in the Caucasian population.\",\n \"Thus, in this population, 40.7% are heterozygous for the Gov alleles, and will not mount an alloimmune response due to Gov incompatibility (not possessing the Gov alloantigen found on platelets received from another).\",\n \"In contrast, 19.8% of Caucasians are homozygous for the Gova allele and thus may mount an immune response due to Gov alloantigen incompatibility against platelets received from anyone in the 80.5% of the Caucasian population that is not homozygous for the Gova allele, while 39.8% are homozygous for the Govb allele and thus may mount an immune response due to Gov alloantigen incompatibility against platelets received from anyone in the 60.2% of the Caucasian population that is not homozygous for the Govb allele.\",\n \"As indicated above, alloimmunization based on Gov incompatibility (the introduction into the blood stream of donor platelets bearing a Gov alloantigen not carried by the recipient) can result in bleeding disorders due to platelet destruction, including NATP, PTPR, and PTP.\",\n \"The location of the Gov antigens within the CD109 molecule, and the nature of the CD109 polymorphism which underlies the Gova/Govb alloantigen (both at the protein and at the gene level), have not heretofore been known.\",\n \"Furthermore, it has not heretofore been possible to generate non-human antibody (polyclonal or monoclonal), as from a rat, mouse, goat, chicken, or the like, with specificity for the Gova alloantigen but not the Govb alloantigen (or vice-versa) sufficient for use in an immunoassay, for typing for Gov phenotype using platelets or CD109 molecules.\",\n \"Previously developed technology, involving gene-specific amplification of platelet RNA-derived cDNA, followed by the determination of the nucleotide sequence of the amplified DNA, has been applied successfully to the elucidation of the molecular basis of other biallelic platelet alloantigen systems (Newman et al., J. Clin.\",\n \"Invest.\",\n \"82,739-744 (1988); Newman et al., J. Clin.\",\n \"Invest.\",\n \"83, 1778-1781 (1989)(P1A or HPA-1 system); Lyman et al., Blood 75, 2343-2348 (1990)(Bak or HPA-3system); Kuijpers et al., J. Clin.\",\n \"Invest.\",\n \"89, 381-384 (1992)(HPA-2 or Ko system); Wang et al., J. Clin.\",\n \"Invest.\",\n \"90, 2038-2043 (1992)(Pen system).\",\n \"With one exception, it has been found in each case that a single amino acid difference at a single position differentiates the amino acid sequences of the two alleles, and that this difference arises from a single allele-specific nucleotide substitution in the coding region of the mRNA and gene.\",\n \"There remains a need to elucidate the molecular basis of the biallelic Gov platelet alloantigen system.\",\n \"SUMMARY OF THE INVENTION The Gov a/Gov b CD109 Single Nucleotide Polymorphism We have now discovered that a single amino acid difference in the CD109 glycoprotein distinguishes the Gova and Govb allelic forms.\",\n \"The two alleles differ at amino acid position 703 of the full-length 1445 amino acid CD109 molecule, with the Gova allele [SEQ ID NO:2] containing a Tyr at this position, while the Govb allele [SEQ ID NO:4] contains Ser.\",\n \"Further, we have discovered that this difference in amino acid sequence between the allelic forms of CD109 is due to a single nucleotide polymorphism at position 2108 of the coding portion of full-length mRNA encoding CD109, or of the corresponding coding strand of the cDNA corresponding to this mRNA.\",\n \"Specifically, the Gova allele [SEQ ID NO:1] contains adenine at position 2108, the second nucleotide of the codon encoding the amino acid at position 703 of the full-length CD109 protein, while the Govb allele contains cytosine at position 2108, as shown in SEQ ID NO:3 The Gova/Govb single nucleotide polymorphism of CD109, lies at position 2108 in SEQ ID NO:1.SEQ ID NO:1 is the cDNA sequence encoding the full-length 1445 amino acid CD109 precursor encoding the Gova alleleln the Govb allele form [SEQ ID NO:3], C occurs at position 2108, rather than A.\",\n \"The ATG at the 5′-end of the sequence in SEQ ID NO:1 corresponds to the translation start of the full-length precursor form (including leader peptide) of CD109.The triplet corresponding to the N-terminal amino acid of the mature CD109 protein is at positions 64-66 in SEQ ID NO:1.The Gova/Govb single nucleotide polymorphism of CD109, lies at position 954 in SEQ ID NO:5.SEQ ID NO:5 is the genomic DNA sequence of human CD109 exon 19 and the contiguous introns, introns 18 and 19.The Gova/Govb single nucleotide polymorphism of CD109 is found within CD109 exon 19, and specifically is located at position 3 of CD109 exon 19.The sequence presented in SEQ ID NO:5 contains A at position 954, and thus corresponds to the Gova allele.\",\n \"The corresponding Govb sequence contains C at position 954 of SEQ ID NO:5 (nucleotide position 3 of exon 19).\",\n \"In view of this discovery, it will be readily apparent to the skilled what the present invention provides: Gov allele-specific oligonucleotides and polynucleotides: Based on the discovery, the present invention provides oligonucleotides and polynucleotides (seems repetitive), including (but not limited to) probes which can be used to determine whether a person is homozygous for one or the other of the Gov alleles, or heterozygous for these alleles, thereby to determine that person's Gov genotype, and by extension, their Gov phenotype (i.e., the Gov alloantigen(s) which their cells express).\",\n \"Further, the invention provides methods of using such oligonucleotides, and test kits to facilitate their use, in such Gov genotype and phenotype determinations.\",\n \"These oligonucleotides of the invention can be used to determine whether, in the CD109 gene, or in the mRNA encoding CD109, the internal nucleotide (nucleotide 2108) of the codon (in CD109 gene or in the mRNA encoding CD109) which corresponds to the amino acid at position 703 in the sequence of full-length CD109 is adenine or cytosine.\",\n \"Such probes will typically be cDNA but may be genomic DNA, mRNA or RNA, and may be labelled for detection.\",\n \"The oligonucleotides of the invention can be used as probes to detect nucleic acid molecules according to techniques known in the art (for example, see U.S. Pat.\",\n \"Nos.\",\n \"5,792,851 and 5,851,788).\",\n \"For example, an oligonucleotide of the invention may be converted to a probe by being end-labelled using digoxigenin-11-deoxyuridine triphosphate.\",\n \"Such probes may be detected immunologically using alkaline-phosphate-conjugated polyclonal sheep antidigoxigenin F(ab) fragments and nitro blue tetrazolium with 5-bromo-4-chloro-3-indoyl phosphate as chromogenic substrate.\",\n \"Gov allele-specific antibodies: Still further, based on the discovery, which underlies the invention, of the molecular basis for the Gova/Govb alloantigen system, the invention provides non-human polyclonal and monoclonal antibodies, which can be used to distinguish one Gov allelic form of CD109 from the other, whether the CD109 is part of a complex embedded in or isolated from a membrane or is isolated.\",\n \"These antibodies of the invention, which are preferably provided in an aqueous buffer solution, and the immunoassays of the invention which employ such antibodies, are useful for determining whether a person has one or both of the Gov alloantigens and for Gov phenotyping.\",\n \"Methods of using the antibodies of the invention in the immunoassays of the invention, and in such determinations, are also encompassed by the invention.\",\n \"The invention also provides test kits to facilitate carrying out such immunoassays and determinations.\",\n \"Gov allele-specific peptides and polypeptides: Again, based on the discovery that underlies the invention, of the molecular basis for the Gova/Govb alloantigen system, the invention provides peptides or polypeptides, which are useful for various purposes.\",\n \"These peptides or polypeptides are typically between 4 and 100, and more typically between 7 and 50, amino acids in length, and have amino acid sequences identical or having sequence identity to those of segments of the CD109 sequences, that include the amino acid at position 703 of full-length mature CD109.This amino acid (position 703) corresponds the triplet at positions 2107-2109 in the CD109 cDNA sequence presented in SEQ ID NO:1, or in the corresponding sequence for the CD109 cDNA that encodes the Govb allelic form [SEQ ID NO:3].\",\n \"These peptides or polypeptides may be synthetic, may be purified from native CD109 or may be prepared by recombinant means.\",\n \"For guidance, one may consult the following U.S. Pat.\",\n \"Nos.\",\n \"5,840,537, 5,850,025, 5,858,719, 5,710,018, 5,792,851, 5,851,788, 5,759,788, 5,840,530, 5,789,202, 5,871,983, 5,821,096, 5,876,991, 5,422,108, 5,612,191, 5,804,693, 5,847,258, 5,880,328, 5,767,369, 5,756,684, 5,750,652, 5,824,864, 5,763,211, 5,767,375, 5,750,848, 5,859,337, 5,563,246, 5,346,815, and WO9713843.Many of these patents also provide guidance with respect to experimental assays, probes and antibodies, methods, transformation of host cells, which are described below.\",\n \"These patents, like all other patents, publications (such as articles and database publications) in this application, are incorporated by reference in their entirety.\",\n \"Gov allele-specific peptides and polypeptides as antigens and immunogens, and Gov allele-specific polyclonal and monoclonal antibodies: These peptides or polypeptides are useful as antigens (usually coupled to a larger, immunogenic carrier [proteinaceous or otherwise], as known in the art) for making the polyclonal or monoclonal antibodies of the invention.\",\n \"The peptides or polypeptides are also useful in screening monoclonal antibody-producing cultures (hybridoma cultures/E.\",\n \"coli cultures or so-called V gene phage antibodies) to identify those that produce monoclonal antibodies of the invention.\",\n \"The invention also encompasses immunogenic compositions which comprise a peptide, polypeptide or fusion compound of the invention and which are immunogenic in a bird, including, without limitation, a chicken, or a mammal, such as, a mouse, rat, goat, rabbit, guinea pig, sheep or human.\",\n \"The compositions may include an immunogenicity-imparting “carrier” which may be but is not necessarily a protein as known in the art, that is immunogenic in a bird or mammal, coupled to at least one peptide or polypeptide of the invention, which has an amino acid sequence that is the same as that of a segment of the sequence for CD109, that includes the amino acid at position 703 of the full length CD109 molecule.\",\n \"The present invention also provides methods of using the peptides, polypeptides and immunogenic compositions of the invention for making antibodies of the invention, and methods of using the peptides and polypeptides of the invention in screening monoclonal antibody-producing hybridoma cultures or bacterial clones for those that produce monoclonal antibodies or fragments thereof of the invention.\",\n \"Therapeutic and diagnostic application of Gov allele-specific peptides, polypeptides, and antibodies: These peptides or polypeptides, as well as antibodies, which are specific for the Gova [SEQ ID NO:2] or Govb [SEQ ID NO:4], but not both, allelic forms of CD109 in the platelet membrane, and which can be produced by a mammal (including an human) immunized with the peptides or polypeptides, which themselves happen to be immunogenic, or the immunogenic compositions of the invention, are also useful both therapeutically and diagnostically.\",\n \"The invention also provides the methods of using the peptides and polypeptides of the invention, and antibodies made using the peptides that are immunogenic and the immunogenic compositions of the invention, in therapeutic and diagnostic applications.\",\n \"The Gov allele-specific peptides or polypeptides can also be used diagnostically to detect the presence of Gova or Govb specific antibodies in human plasma or serum samples, using methods that are readily apparent to those skilled in the art.\",\n \"Such analyses would be useful in the investigation of cases of acquired alloimmune thrombocytopenia, including PTP, PTPR, and NATP.\",\n \"In the latter case, this approach could also be used to detect the presence of Gov allele-specific antibodies in the mother of the affected fetus or newborn.\",\n \"The presence of Gov allele-specific antibodies can also be detected using platelets of known Gov phenotype.\",\n \"However, this approach has numerous technical disadvantages that are eliminated by the use of Gov allele-specific peptides or polypeptides for Gov allele-specific antibody detection.\",\n \"Administration to a person, who is suffering from, or at risk for, for example, PTP or PTPR, or a mother at risk for passing NATP-causing alloantibodies to her fetus, of one of the peptides or polypeptides, that would be bound by the anti-Gov alloantibodies in such a person, would inhibit the binding of the alloantibodies to the person's (or the fetus's platelets and thereby inhibit the platelet destruction and abnormal bleeding associated with the disorders.\",\n \"Alternatively, administration to such a person of antibodies (particularly human antibodies), which are produced using a peptide or polypeptide of the invention, which is immunogenic by itself, or an immunogenic composition of the invention, and which are specific for the Gov allelic form of the CD109 on the person's platelets which is associated with the PTP or PTPR, from which the person is suffering or may suffer, would induce the production of anti-idiotypic antibodies, which, in turn, would inhibit the platelet-destructive effects of the anti-Gov alloantibodies, which are generated by the person's own immune system and which are causing or threatening to cause the PTP, PTPR or NATP.\",\n \"These therapeutic applications of peptides and polypeptides of the invention would be especially useful in treating NATP in a newborn, because the alloantibody giving rise to NATP in the newborn is not continuously produced by the immune system of the newborn, but rather is acquired passively, and therefore in limited, non-replenished quantity, by the newborn from its mother.\",\n \"Thus, in accordance with one aspect of the present invention, an oligonucleotideprobe is provided that hybridizes to a portion of the CD109 gene, or a portion of CD109-encoding mRNA or cDNA prepared from such mRNA, which portion includes a nucleotide corresponding to the internal nucleotide of the codon for the amino acid at position 703 of the full-length CD109 molecule, and that is capable of distinguishing one Gov allele from the other through the ability to hybridize under stringent conditions to the portion in question only when the nucleotide in question is A (or dA), when the probe is to detect the Gova allele, or C (or dC), when the probe is to detect the Govb allele.\",\n \"The nucleotide in question is at position 2108 of the coding region of the CD109 cDNA sequence and lies at position 2108 in SEQ ID NO:1.The cDNA sequence has A at this position, and so is the sequence corresponding to the Gova allele.\",\n \"The nucleotide in question lies at position 954 of the sequence presented as SEQ ID NO:5 and contains an A in this position, and thus also corresponds to Gova allele.\",\n \"The Gov allele-specific oligonucleotide hybridization probes of the invention may comprise genomic DNA, cDNA, or RNA, although preferably it is DNA.\",\n \"Such oligonucleotide probes can be synthesised by automated synthesis and will preferably contain about 10-30 bases, although as understood in the oligonucleotide probe hybridization assay art, as few as 8 and as many as about 50 nucleotides may be useful, depending on the position within the probe where the potential mismatch with the target is located, the extent to which a label on the probe might interfere with hybridization, and the physical conditions (e.g., temperature, pH, ionic strength) under which the hybridization of probe with target is carried out.\",\n \"In accordance with another aspect of the present invention, a test kit for Gov alloantigen typing is provided comprising: (a) means for amplifying nucleic acid that comprises at least a portion of a CD109 gene, a CD109-encoding mRNA, or a CD109 cDNA made from such RNA, wherein the portion includes a nucleotide (nucleotide 2108 in SEQ ID NO:1, or nucleotide 954 in SEQ ID NO:5) corresponding to the internal nucleotide of the codon encoding amino acid 703 of the full length CD109 protein.\",\n \"(b) an oligonucleotide probe of the invention, that distinguishes one Gov allele from the other.\",\n \"The “means for amplifying” will, as the skilled will readily understand, depend on the amplification method to be used.\",\n \"Thus, for example, these means might include suitable primers, a suitable DNA polymerase, and the four 2′-deoxyribonucleoside triphosphates (dA, dC, dG, dT), if amplification is to be by the PCR method.\",\n \"To cite another example, if the amplification is to be by a method relying on transcription, such as the 3SR method, the means will include two primers, at least one of which, when made double-stranded, will provide a promoter, an RNA polymerase capable of transcribing from that promoter, a reverse transcriptase to function in primer-initiated, DNA-directed and RNA-directed, DNA polymerization and possibly also in RNAse H degradation of RNA to free DNA strands from RNA/RNA hybrids, the four ribonucleoside triphosphates (A, C, G and U), and the four 2′-deoxyribonucleoside triphosphates.\",\n \"In another example, if the amplification is by the ligase chain reaction, the means will include two oligonucleotides (DNAs) and a suitable DNA ligase that will join the two if a target, to which both can hybridize adjacent to one another in ligatable orientation, is present.\",\n \"The oligonucleotide probes of the invention will preferably be labelled.\",\n \"The label may be any of the various labels available in the art for such probes, including, but not limited to 32P; 35S; biotin (to which a signal generating moiety, bound to or complexed with avidin can be complexed); a fluorescent moiety; an enzyme such as alkaline phosphatase (which is capable of catalysing a chromogenic reaction); digoxigenin, as described above; or the like.\",\n \"As indicated in the examples, RFLP analysis can be employed, using BstNI (or isoschizomers thereof), in analysing cDNA or genomic DNA (with or without amplification) to determine Gov genotype.\",\n \"As indicated further in the examples, electrophoretic SSCP analysis may be used to determine Gov genotype.\",\n \"And as indicated in the examples, the hybridization studies outlined above may use fluorescent probes, and may be directly coupled to the DNA amplification step, as in “Real-Time PCR” or related methods.\",\n \"There has also been provided, in accordance with another aspect of the present invention, a method of typing for Gov allele-specific target sequence in a CD 109 nucleic acid derived from a subject, comprising the steps of, (a) obtaining, by a target nucleic acid amplification process applied to mRNA from human platelets, endothelial cells, or T cells, an assayable quantity of amplified nucleic acid with a sequence that is that of a subsequence (or the complement of a subsequence) of the mRNA that encodes a CD109 said subsequence including the nucleotide at the position in the mRNA corresponding to position 2108 in SEQ ID NO:1 or to nucleotide 954 in SEQ ID NO:5; and (b) analyzing (e.g., in a nucleic acid probe hybridization assay employing an oligonucleotide probe or probes according to the invention) the amplified nucleic acid obtained in step (a) to determine the base or bases at the position in the amplified nucleic acid that corresponds to position 2108 in SEQ ID NO:1 or to nucleotide 954 in SEQ ID NO:5.It is noteworthy that, if the product of the amplification is double-stranded DNA, analysis for Gov genotype can be carried out by a RFLP (restriction fragment length polymorphism) analysis comprising exposing the amplified DNA to the restriction endonuclease BstNI (or isoschizomer thereof) under conditions whereby the DNA will be cleaved if it includes a site for cleavage by that enzyme.\",\n \"Such DNA, prepared from mRNA encoding the Govb alloantigen, containing a C rather than an A at the position corresponding to nucleotide 2108 in SEQ ID NO:1 (or to nucleotide 954 in SEQ ID NO:5), includes a recognition site for that endonuclease, while such DNA prepared from mRNA encoding the Gova alloantigen, does not If the analysis, by whatever method, of the amplified nucleic acid reveals that there is only an A (or dA) at the position corresponding to position 12108, the platelets (and blood from which they came) have only the Gova alloantigen, and the individual from whom the platelets came, is homozygous for Gova.\",\n \"Alternatively, if the analysis of the amplified nucleic acid reveals that there is only a C (or dC) at the position corresponding to position 2108, the platelets (and blood from which they came) have only the Govb alloantigen and the individual, from whom the platelets came, is homozygous for the Govb allele.\",\n \"Finally, if the analysis indicates that there is either an A (or dA) or a C (or dC) at that position, the platelets (and blood from which they came) have both Gov alloantigens, and the individual from whom the platelets came, is heterozygous for Gov alloantigen.\",\n \"In one application of the typing methods of the invention, the methods are applied to two individuals to determine whether blood or platelets from one would provoke an alloimmune response, and possibly PTP or PTPR, in the other.\",\n \"The typing method can be applied with a man and a woman, who are contemplating conceiving or have conceived a child together, to determine the risk that the child would be at risk for NATP and the risk that the woman would be at increased risk for PTP or PTPR.\",\n \"If the woman were heterozygous for the Gov alloantigens there would be, due to Gov alloantigen incompatibility, no risk of NATP and no increased risk for the woman of PTP or PTPR.\",\n \"If, however, the woman were homozygous for one of the Gov alloantigens, there would be, due to Gov alloantigen incompatibility, risk of NATP in a child and increased risk of PTP or PTPR for the woman, unless the man is homozygous for the same Gov alloantigen as is the woman.\",\n \"In accordance with yet another aspect of the present invention, a method of typing an individual for Gov alloantigen is provided that comprises analyzing the genomic DNA of the individual to determine the Gov alloantigen(s) of the individual.\",\n \"Applications of this method are substantially the same as those of the method of the invention for typing for Gov alloantigen that begins with platelet, endothelial cell, or T cell mRNA.\",\n \"This method of the invention, entailing analysis of genomic DNA, can be carried out in substantially the same way as outlined above for analysis of mRNA, namely first amplifying the genomic DNA and then analyzing to product of the amplification to ascertain whether there is only dA, only dC, or both dA and dC, at the position in the coding region of the genomic DNA corresponding to position 2108 in SEQ ID NO:1, or to nucleotide 954 in SEQ ID NO:5.In accordance with a further aspect of the present invention, a test kit for Gov alloantigen typing is provided comprising a non-human antibody (or antibodies) that distinguishes the two allelic forms of CD109.The antibody (or antibodies) of the kit may be polyclonal, or preferably monoclonal, and in addition to its (their) specificity for either but not both Gov alloantigens (on the surface of platelets or separated therefrom) or the CD109 subunit of one but not both of such alloantigens, typically will recognise a polypeptide molecule encoded by a nucleotide sequence encoding at least amino acid 703 of a CD109 polypeptide (the amino acid at the position corresponding to nucleotides 2107-2109 in SEQ ID NO:1, or to nucleotides 953-955 in SEQ ID NO:5).\",\n \"DETAILED DESCRIPTION OF THE INVENTION Definitions The following definitions are provided in order to aid those skilled in the art in understanding the detailed description of the present invention.\",\n \"The term “alloantigens” refers to antigens of an individual that are responsible for eliciting an alloimmune response.\",\n \"The phrase “alloimmune response” refers to an immune response, which occurs when antibodies from one individual react against antigens of a different individual of the same species.\",\n \"The phrase “anti-idiotypic antibodies” refers to antibodies which can bind endogenous or foreign idiotypic antibodies and which can be used to treat or prevent pathological conditions associated with an immune response to a foreign alloantigen.\",\n \"The phrase “Gova/Govb biallelic system” refers to a system of human platelet alloantigens in which an individual can be homozygous for either Gova or Govb allelic forms of CD 109, or an individual can be Gova/Govb heterozygous for CD109.“GPI” refers to glycosylphosphatidylinositol.\",\n \"The term “NATP” refers to neonatal alloimmune thrombocytopenia.\",\n \"“Nucleic acid” includes DNA and RNA, whether single or double stranded.\",\n \"The term is also intended to include a strand that is a mixture of nucleic acids and nucleic acid analogs and/or nucleotide analogs, or that is made entirely of nucleic acid analogs and/or nucleotide analogs.\",\n \"“Nucleic acid analogue” refers to modified nucleic acids or species unrelated to nucleic acids that are capable of providing selective binding to nucleic acids or other nucleic acid analogues.\",\n \"As used herein, the term “nucleotide analogues” includes nucleic acids where the internucleotide phosphodiester bond of DNA or RNA is modified to enhance biostability of the oligomer and “tune” the selectivity/specificity for target molecules (Ulhmann, et al., 1990, Angew.\",\n \"Chem.\",\n \"Int.\",\n \"Ed.\",\n \"Eng., 90: 543; Goodchild, 1990, J. Bioconjugate Chem., I: 165; Englisch et al., 1991, Angew, Chem.\",\n \"Int.\",\n \"Ed.\",\n \"Eng., 30: 613).\",\n \"Such modifications may include and are not limited to phosphorothioates, phosphorodithioates, phosphotriesters, phosphoramidates or methylphosphonates.\",\n \"The 2′-O-methyl, allyl and 2′-deoxy-2′-fluoro RNA analogs, when incorporated into an oligomer show increased biostability and stabilization of the RNA/DNA duplex (Lesnik et al., 1993, Biochemistry, 32: 7832).\",\n \"As used herein, the term “nucleic acid analogues” also include alpha anomers (α-DNA), L-DNA (mirror image DNA), 2′-5′ linked RNA, branched DNA/RNA or chimeras of natural DNA or RNA and the above-modified nucleic acids.\",\n \"For the puposes of the present invention, any nucleic acid containing a “nucleotide analogue” shall be considered as a nucleic acid analogue.\",\n \"Backbone replaced nucleic acid analogues can also be adapted to for use as immobilised selective moieties of the present invention.\",\n \"For purposes of the present invention, the peptide nucleic acids (PNAs) (Nielsen et al, 1993, Anti-Cancer Drug Design, 8: 53; Engels et al., 1992, Angew, Chem.\",\n \"Int.\",\n \"Ed.\",\n \"Eng., 31: 1008) and carbamate-bridged morpholino-type oligonucleotide analogs (Burger, D. R., 1993, J.\",\n \"Clinical Immunoassay, 16: 224; Uhlmann, et al., 1993, Methods in Molecular Biology, 20,.\",\n \"“Protocols for Oligonucleotides and Analogs,” ed.\",\n \"Sudhir Agarwal, Humana Press, NJ, U.S.A., pp.\",\n \"335-389) are also embraced by the term “nucleic acid analogues”.\",\n \"Both exhibit sequence-specific binding to DNA with the resulting duplexes being more thermally stable than the natural DNA/DNA duplex.\",\n \"Other backbone-replaced nucleic acids are well known to those skilled in the art and may also be used in the present invention (See e.g., Uhlmann et al 1993, Methods in Molecular Biology, 20, “Protocols for Oligonucleotides and Analogs,” ed.\",\n \"Sudhir Agrawal, Humana Press, NJ, U.S.A., pp.\",\n \"335).\",\n \"The term “PTP” refers to post-transfusion purpura.\",\n \"The term “PTPR” refers to post-transfusion platelet refractorines.\",\n \"“SNP” refers to single nucleotide polymorphism.\",\n \"The standard, one-letter codes “A,” “C,” “G, ” and “T” are used herein for the nucleotides adenylate, cytidylate, guanylate, and thymidylate, respectively.\",\n \"The skilled will understand that, in DNAs, the nucleotides are 2′-deoxyribonucleotide-5′-phosphates (or, at the 5′-end, possibly triphosphates) while, in RNAs, the nucleotides are ribonucleotide-5′-phosphates (or, at the 5′-end, possibly triphosphates) and uridylate (U) occurs in place of T. “N” means any one of the four nucleotides.\",\n \"On occasion herein, dA, dC, dG and dT might be used for the respective 2′-deoxyribonucleotides.\",\n \"Unless otherwise specified or required by the context, “nucleic acid” means DNA or RNA and “nucleotide” means ribonucleotide or 2′-deoxyribonucleotide.\",\n \"Reference herein to a “full-length” CD109 molecule or protein means the 1445-amino acid-long polypeptide, for which the amino acid sequence, deduced from a cDNA sequence, is provided in SEQ ID NO:1 and in SEQ ID NO:3 and which is denoted as the full-length translated product (i.e., including the amino-terminal leader peptide, and excluding carboxyl-terminal processing associated with GPI anchor addition).\",\n \"The Gova alloantigen bearing form of CD109 may be referred to herein as 703Tyr CD109.The Govb alloantigen bearing form of CD109 may be referred to herein as 703Ser CD109.It has been determined that a single nucleotide of the CD109 gene is responsible for the Gov polymorphism in CD109.Extensive serological studies initially demonstrated that the polymorphism underlying the Gov system resides solely on the CD109 molecule [Sutherland, D. R. (1991); Smith et al.\",\n \"(1995)].\",\n \"Further, extensive deglycosylation of CD109 does not affect the binding the anti-Gova and anti-Govb antibodies to molecules of the appropriate phenotype, or to cells bearing the appropriate CD109 variant, indicating that carbohydrate residues are not involved in the formation of Gov antigenic epitopes.\",\n \"Further work has indicated that the Gov allele-specific antibody binding can however, be abrogated by denaturation of CD109 with the detergent SDS [Smith et al.\",\n \"(1995)].\",\n \"Taken together, these observations indicate that the Gov alleles of CD109 are protein epitopes that are likely defined by the primary amino acid sequence of CD109.Following the isolation of a CD109 cDNA the nature of the two Gov alleles was characterised further using platelet RNA-derived cDNA in the polymerase chain reaction (“PCR”).\",\n \"Platelet mRNA transcripts were obtained from serologically defined Gova/a, Gova/b and Govb/b individuals.\",\n \"The RNA was then converted to cDNA, and the entire CD109 cDNA coding region was then amplified as a series of overlapping PCR products.\",\n \"The Gova [SEQ ID NO:1] and Govb [SEQ ID NO:3] alleles differ by an A to C substitution at position 2108 of the coding region of the CD109 cDNA.\",\n \"This single nucleotide polymorphism also results in a BstNI restriction site in the Govb allele that is not present in its Gova counterpart.\",\n \"On the basis of this BstNI site, Gova can by distinguished from Govb by restriction fragment length polymorphism (RFLP) analysis.\",\n \"This single nucleotide polymorphism can also be detected by SSCP analysis, and by allele-specific hybridization studies, including “Real-Time” PCR analyses.\",\n \"As a result of this A2108C single nucleotide polymorphism, the Gova allele [SEQ ID NO:2] of CD109 contains a Tyr at position 703 of the full-length protein, while the Govb allele [SEQ ID NO:4] contains a Ser in this position.\",\n \"The polymorphism does not alter the ability of Gova and Govb homozygous platelets to adhere to collagen types I, III and V. Additionally, the binding of anti-Gova and ant-Govb antibodies to platelets of the appropriate phenotype did not interfere with platelet adhesion to any of the above collagen types.\",\n \"Thus, while the Tyr703Ser results in the formation of the Gov alloantigen epitopes, it does not appear to impair platelet function.\",\n \"Identification and characterisation of the Gov alloantigen system permits pre- and post-natal diagnosis of the Gov phenotype of an individual, providing a warning for the possibility of NATP, PTP and PTPR.\",\n \"Allelic Gov typing of CD109 equates with the Gov status of the CD109 protein of an individual.\",\n \"The Gov system led to diagnostic and therapeutic strategies to avoid or control diseases that result from Gov incompatibility.\",\n \"The present invention can be applied to these tasks and goals in a variety of ways, illustrative examples of which are discussed below.\",\n \"For example, an oligonucleotide probe can be synthesized, in accordance with the present invention, that will hybridize to a cDNA segment, derived from CD109 mRNA, that contains the nucleotide G at polymorphic nucleotide 2108 (nucleotide=guanylate).\",\n \"Alternatively, an oligonucleotide probe can be synthesized that will hybridize with a CD109 cDNA segment containing the base adenine at nucleotide 2108.(nucleotide=adenylate).\",\n \"These allele-specific probes can be appropriately labelled and added to the generated cDNA segments under annealing conditions, such that only one of the allele-specific probes hybridizes and can be detected, thereby identifying the specific Gova or Govb allele.\",\n \"In accordance with conventional procedures, the design of an oligonucleotide probe according to the present invention preferably involves adjusting probe length to accommodate hybridization conditions (temperature, ionic strength, exposure time) while assuring allele-specificity.\",\n \"A length of ten to thirty nucleotides is typical.\",\n \"Diagnostic kits can also be used, in accordance with the present invention, for the determination and diagnosis of alloantigen phenotypes via the procedures described herein.\",\n \"Such a kit can include, among others, antibodies or antibody fragments to an antigenic determinant expressed by either of the above-described Gova- and Govb-encoding sequences.\",\n \"These antibodies would react with the blood sample of an individual so as to indicate whether that individual has a Gova or Govb phenotype.\",\n \"Alternatively, all the reagents required for the detection of nucleotide(s) that distinguish the Gov alloantigens, by means described herein, can be provided in a single kit that uses isolated genomic DNA, platelet (or other cellular) mRNA or total RNA, or corresponding cDNA from an individual.\",\n \"A kit containing a labelled probe that distinguishes, for example, nucleotide 2108 of CD109 can be utilised for Gov alloantigen genotyping and phenotyping.\",\n \"A further beneficial use of the nucleotide sequences that distinguish the Gova allele from the Govb allele is to obtain or synthesize the respective expression product, in the form of a peptide or polypeptide, encoded by these nucleotide sequences.\",\n \"These polypeptides can be used to generate antibodies for diagnostic and therapeutic uses, for example, with regard to pathological conditions such as PTP, PTPR or NATP.\",\n \"These polypeptides can also be used diagnostically to detect the presence of Gova or Govb specific antibodies in patient plasma or serum, or used therapeutically (see below; assays may be adopted, for example, from U.S. Pat.\",\n \"No.\",\n \"5,851,788).\",\n \"A polypeptide within the present invention which can be used for the purpose of generating such antibodies preferably comprises an amino-acid sequence that corresponds to (i.e., is coincident with or functionally equivalent to) a fragment of the CD109 molecule that includes amino acid 703.When amino acid 703 is Tyrosine, the polypeptide can be used, as described above, to produce antibodies that specifically bind the Gova form of CD109; in contrast, when it is Serine, antibodies can be obtained that specifically recognise the Govb form.\",\n \"The class of polypeptides thus defined, in accordance with the present invention, is not intended to include the native CD109 molecule, but does encompass fragments of the molecule, as well as synthetic polypeptides meeting the aforementioned definition.\",\n \"Although the length of a polypeptide within this class is not critical, the requirement for immunogenicity may require that the polypeptide be attached to an immunogenicity-imparting carrier.\",\n \"Such carriers include a particulate carrier such as a liposome or a soluble macromolecule (protein or polysaccharide) with a molecular weight in the range of about 10,000 to 1,000,000 Daltons Additionally, it may be desirable to administer the polypeptide with an adjuvant, such as complete Freund's adjuvant For artificial polypeptides, as distinguished from CD109 fragments, maximum length is determined largely by the limits of techniques available for peptide synthesis, which are currently about fifty amino acids.\",\n \"Thus, a synthetic polypeptide of the present invention is preferably between four to about fifty amino acids in length.\",\n \"In the context of the present invention, the term “antibody” encompasses monoclonal and polyclonal antibodies produced by any available means.\",\n \"Such antibodies can belong to any antibody class (IgG, IgM, IgA, etc.)\",\n \"and may be chimeric.\",\n \"Examples of the preparation and uses of polyclonal antibodies are disclosed in U.S. Pat.\",\n \"Nos.\",\n \"5,512,282, 4,828,985,5,225,331 and 5,124,147 which are incorporated by reference in their entirety The term “antibody” also encompasses antibody fragments, such as Fab and F(ab′)2 fragments, of anti-Gova or anti-Govb antibodies, conjugates of such fragments, and so-called “antigen binding proteins” (single-chain antibodies) which are based on anti-Gova or anti-Govb antibodies, in accordance, for example, with U.S. Pat.\",\n \"No.\",\n \"4,704,692, the contents of which are hereby incorporated by reference.\",\n \"Alternatively, monoclonal antibodies or fragments thereof within the present invention can be produced using conventional procedures via the expression of isolated DNA that encodes variable regions of such a monoclonal antibody in host cells such as E. coli (see, e.g., Ward et al., Nature, 341:544-546 (1989)) or transfected murine myeloma cells (see Gillies et al., Biotechnol.\",\n \"7:799-804 (1989); Nakatani et al., Biotechnol.\",\n \"7:805-810 (1989)).\",\n \"For additional examples of methods of the preparation and uses of monoclonal antibodies, see U.S. Pat.\",\n \"Nos.\",\n \"5,688,681, 5,688,657, 5,683,693, 5,667,781, 5,665,356, 5,591,628, 5,510,241, 5,503,987, 5,501,988, 5,500,345 and 5,496,705 that are incorporated by reference in their entirety.\",\n \"While human alloantisera currently used for serological typing are specifically excluded from this definition, the use of CD109 or Gov allele-specific peptides to detect anti-Gov antibodies in human plasma or serum, or to determine the specificity of such alloantibodies, are specifically included.\",\n \"Similarly, the use of such CD109 peptides or Gov allele-specific peptides to purify CD109 antibodies, or allele-specific CD109 antibodies from human serum is specifically included.\",\n \"Similarly, the use in vitro of such CD109 peptides or Gov allele-specific peptides to deplete allele-specific antibody activity from human serum samples, or to block CD109 antibody binding, or allele-specific antibody binding, is specifically included.\",\n \"Diagnostic applications of these antibodies are exemplified, according to the present invention, by the use of a kit containing an anti-Gova or an anti-Govb antibody, which undergoes a reaction with a sample of an individual's blood to determine a Gova or Govb platelet phenotype.\",\n \"Such a reaction involves the binding of anti-Gova antibody to Gova antigen or the binding of anti-Govb antibody to Govb antigen.\",\n \"The observation of antibody-antigen complex in a blood sample would indicate a positive result.\",\n \"A kit of this type could be used to diagnose, or to help prevent the occurrence of pathological conditions like PTP, PTPR, or NATP.\",\n \"A polypeptide of the present invention that is recognised specifically by anti-Gova or ant-Govb antibodies can also be used therapeutically.\",\n \"Thus, antibodies raised against such a polypeptide can be employed in the generation, via conventional methods, of anti-idiotypic antibodies, that is, antibodies that bind an anti-Gova or anti-Govb antibody.\",\n \"See, e.g., U.S. Pat.\",\n \"No.\",\n \"4,699,880, the contents of which are hereby incorporated by reference.\",\n \"Such anti-idiotypic antibodies would bind endogenous or foreign anti-Gov antibodies in the blood of an individual, which would treat or prevent pathological conditions associated with an immune response to a “foreign” Gov alloantigen.\",\n \"Alternatively, a polypeptide within the present invention can be administered to an individual, with a physiologically-compatible carrier, to achieve the same qualitative effect, namely, the selective reduction or elimination of circulating anti-Gov antibodies from a patient suffering or at risk from an immune response, or the abrogation by competitive binding to administered peptide, of the binding of Gov-specific antibodies to the platelets of such an individual The present invention is further described below by reference to the following, illustrative examples.\",\n \"EXAMPLE 1 PCR Amplification and Analysis of PCR Products Platelet total RNA was isolated from EDTA anticoagulated blood of Govaa and Govbb individuals in the manner described in Lymann et al., Blood 75:234348 (1990).\",\n \"First, platelet mRNA in 10 μl aliquots was heated to 70° C. for 10 minutes and quickly cooled on ice before reverse transcription.\",\n \"The first strand cDNA was then synthesized using 10 μM oligo dT, 40 units RNAsin (Promega), 2 mM of each dNTP (dN triphosphate) (Pharmacia), 500 units of cloned MMLV reverse transcriptase and 5× enzyme buffer (Gibco) in a total volume of 50 μl.\",\n \"The cDNA synthesis was carried out at 42° C. for 45 minutes and was stopped by chilling to 0° C. Overlapping sets of oligonucleotide primers (Table 2) based on the sequence of CD109 were then used to amplify by PCR the entire coding region of platelet CD109 in 8 overlapping segments that spanned the entire CD109 open reading frame.\",\n \"TABLE 2 Anneal- ing Temper- Frag- Size ature ment Sense Primer Antisense Primer (bp) (° C.) 1 K1-80 (−24) K1-650 544 568 59 5′ GTAGCCCAGGCAGACGCC 3′ 5′ GTGACAACCACTGTTGGATCAA 3′ 2 K1-1 445 K1-1120 1014 570 50 5′ CGCATTGTTACACTCTTCTC 3′ 5′ TACATTTCTTGAAATACCTG 3′ 3 K1-1022 910 K1-REV-1 1747 838 50 5′ GATTCTTCAAATGGACTTT 3′ 5′ GGCTGTGTCACAGAGATC 3′ 4 KI-1400 1291 GSP3 2165 875 55 5′ TGAATTCCCAATCCTGGAGGA 3′ 5′ GCCACCCAAGAAGTGATAGA 3′ 5 K1-M43 1898 6R4N 2998 1101 56 5′ TTCAGGAATGTGGACTCTGG 3′ 5′ CGGCTTCAAGGAAACATCT 3′ 6 K1-3080 2948 1-SN 3859 912 56 5′ CTGGGAGCACTTGGTTGTCA 3′ 5′ CAGCAACATCTAAATCAAAGGC 3′ 7 K1-3570 3462 7U3N 4337 876 50 5′ ACAATTTCAGACTTCTGAGG 3′ 5′ CACAGCCAAAGTTCCATA 3′ 8 K1-3920 3812 K1-4600 4489 678 55 5′ GACGAAGATCTATCCAAAATC 3′ 5′ GCTAGGACCTGTTGTACACC 3′ Table 2 lists the position of the 5′ end of each oligonucleotide with respect to the CD109 cDNA sequence, which includes both 3′ and 5′ untranslated regions, is noted in parentheses.\",\n \"The CD109 ORF encompasses nucleotides 1-4335 of the published CD109 cDNA, and corresponds exactly to the CD109 cDNA sequence presented in SEQ ID NO:1.The size of each PCR product, and the annealing temperature used for the corresponding primer pair, is listed.\",\n \"PCR reactions (50:I) containing 1×PCR buffer (Gibco Life Technologies), 1.5 mM MgCl2, 200:M of each dNTP, 1:M of each primer, 1.25 units Taq polymerase (Gibco Life Technologies), and 3:I cDNA underwent 40 cycles of 94° C. (45 seconds), primer-specific annealing temperature (Table 2; 45 seconds), and 72° C. (45-60 seconds), using a Perkin Elmer 2400 thermocycler.\",\n \"PCR products (30:I) were subsequently size-separated electrophoretically on a 1.2% agarose/TAE gel containing 1:g/ml ethidium bromide.\",\n \"Bands were subsequently excised and purified (50:I) using the QIAquick (Qiagen) kit for direct sequencing and subcloning.\",\n \"Sequencing reactions (3-5:1 purified product per reaction) were carried out using the Thermosequenase Cy5.5 dye terminator sequencing kit (Amersham Pharmacia Biotech) and the same primers that had been used for initial PCR amplification (Table 2), or selected internal CD109-specific primers as appropriate, and were subsequently analysed using the Open Gene automated DNA sequencing system (Visible Genetics).\",\n \"In parallel, PCR products were cloned into PmeI-digested pMABI, a pBS SK(−) (Stratagene) derivative containing a PmeI restriction site within the polylinker.\",\n \"Resultant plasmid clones were analysed by alkaline lysis/restriction digestion, and as appropriate (and following an additional overnight 13% PEG/ 1.6 M NaCl precipitation), by DNA sequence analysis as above.\",\n \"By combining direct PCR sequencing and the analysis of subcloned fragments, it was ensured that the DNA sequence of each PCR-derived cDNA fragment was obtained independently at least twice, with each fragment being sequenced in both directions in its entirety.\",\n \"This analysis revealed that the CD109 cDNA sequences of Govaa and Govbb individuals differed by a single nucleotide at position 2108 of the sequence shown in SEQ ID NO:1.Gova/a individuals have an A at position 2108, whereas Govb/b individuals have a C at the same position.\",\n \"This change results in a Tyr-Ser amino acid polymorphism at residue 703 of the full-length CD109 polypeptide chain.\",\n \"This single nucleotide polymorphism also results in a BstNI restriction site in the Govb allele that is not present in the Gova allele.\",\n \"Analysis of the other regions of the CD109 cDNA in their entirety revealed no other nucleotide differences that segregated with Gov phenotype (i.e., that could be used to distinguish the Gova allele from the Govb allele).\",\n \"To facilitate subsequent genomic DNA analyses of the Gova/b alleles, the intron/exon junctions of the exon bearing the putative Gov-specific nucleotide substitution identified above, as well as the DNA sequence of the flanking introns, were determined.\",\n \"CD109 cDNA-specific oligonucleotides binding in the vicinity of this substitution were used for the direct sequencing of p4L10, a pCYPAC—1-derived PAC clone bearing the human CD109 locus using the Open Gene system (Visible Genetics) as above.\",\n \"The nucleotide sequence of the Gov polymorphism-containing exon, as well as of the flanking introns, is presented in SEQ ID NO:5.The Gov polymorphism lies at nucleotide position 954 in SEQ ID NO:5.Subsequent work has mapped the intron-exon structure of the entire human CD109 locus, and has determined that the Gov single nucleotide polymorphism of CD109 lies in exon 19 of the CD109 gene.\",\n \"EXAMPLE 2 RFLP Analysis of PCR Amplified Genomic DNA The A-C Gov CD109 polymorphism corresponds to the internal nucleotide of the first complete codon of exon 19 of the CD109 gene.\",\n \"As this exon comprises only 118 nucleotides, and the Gov polymorphism lies almost at the extreme 5′ end of this exon, we determined the nucleotide sequence of both introns flanking this exon to facilitate subsequent genomic DNA analyses of the Gova/b alleles.\",\n \"The DNA sequence of CD109 exon 19 and its flanking introns (CD109 introns 18 and 19) is presented as SEQ ID NO:5.To confirm that the A to C polymorphism at position 2108 of the CD109 open reading frame (nucleotide 2108, SEQ ID NO: 1; nucleotide 954, SEQ ID NO:5) segregates with the Gov phenotype, RFLP analysis was carried out on PCR amplified genomic CD109 DNA using the BstNI restriction endonuclease, which recognises the DNA sequence 5′CCAGG 3′ found in the Govb cDNA (nucleotides position 2108-2112 in SEQ ID NO:3; the corresponding Gova sequence, 5′ ACAGG 3′, is nucleotides 2108-2112 in SEQ ID NO:1).\",\n \"This enzyme does not cleave at 5′ ACAGG 3′ (found in Gova; nucleotides 2108-2112 in SEQ ID NO: 1).\",\n \"A 448 bp genomic fragment was PCR-amplified from Govaa, Govab, and Govbb individuals using the pair of oligonucleotides SEQ ID NO:9 and SEQ ID NO:10.These oligonucleotides flank exon 19.The former binds within intron 18 (nucleotides 875-892 SEQ ID NO:5), while the latter binds within intron 19 to the sequence complementary to nucleotides 1305-1322 of SEQ ID NO:5).\",\n \"The resultant 448 bp PCR product, when digested with BstNI, yielded the restriction fragments predicted on the basis that the A to C polymorphism at position 2108 (SEQ ID NO: 1) segregates with the Gov phenotype.\",\n \"EXAMPLE 3 Hybridization Analysis of PCR Amplified Genomic DNA To further confirm that the A to C polymorphism at position 2108 of the CD109 open reading frame (nucleotide 2108, SEQ ID NO:1; nucleotide 954, SEQ ID NO:5) segregates with the Gov phenotype, we also performed an alternative analysis involving the selective hybridization of Gov allele-specific DNA probes to PCR amplified genomic CD109 DNA.\",\n \"Two primers flanking the polymorphic A-C site at position 2108 (SEQ ID NO:1; position 954, SEQ ID NO:5) were designed to amplify by PCR a 105 bp genomic DNA fragment containing the polymorphic site from genomic DNA isolated from Govaa, Govab, and Govbb individuals.\",\n \"The first primer (SEQ ID NO:11) binds within intron 18 to nucleotides 902-928 of SEQ ID NO:5.The second primer (SEQ ID NO:12) binds within exon 19 to the sequence complementary to nucleotides 977-1106 of SEQ ID NO:5.Two additional nucleotide probes were designed—one specific for the target sequence of the Gova allele of the CD109 gene, and the other for the Govb allele of the CD109 gene.\",\n \"The first probe (SEQ ID NO:13) overlaps the CD109 intron 18/exon 19 junction, binds to the Gova allele at nucleotides 935-974 of SEQ ID NO:5, and was tagged with the fluorescent dye 6-FAM.\",\n \"The second probe (SEQ ID NO:14), also overlapping the CD109 intron 18/exon 19 junction, binds to the Govb allele at the position corresponding to nucleotides 935-971 of SEQ ID NO:5, and was tagged with the fluorescent dye VIC.\",\n \"Genomic DNA was isolated from Gov phenotyped human peripheral blood leukocytes, and PCR/hybridization analysis was carried out using Taqman real-time PCR technology (Perkin Elmer).\",\n \"Genomic DNA was amplified using primers SEQ ID NO:11 and SEQ ID NO:12, with each reaction additionally containing 100 nM FAM-labelled Gova probe and 200 nM VIC-labelled Govb probe.\",\n \"Allelic discrimination, based on allele-specific fluorescence, was then determined using a post-PCR plate reader (Perkin Elmer).\",\n \"In all cases, PCR/fluorescence-based Gov genotyping correlated with the Gov phenotype, indicating that the A to C polymorphism at position 2108 (SEQ ID NO: 1) does indeed segregate with the Gov phenotype.\",\n \"EXAMPLE 4 SSP Analysis of PCR Amplified Genomic DNA To further confirm that the A to C polymorphism at position 2108 of the CD109 open reading frame (nucleotide 2108, SEQ ID NO:1; nucleotide 954, SEQ ID NO:5) segregates with the Gov phenotype, we also performed an alternative analysis involving SSCP analysis of PCR amplified genomic CD109 DNA.\",\n \"Two Gov allele-specific antisense oligonucleotides—SEQ ID NO:6 and SEQ ID NO:7—differing by a single 3′ nucleotide (and binding to sequence complementary to nucleotides 954-976 of SEQ ID NO:5, and of the Govb counterpart of SEQ ID NO:5, respectively), were combined with a common sense primer—SEQ ID NO:8 binds within intron 18 and which corresponds to nucleotides 752-773 of SEQ ID NO:5, to amplify a 225 bp genomic DNA fragment containing the Gov polymorphic site from genomic DNA isolated from Govaa, Govab, and Govbb individuals.\",\n \"In all cases, complete concordance between PCR-SSP analysis and Gov phenotyping was observed.\",\n \"Sequences: SEQ ID NO: 1 consists of the entire 4335 nucleotide CD109 cDNA open reading frame encoding the Gova allele.\",\n \"The Gova allele comprises an A at nucleotide position 2108.SEQ ID NO:2 consists of the entire 1445 aa protein sequence produced from CD109 Gova cDNA.\",\n \"The Gova allele comprises a Tyr at amino acid 703.SEQ ID NO: 3 consists of the entire 4335 nucleotide CD109 cDNA open reading frame encoding the Govb allele.\",\n \"The Govb allele comprises a C at nucleotide position 2108.SEQ ID NO: 4 consists of the entire 1445 aa protein sequence produced from the CD109 Govb cDNA.\",\n \"The Govb allele comprises a Ser at amino acid 703.SEQ ID NO: 5 consists of the CD109 genomic DNA comprising CD109 exon 19 and the flanking introns, introns 18 and 19.The 118 nucleotide exon 19, comprising nucleotides 952-1069 of SEQ ID NO:5, corresponds to nucleotides 2106-2223 of SEQ ID NO: 1.The A to C Gov polymorphism of CD109 (corresponding to nucleotide 2108 of SEQ ID NO: 1) therefore corresponds to nucleotide 954 of SEQ ID NO:5.In the Gova allele, nucleotide 954 is A, while in the Govb allele nucleotide 954 is C. Thus, SEQ ID NO:5 corresponds to the Gova allele of CD109.Within SEQ ID NO:5, nucleotides 1-951 correspond to CD109 intron 18, while nucleotides 1070-2608 correspond to intron 19.We note that nucleotides 2108-2112 of SEQ ID NO: 1, and the corresponding nucleotides 954-958 of SEQ ID NO:5, which consist of the sequence 5′ ACAGG 3′ (and which contains the Gova allele-specific polymorphic nucleotide at its 5′ end), is not cleavable by the restriction endonuclease BstNI.\",\n \"However, in the corresponding Govb allele, the corresponding sequence—5′ CCAGG 3′—is cleavable by BstNI, and that the two Gov alleles can be discriminated on this basis.\",\n \"We note also that a group of restriction endonucleases—Bst2UI, BstNI, BstOI, EcoRII, MaeIII, MspR91, MvaI, or ScrFI (or one of their isoschizomers)—is capable of differentiating between the Gova and Govb alleles on this basis.\",\n \"SEQ ID NO:6- SEQ ID NO:14 comprise oligonucleotides for the PCR amplification of Gov polymorphism containing CD109 sequence from RNA, cDNA derived from RNA, or from genomic DNA, and for the Gov typing analyses of such amplified DNA fragments.\",\n \"SEQ ID NO:6.SEQ ID NO: 3, an antisense oligonucleotide specific for the Gova allele, binds to exon 19 sequence complementary to nucleotides 954-976 of SEQ ID NO:5.SEQ ID NO:6 and SEQ ID NO: 7 (see below) differ by a single allele-specific 3′ nucleotide SEQ ID NO:7.SEQ ID NO:7, an antisense oligonucleotide specific for the Govb allele, binds to exon 19 sequence complementary to nucleotides 954-976 of the Govb counterpart of SEQ ID NO:5.SEQ ID NO:6 (see above) and SEQ ID NO:7 differ by a single allele-specific 3′ nucleotide.\",\n \"SEQ ID NO:8.SEQ ID NO:8 binds within intron 18, and corresponds to nucleotides 752-773 of SEQ ID NO:5.SEQ ID NO:9.SEQ ID NO:9 binds within intron 18 (nucleotides 875-892 SEQ ID NO:5).\",\n \"SEQ ID NO:10.SEQ ID NO:10 binds within intron 19 to the sequence complementary to nucleotides 1305-1322 of SEQ ID NO:5.SEQ ID NO:11 SEQ ID NO:1 binds within intron 18 to nucleotides 902-928 of SEQ ID NO:5.SEQ ID NO:12.SEQ ID NO:12, binds within exon 19 to the sequence complementary to nucleotides 977-1006 of SEQ ID NO:5.SEQ ID NO:13.SEQ ID NO:13, specific for the Gova allele, overlaps the CD109 intron 18/exon 19 junction, and binds to the Gova allele at nucleotides 935-974 of SEQ ID NO:5.SEQ ID NO:14.SEQ ID NO:14, specific for the Govb allele, overlaps the CD109 intron 18/exon 19 junction, and binds to the Govb allele at the position corresponding to nucleotides 935-971 of SEQ ID NO:5.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471327"}}},{"rowIdx":589,"cells":{"text":{"kind":"list like","value":[["Alkylated c-sugars and uses thereof","The present invention relates to alkylated C-sugars which are new carbohydrate derivatives based on the “C-Sugar” platform.","These alkylated C-sugars are converted from hydrophilic, hydrogen-bonded saccaride derivatives and are very stable, highly soluble and relatively low molecular weight structures.","These alkylated C-sugars can serve as effective bioconjugates and pharmaceutical carriers.","The alkylated C-sugar have the formula wherein n=0, 1, 2, 3, 4, Rx=aryl, alkyl, or halogen-substituted aryl or alkyl, Y=(CH2)mNH2,(CH2)mCO2H,(CH2)mOH,(CH2)mCH, or (CH2)mCl, and m=0, 1, 2 or 3."],["1.An alkylated C-sugar of the formula wherein n=0, 1, 2, 3 or 4, Rx=aryl, alkyl, or halogen-substituted aryl or alkyl, 2.An alkylated C-sugar of the formula wherein X═CO2H, CHO, OH, NH2, Cl, Br or CH2OH, and R=alkyl or aryl.","3.A method of making the alkylated C-sugar according to claim 1, comprising alkylating a C-sugar to eliminate hydroxyl groups and obtain ether linkages.","4.A method of making the alkylated C-sugar according to claim 2, comprising alkylating a C-sugar to eliminate hydroxyl groups and obtain ether linkages.","5.The method according to claim 3, wherein the C-sugar to be alkylated is derived from mannose, glucose or galactose.","6.The method according to claim 4, wherein the C-sugar to be alkylated is derived from mannose, glucose or galactose.","7.The method according to claim 3, wherein the alkylated C-sugar is structured to comprise a free carboxylic acid, amine or other functional groups which facilitate coupling of said alkylated C-sugar to a drug or peptide.","8.The method according to claim 4, wherein the alkylated C-sugar is structured to comprise a free carboxylic acid, amine or other functional groups which facilitate coupling of said alkylated C-sugar to a drug or peptide.","9.A composition comprising a drug and the alkylated C-sugar according to claim 1.10.A composition comprising a drug and the alkylated C-sugar according to claim 2.11.The composition according to claim 9, wherein the alkylated C-sugar serves as a bioconjugate.","12.The composition according to claim 10, wherein the alkylated C-sugar serves as a bioconjugate.","13.The composition according to claim 9, wherein the drug is a peptide or polypeptide.","14.The composition according to claim 10, wherein the drug is a peptide or polypeptide.","15.The composition according to claim 9, wherein the alkylated C-sugar is coupled to said drug.","16.The composition according to claim 10, wherein the alkylated C-sugar is coupled to said drug.","17.A method of making the composition according to claim 9, comprising coupling said alkylated C-sugar to said drug.","18.A method of making the composition according to claim 10, comprising coupling said alkylated C-sugar to said drug.","19.A bioconjugate comprising the alkylated C-sugar according to claim 1.20.A bioconjugate comprising the alkylated C-sugar according to claim 2.21.A glycopeptide comprising the alkylated C-sugar according to claim 1, said alkylated C-sugar being coupled to the N-terminal, C-terminal and/or side chain of a peptide or polypeptide.","22.A glycopeptide comprising the alkylated C-sugar according to claim 2, said alkylated C-sugar being coupled to the N-terminal, C-terminal and/or side chain of a peptide or polypeptide.","23.A method of making the glycopeptide according to claim 21, comprising coupling said alkylated C-sugar to the N-terminal, C-terminal and/or side chain of the peptide or polypeptide.","24.A method of making the glycopeptide according to claim 22, comprising coupling said alkylated C-sugar to the N-terminal, C-terminal and/or side chain of the peptide or polypeptide."],["<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to alkylated C-sugars which are new carbohydrate derivatives based on the “C-Sugar” platform.","These alkylated C-sugars are converted from hydrophilic, hydrogen-bonded saccharide derivatives and are very stable, highly soluble and relatively low molecular weight structures.","These alkylated C-sugars can serve as effective bioconjugates and pharmaceutical carriers.","2.Description of the Related Art Advances in the synthetic chemistry of oligosaccharides, the most structurally diverse biopolymers, have been driven principally by the growing understanding of their role as encoders of biological information.","Glycoconjugates on cell surfaces have a major role in biological phenomena such as the immune response, intercellular recognition, cellular adhesion, intracellular targeting, cell growth regulation, metastasis, and inflammation (Dwek, 1996; Kahane, 1996; Garegg, 1993).","Glycosylation of proteins affects a variety of properties such as folding, packing, proteolytic resistance, conformational stability, quaternary structure, and water structure (Dwek, 1996; St. Hilaire, 1998).","For these reasons, interest is strong in the creation and biological evaluation of oligosaccharide and glycopeptide libraries (Armstrong, 1996; Seeberger, 1996; Sofia, 1998).","While cell surface carbohydrates are, typically, oligosaccharides, recognition by a protein typically involves a monosaccharide or disaccharide unit of the oligosaccharide.","Consequently, monoglycosylated amino acids (and analogs) and a few glycosyl-glycosylated amino acids have been the building blocks of glycopeptide libraries.","More interest has focused on the glycopeptide libraries, principally because cell surface carbohydrates, while they bind to proteins with high specificity, typically have weak binding constants (and limited bioavailability due to their biological instability).","With glycopeptide libraries, the principal aim has been to create glycomimetics targeted for lectins, enzymes, and other cell surface receptors which bind carbohydrates (St. Hilaire et al., 1998; Sutherlin et al., 1996).","For example, considerable interest has focused on E-selectin and its ligand, the sialyl Lewis X antigen because of their role in inflammation.","Glycomimetics of sialyl Lewis X, a tetrasaccharide, have been found with binding potencies higher than the natural compound (Bertozzi, 1992) and glycosylation of peptides is known to enhance binding to this receptor (St. Hilaire, 1998).","Various methods are available for developing small molecule therapeutics ranging from structure-based combinatorial techniques (Ellman, 1996; Gallop, 1994), computer screening approaches (Li et al., 1997), and various peptidomimetic strategies (Hruby, 1993; Spatola, 1983; Sawyer, 1997).","Once a lead is identified, the developmental emphasis is directed toward optimizing potency, enhancing receptor or target specificity, and ensuring bioavailability with growing emphasis on nuclear-based therapies.","It is clear that entry into the nucleus through membrane barriers becomes a formal obstacle.","Various workers have reported success with pro-drugs, other bioconjugates (Niidome, 1999; Prouillat, 1998), and tactics designed to mask difficulties such as desolvation penalties by using, for example, N-methyl amides to enhance the solubility of polyamide species such as peptides (Conradi, 1992).","More recently, there has been renewed interest in stabilized glycopeptides as drug conjugates.","Currently, polyethylene glycol (PEG) is used as an effective bioconjugate because of its global solubility, enhanced stability through the ester linkage, and an optimum blend of non-reactivity and non-immunogenicity (Greenwald, 2000, Zalipsky, 1995).","This knowledge and the benefits of glycoconjugation has led the present inventors to research C-sugars as bioconjugates and determine their effect when coupled to peptides and drugs.","Through their research, the present inventors believe that the alkylated C-sugars of the present invention are particularly effective as bioconjugates.","The presently claimed alkylated C-sugars possess some of the beneficial attributes of PEG and additionally provides stereochemical versatility via five stereocenters.","The alkylated C-sugars can be easily and covalently linked to either a backbone or side chain peptide or pseudopeptide functionality or a drug.","Thus, the invention has been completed based on this understanding and research."],["<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is directed to alkylated C-sugars of the following formula: wherein n=0, 1, 2, 3 or 4, R x =aryl, alkyl, or halogen-substituted aryl or alkyl, The present invention is also directed to compositions comprising a drug and the alkylated C-sugars, as well as methods of making and using said alkylated C-sugars and compositions.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["This application is a U.S. non-provisional application which claims the benefit of U.S. provisional application Ser.","No.","60/274,860 filed Mar.","9, 2001, the teachings of which are hereby incorporated by reference.","BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to alkylated C-sugars which are new carbohydrate derivatives based on the “C-Sugar” platform.","These alkylated C-sugars are converted from hydrophilic, hydrogen-bonded saccharide derivatives and are very stable, highly soluble and relatively low molecular weight structures.","These alkylated C-sugars can serve as effective bioconjugates and pharmaceutical carriers.","2.Description of the Related Art Advances in the synthetic chemistry of oligosaccharides, the most structurally diverse biopolymers, have been driven principally by the growing understanding of their role as encoders of biological information.","Glycoconjugates on cell surfaces have a major role in biological phenomena such as the immune response, intercellular recognition, cellular adhesion, intracellular targeting, cell growth regulation, metastasis, and inflammation (Dwek, 1996; Kahane, 1996; Garegg, 1993).","Glycosylation of proteins affects a variety of properties such as folding, packing, proteolytic resistance, conformational stability, quaternary structure, and water structure (Dwek, 1996; St. Hilaire, 1998).","For these reasons, interest is strong in the creation and biological evaluation of oligosaccharide and glycopeptide libraries (Armstrong, 1996; Seeberger, 1996; Sofia, 1998).","While cell surface carbohydrates are, typically, oligosaccharides, recognition by a protein typically involves a monosaccharide or disaccharide unit of the oligosaccharide.","Consequently, monoglycosylated amino acids (and analogs) and a few glycosyl-glycosylated amino acids have been the building blocks of glycopeptide libraries.","More interest has focused on the glycopeptide libraries, principally because cell surface carbohydrates, while they bind to proteins with high specificity, typically have weak binding constants (and limited bioavailability due to their biological instability).","With glycopeptide libraries, the principal aim has been to create glycomimetics targeted for lectins, enzymes, and other cell surface receptors which bind carbohydrates (St. Hilaire et al., 1998; Sutherlin et al., 1996).","For example, considerable interest has focused on E-selectin and its ligand, the sialyl Lewis X antigen because of their role in inflammation.","Glycomimetics of sialyl Lewis X, a tetrasaccharide, have been found with binding potencies higher than the natural compound (Bertozzi, 1992) and glycosylation of peptides is known to enhance binding to this receptor (St. Hilaire, 1998).","Various methods are available for developing small molecule therapeutics ranging from structure-based combinatorial techniques (Ellman, 1996; Gallop, 1994), computer screening approaches (Li et al., 1997), and various peptidomimetic strategies (Hruby, 1993; Spatola, 1983; Sawyer, 1997).","Once a lead is identified, the developmental emphasis is directed toward optimizing potency, enhancing receptor or target specificity, and ensuring bioavailability with growing emphasis on nuclear-based therapies.","It is clear that entry into the nucleus through membrane barriers becomes a formal obstacle.","Various workers have reported success with pro-drugs, other bioconjugates (Niidome, 1999; Prouillat, 1998), and tactics designed to mask difficulties such as desolvation penalties by using, for example, N-methyl amides to enhance the solubility of polyamide species such as peptides (Conradi, 1992).","More recently, there has been renewed interest in stabilized glycopeptides as drug conjugates.","Currently, polyethylene glycol (PEG) is used as an effective bioconjugate because of its global solubility, enhanced stability through the ester linkage, and an optimum blend of non-reactivity and non-immunogenicity (Greenwald, 2000, Zalipsky, 1995).","This knowledge and the benefits of glycoconjugation has led the present inventors to research C-sugars as bioconjugates and determine their effect when coupled to peptides and drugs.","Through their research, the present inventors believe that the alkylated C-sugars of the present invention are particularly effective as bioconjugates.","The presently claimed alkylated C-sugars possess some of the beneficial attributes of PEG and additionally provides stereochemical versatility via five stereocenters.","The alkylated C-sugars can be easily and covalently linked to either a backbone or side chain peptide or pseudopeptide functionality or a drug.","Thus, the invention has been completed based on this understanding and research.","SUMMARY OF THE INVENTION The present invention is directed to alkylated C-sugars of the following formula: wherein n=0, 1, 2, 3 or 4, Rx=aryl, alkyl, or halogen-substituted aryl or alkyl, The present invention is also directed to compositions comprising a drug and the alkylated C-sugars, as well as methods of making and using said alkylated C-sugars and compositions.","DESCRIPTION OF THE PREFERRED EMBODIMENT C-sugars are derivatives of carbohydrates in which the anomeric carbon is replaced by a methylene unit.","The presently claimed alkylated C-sugars utilizes C-sugars instead of carbohydrates or polymers as the core conjugate.","Through alkylation, the inventors have eliminated hydroxyl groups in favor of ether linkages.","As background, the methods for making the alkylated C-sugars of the present invention starts with the basic approaches used in the prior art.","As an example, the stereoselective synthesis of alpha and beta C-glucosides in Scheme 1 (see below) is representative of an approach used in the prior art (Lewis, 1982).","The approach to Sa and 6a of Scheme 2 works equally well for the synthesis of alpha-mannoside 7a, and this is readily converted to 7b in high yield (Tang, 1998).","The beta-linked C-glycoside 8a (Scheme 2) was obtained by Bertozzi employing free radical chemistry and the subtle manipulation of phthalimido N-protecting groups (Roe, 1996), while 8b was obtain by Hollingsworth using direct SN2 alkylation of a halo sugar (Kim, 1994).","These methods should be adaptable to the synthesis of 9a and 9b (Scheme 2) as well.","Compounds 7, 8, and 9 can be the starting points for the construction of an initial set of C-glycosylated amino acid building blocks which is an embodiment of the present invention.","The key step in the synthesis of beta-GlcNAc—CH2CO—NH-Lys (Scheme 2) is the carbodiimide-mediated coupling of protected GlcNAc derivative 10 with lysine derivative 11 (P=Boc or Fmoc).","While Hollingsworth and Bertozzi used acetyl protection of OH groups for their synthesis of 8, either route is adaptable employing benzyl ether protection.","Benzyl ether protection has been employed in the synthesis of the analogous GlcNAc—C-glycoside 12 (Liang, 1996).","Benzyl ether protection is preferable to acetyl in this case because it offers orthogonality of conditions for deprotection and enhanced stability for solid phase synthesis of oligopeptides.","Further, if HF deprotection conditions result in the Friedel Crafts alkylation of Tyr residues that might be present in the glycopeptides, then 2,6-dichloro-benzyl ether protection could be employed.","For the synthesis of alpha-Man-CH2CO—NH-Lys, alpha-Man-CH2CO—NH-Lys can be prepared by an analogous coupling of benzyl ether-protected 7b (R═H) (Tang, 1998).","Both D and L amino acids would be used affording both diastereoisomers for use in glycopeptide assembly.","The basic approach for the synthesis of the alpha and beta-linked C-serine analogs can be illustrated by reference to the case of alpha-Man-CH2Ser.","Scheme 2 outlines the synthesis of alpha-Man-CH2Ser.","Those skilled in the art can follow the teachings of Scheme 2 below to make any desired C-Sugar.","The ester group of 7b (or the aldehyde group from the ozonization of 7a) will be reduced and the resulting alcohol converted to a sulfonate or some other leaving group.","A glycine anion 13 (Luxen, 1993) can be used for amino acid synthesis and it can be employed as shown in Scheme 2, Reaction (1) above.","The reaction will give a mixture of L and D diastereoisomers which is what is desired since both will be employed in glycopeptide formation.","The diastereoisomers can be separated by chromatography.","In the case of the GlcNAc— and GalNAc—CH2Ser derivatives, phthalimido protection may have to be employed for N in the event of H abstraction by the nucleophile or base.","An alternative approach to C-serine derivatives has been developed by Bertozzi (Bertozzi, 1992).","This approach is summarized as Reaction (2) of Scheme 2 above.","One skilled in the art should be familiar with the synthesis of Cl aldehyde derivatives such as 14.For example, the alpha and beta mannose derivative 15 has been the starting point for a number of C-mannosides (Zhang, 1998).","An advantage of the approach of reaction pathway (2) is that it allows for the selection of the amino acid stereoisomer, either L or D. The disadvantage is that isomerization of an aldehyde a isomer to the more thermodynamically stable b isomer is facile, often occurring under conditions of reaction at the aldehyde center.","Consequently, this pathway appears to be limited to the synthesis of only b-linked C-glycosyl derivatives.","Nonetheless, it remains an alternative approach that has been demonstrated to work.","Scheme 3 The following scheme teaches the synthesis of an alkylated mannosylacetic acid which is a specific embodiment of the present invention: Synthesis of Permethylated Mannosylacetic Acid a. Allylation A. Hosami, Y. Sakata, H. Sakurai, “Highly Stereoselective C-allylation of Glycopyranosides with Allylsilanes Catalyzed by Silyltriflate or Iodosilane,” Tetrahedron Lett., 1984, 25, 2383.b.","Oxidation P. A. Aristoff, P. D. Johnson, and A. W. Harrison, “Total Synthesis of a Novel Antiulcer Agent via a Modification of the Intramolecular Wadsworth-Emmons-Wittig Reaction,” JACS, 1985, 107, 7967-7974.As described earlier, the C-sugars are typically prepared in an O-benzyl protected form.","The derivatives can then be coupled to a peptide-free amine using standard condensation procedures and the remaining peptide and carbohydrate protecting groups are then removed, usually using catalytic hydrogenation.","This leaves a hydrophilic compound that can increase aqueous solubility and balance excessive peptide hydrophobic character.","Analogously, the alkylated C-sugars of the present invention can thus be readily prepared as, for example, an O-methyl rather than O-benzyl derivative.","But following peptide condensation, the O-methylation will be retained.","These ether compounds should now have solubility properties comparable to polyethylene glycol (PEG), the well-known bioconjugate.","After synthesis of a protected C-glycosyl residue or its desired amino acid derivative, conditions for its use in solid phase peptide construction will be optimized.","This will be a necessary step, one which can be performed based on the teachings of others (Bertozzi, 1992; St. Hilaire, 1998; Kutterer, 1999).","Once the alkylated C-sugars are prepared, appropriate coupling reagents should be selected.","The alkylated C-sugars, with, for example, a free carboxylic acid residue, will be condensed to the N-terminus of the appropriate linear peptides or a pseudopeptide using traditional peptide condensing agents.","Deprotection and cleavage from the solid support will be accomplished with anhydrous hydrogen fluoride (HF) (Sakakibara, 1971) singly or in groups (Houghten, 1986).","Alternatively, if HF proves unusually harsh for the carbohydrate component, inventors advise utilizing a two step cleavage and deprotection approach using two methods previously developed: phase transfer catalysis for peptide-solid phase resin cleavage (Anwer and Spatola, 1992) or by ammonium formate catalytic transfer hydrogenolysis (Anwer et al., 1983).","Alternatively, traditional hydrogenation at elevated temperature and pressure can be used if any of the hydroxylbenzyl functions prove refractory to the standard deprotection methods.","As an additional example of a functionalized alkylated C-sugar, a simplified method of synthesis for an embodiment of the present invention can be set forth as follows in Scheme 4: Thus, the alkylated C-sugars of the present invention can be structured to comprise a free carboxylic acid, amine group or any other appropriate functional group which facilitates the coupling of the C-sugar bioconjugate to a drug or peptide.","Also, the C-sugars may thus be coupled to a wide variety of drugs and/or peptides for the purpose of improving their solubility, membrane transport, and overall bioavailability.","Such variability in the types of coupling partners is shown in Scheme 5 below: Among the other variables for the claimed alkylated C-sugars in addition to a free functional group are the ring size, nature, and stereochemistry of the parent carbohydrates (such as mannose, glucose, and galactose) and the nature of the O-alkyl derivatives (O-methyl: Rx═CH3; O-ethyl: Rx═CH2CH3; trifluoromethyl, R═CF3; D-2,2,2-trifluoroethyl, R═CH2CF3, as well as other alkyl, substituted alky, aryl and other heterocyclic variants).","Moreover, both linear and cyclic peptide drug candidates may be enhanced by the attachment of the bioconjugates of the present invention.","Beneficial properties of cyclic peptides, are also set forth in our co-pending provisional application Ser.","No.","60/274,846 entitled STABILIZED L-Xxx-Yyy-L-L PEPTIDOMIMETICS are further enhanced by the stabilization of the carbohydrate moiety, as is described herein.","It is important to note that although the above disclosure primarily teaches the synthesis of N-terminal and side chain modified glycopeptides, the alkylated C-sugars taught herein can be linked to many desired molecules and as such, are considered to be novel bioconjugates.","One skilled in the art can easily link the claimed alkylated C-sugars to many desired molecules based on the knowledge readily possessed and available to one skilled in the art.","The desired molecules can be any substance (i.e.","drug) which is intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, and have an effect in the structure or function of the body so as to achieve its intended use.","Thus, the present invention should not be limited to the synthesis of N-terminal, C-terminal and side chain modified glycopeptides disclosed above.","Instead, the present invention should be interpreted to encompass any composition comprising a drug and the presently claimed alkylated C-Sugars.","As stated above, addition of a permethylated C-sugar to peptides can be conveniently performed through standard amide coupling reactions to the N-terminus, the C-terminus, or even to side chain functional groups.","In the latter case, an acid functionalized C-sugar is attached to lysine while an amine substituted C-sugar may be coupled to side chain carboxylic acids such as those found in aspartic acid or glutamic acid.","By selecting other functional group combinations, additional linkages to either linear or cyclic peptides may be effected using techniques well known to those skilled in the art.","In the present example, a C-sugar with a carboxylic acid group is added to the N-terminus of a cyclic peptide that has been prepared by solid phase methods of synthesis.","The final product retains the major structural characteristics required for its biological action but now is modified by a bioconjugate that provides important solubility advantages.","In a similar fashion, other drug candidates may be appropriately modified by one or more alkylated C-sugars to assist in the transport and bioavailability of the derivatized compound.","The present invention is further specifically described according to the following examples.","However, the Examples are merely illustrative in nature and should not be construed to limit the scope of the claims.","EXPERIMENTAL Synthetic and Analytical Conditions: KMnO4, NaIO4, a methyl D mannopyranose, CH3I, NaH, allyltrimethylsilane, and TMSOTf were purchased from Aldrich, and Silica gel (60 Å 70-230/mesh) was purchased from Whatman.","Thin layer chromatography (TLC) was performed on Merck-254 silica plates in the following systems (V/V): A. Ethyl acetate:hexane (1:1) B. Ethyl acetate:hexane (1:2) The Rf values are expressed with appropriate parenthetical abbreviations.","The plates were sprayed with a mixture of H2SO4/HCl and heated to be visualized.","Purification was performed by column chromatography using silica gel.","Molecular weight determinations were made on a Matrix-Assisted-Laser Desorption Ionization-Time of flight (MALDI-TOF) Mass Spectrometer Model, DE-Pro, made by Perceptive Biosystems (now Applied Biosystems) as well as on a QUATTRO-LCZ, Electron-Spray Ionization Quadripole Mass Analyzer, made by Micromass.","Example 1 Synthesis of a Methylated D-Mannosylacetic Acid and Coupling to a Peptide A. Synthesis of the Permethylated Mannoside The 5.84 g (0.03 mole) of a methyl D mannopyranoside was dissolved in freshly distilled DMF in a 3 neck round bottom flask equipped with a condenser under a flow of argon.","A two fold excess NaH (1.45 g, 0.06 moles) was added and the temperature was increased to 75° C. After two hours, the mixture was cooled and a two fold excess of CH3I (3.76 ml, 0.06 mole) was added.","After an hour, the process was repeated three to four times depending on the TLC of the mixture.","The mixture was then quenched by the dropwise addition of methanol and extracted with EtOAc and brine.","The organic layer was dried over Na2CO3 and the solvent was removed by rotary evaporation.","The oil was dried overnight under high vacuum and 5.21 g (69%) was obtained.","The compound was purified on a silica column with a solvent mixture of EtOAc:hexane (1:2) then (1:1).","3.2 g (43%) of pure product was obtained.","The compound was characterized by TLC, 1H and 13C NMR.","Rf=0.4 in A.","B. Allylation of the Permethylated Mannoside The 2.82 g (0.011 mole) of permethylated mannoside was dissolved in CH3CN at 0° C. under argon.","The allyltrimethylsilane 3.5 ml (0.022 moles) was added at once and then 2.03 ml (0.011 mole) TMSOTf was slowly added.","Completion of the reaction was checked after two hours.","The reaction was quenched by the dropwise addition of methanol and extracted with EtOAc and brine.","The organic layer was dried over Na2CO3 and the solvent was removed by rotary evaporation.","The oil was dried overnight under high vacuum and a yellowish oil was obtained.","The compound was purified by column chromatography with a mixture of EtOAc:hexane (1:3), then (1:2).","2.86 g (97%) of pure compound was obtained.","The alkene was characterized by TLC, 1H and 13C NMR.","Rf=0.14 in system B. C. Oxidation of the Alkene Aristoff's procedure to transform an alkene into an acid was applied.","A solution of 20 g (93 mmol) of sodium metaperiodate in 300 ml of distilled water was treated with 0.33 g (2 mmol) of potassium permanganate, stirred 30 min at 25° C., and treated with 1.6 g (12 mmol) of anhydrous potassium carbonate, and then 2.8 g (11 mmol) of the alkene in 80 ml of tert-butanol was added.","The resulting reddish-purple suspension was stirred for 2 hours at 25° C., heated with 2 ml (30 mmol) of ethylene glycol, stirred for 2 hours, acidified to pH=2 with 1M aqueous hydrochloric acid, and extracted with chloroform.","The combined organic extracts were washed with brine and dried (Na2SO4).","The solvents were removed in vacuum to give 1.9 g of acid as a yellowish oil (64%).","The compound was characterized by 1H, 13C NMR, IR, MALDI-TOF and ionization mass spectrometry.","C12H22O7 mass calculated: 278.29 mass found [M-1]: 277.3 D. Synthesis of an Alkylated C-Sugar Linked at the Amino Terminus of a Cyclic Peptide.","The compound: was assembled by classical methods using solid phase synthesis.","0.1 mmol of peptide loaded on the resin was treated with 0.2 mmol of permethylated mannosylacetic acid in freshly distilled DMF with TFFH (0.4 mmol) as a coupling agent and diisopropylethylamine as base.","After 2 hours the coupling was complete according to a Kaiser (ninhydrin) test.","The resin was washed with CH2Cl2/EtOH/CH2Cl2 and treated with anhydrous HF for 1 hour under standard conditions.","The compound obtained (88% yield) was checked by mass spectrometry and the expected structure was confirmed.","The disulfide bridge was formed quantitatively overnight by mixing the peptide with DMSO (excess).","The final mass of the compound was checked by mass spectrometry and confirmed.","Calculated for M+H: 1347.7 Found: 1347.7 Example 2 Synthesis of an Amine Funtionalized Methylated Mannose C-Sugar Following the methods disclosed above and from knowledge commonly available and possessed by one skilled in the art as reflected in the incorporated references cited herein, an aminomethyl version of an alkylated C-sugar can be synthesized based on the scheme shown below: Example 3 Synthesis of Alkalated C-Sugar Derivatives of Penicillin and Cephalasporin As stated above, the synthesis of N-terminal, C-terminal and side chain modified glycopeptides is only one of many embodiments of the present invention.","One skilled in the art can also easily link the presently claimed alkylated C-sugars to many desired molecules such as a drug based on the knowledge readily possessed and available to one skilled in the art as reflected in the incorporated references cited herein.","As an example, following the methods disclosed above and from knowledge commonly available and possessed by one skilled in the art, a functionalized alkylated C-sugar can be attached to an amino-penicillinic and amino-cephalasporic acid based on the scheme shown below.","Example 4 Peptide and Pseudopeptide Derivatives of Mannosylacetic Acid Following the methods disclosed above and in our co-pending U.S. provisional application Ser.","No.","60/274,846 entitled STABILIZED L-Xxx-Yyy-L-L PEPTIDOMIMETICS by Amo F. Spatola and Anne-Marie Leduc, the teaching of which is hereby incorporated by reference, the following C-sugar glycopeptides have been synthesized: 1.mannosylacetyl-Leu-Glu-Gln-Leu-Leu-OH.","2.mannosylacetyl-Leu-2-Nal φ[CH2NH]Gln-Leu-Leu-OH.","Those skilled in the art will recognize that many modifications, substitutions and extensions of the present invention disclosed herein may be readily made without departing from the teachings and scope of the appended claims.","Therefore, the scope of the claims is not limited to the embodiments herein described in detail, but covers all such modifications, substitutions and extensions of said embodiments.","All references cited herein are hereby incorporated by reference."]],"string":"[\n [\n \"Alkylated c-sugars and uses thereof\",\n \"The present invention relates to alkylated C-sugars which are new carbohydrate derivatives based on the “C-Sugar” platform.\",\n \"These alkylated C-sugars are converted from hydrophilic, hydrogen-bonded saccaride derivatives and are very stable, highly soluble and relatively low molecular weight structures.\",\n \"These alkylated C-sugars can serve as effective bioconjugates and pharmaceutical carriers.\",\n \"The alkylated C-sugar have the formula wherein n=0, 1, 2, 3, 4, Rx=aryl, alkyl, or halogen-substituted aryl or alkyl, Y=(CH2)mNH2,(CH2)mCO2H,(CH2)mOH,(CH2)mCH, or (CH2)mCl, and m=0, 1, 2 or 3.\"\n ],\n [\n \"1.An alkylated C-sugar of the formula wherein n=0, 1, 2, 3 or 4, Rx=aryl, alkyl, or halogen-substituted aryl or alkyl, 2.An alkylated C-sugar of the formula wherein X═CO2H, CHO, OH, NH2, Cl, Br or CH2OH, and R=alkyl or aryl.\",\n \"3.A method of making the alkylated C-sugar according to claim 1, comprising alkylating a C-sugar to eliminate hydroxyl groups and obtain ether linkages.\",\n \"4.A method of making the alkylated C-sugar according to claim 2, comprising alkylating a C-sugar to eliminate hydroxyl groups and obtain ether linkages.\",\n \"5.The method according to claim 3, wherein the C-sugar to be alkylated is derived from mannose, glucose or galactose.\",\n \"6.The method according to claim 4, wherein the C-sugar to be alkylated is derived from mannose, glucose or galactose.\",\n \"7.The method according to claim 3, wherein the alkylated C-sugar is structured to comprise a free carboxylic acid, amine or other functional groups which facilitate coupling of said alkylated C-sugar to a drug or peptide.\",\n \"8.The method according to claim 4, wherein the alkylated C-sugar is structured to comprise a free carboxylic acid, amine or other functional groups which facilitate coupling of said alkylated C-sugar to a drug or peptide.\",\n \"9.A composition comprising a drug and the alkylated C-sugar according to claim 1.10.A composition comprising a drug and the alkylated C-sugar according to claim 2.11.The composition according to claim 9, wherein the alkylated C-sugar serves as a bioconjugate.\",\n \"12.The composition according to claim 10, wherein the alkylated C-sugar serves as a bioconjugate.\",\n \"13.The composition according to claim 9, wherein the drug is a peptide or polypeptide.\",\n \"14.The composition according to claim 10, wherein the drug is a peptide or polypeptide.\",\n \"15.The composition according to claim 9, wherein the alkylated C-sugar is coupled to said drug.\",\n \"16.The composition according to claim 10, wherein the alkylated C-sugar is coupled to said drug.\",\n \"17.A method of making the composition according to claim 9, comprising coupling said alkylated C-sugar to said drug.\",\n \"18.A method of making the composition according to claim 10, comprising coupling said alkylated C-sugar to said drug.\",\n \"19.A bioconjugate comprising the alkylated C-sugar according to claim 1.20.A bioconjugate comprising the alkylated C-sugar according to claim 2.21.A glycopeptide comprising the alkylated C-sugar according to claim 1, said alkylated C-sugar being coupled to the N-terminal, C-terminal and/or side chain of a peptide or polypeptide.\",\n \"22.A glycopeptide comprising the alkylated C-sugar according to claim 2, said alkylated C-sugar being coupled to the N-terminal, C-terminal and/or side chain of a peptide or polypeptide.\",\n \"23.A method of making the glycopeptide according to claim 21, comprising coupling said alkylated C-sugar to the N-terminal, C-terminal and/or side chain of the peptide or polypeptide.\",\n \"24.A method of making the glycopeptide according to claim 22, comprising coupling said alkylated C-sugar to the N-terminal, C-terminal and/or side chain of the peptide or polypeptide.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to alkylated C-sugars which are new carbohydrate derivatives based on the “C-Sugar” platform.\",\n \"These alkylated C-sugars are converted from hydrophilic, hydrogen-bonded saccharide derivatives and are very stable, highly soluble and relatively low molecular weight structures.\",\n \"These alkylated C-sugars can serve as effective bioconjugates and pharmaceutical carriers.\",\n \"2.Description of the Related Art Advances in the synthetic chemistry of oligosaccharides, the most structurally diverse biopolymers, have been driven principally by the growing understanding of their role as encoders of biological information.\",\n \"Glycoconjugates on cell surfaces have a major role in biological phenomena such as the immune response, intercellular recognition, cellular adhesion, intracellular targeting, cell growth regulation, metastasis, and inflammation (Dwek, 1996; Kahane, 1996; Garegg, 1993).\",\n \"Glycosylation of proteins affects a variety of properties such as folding, packing, proteolytic resistance, conformational stability, quaternary structure, and water structure (Dwek, 1996; St. Hilaire, 1998).\",\n \"For these reasons, interest is strong in the creation and biological evaluation of oligosaccharide and glycopeptide libraries (Armstrong, 1996; Seeberger, 1996; Sofia, 1998).\",\n \"While cell surface carbohydrates are, typically, oligosaccharides, recognition by a protein typically involves a monosaccharide or disaccharide unit of the oligosaccharide.\",\n \"Consequently, monoglycosylated amino acids (and analogs) and a few glycosyl-glycosylated amino acids have been the building blocks of glycopeptide libraries.\",\n \"More interest has focused on the glycopeptide libraries, principally because cell surface carbohydrates, while they bind to proteins with high specificity, typically have weak binding constants (and limited bioavailability due to their biological instability).\",\n \"With glycopeptide libraries, the principal aim has been to create glycomimetics targeted for lectins, enzymes, and other cell surface receptors which bind carbohydrates (St. Hilaire et al., 1998; Sutherlin et al., 1996).\",\n \"For example, considerable interest has focused on E-selectin and its ligand, the sialyl Lewis X antigen because of their role in inflammation.\",\n \"Glycomimetics of sialyl Lewis X, a tetrasaccharide, have been found with binding potencies higher than the natural compound (Bertozzi, 1992) and glycosylation of peptides is known to enhance binding to this receptor (St. Hilaire, 1998).\",\n \"Various methods are available for developing small molecule therapeutics ranging from structure-based combinatorial techniques (Ellman, 1996; Gallop, 1994), computer screening approaches (Li et al., 1997), and various peptidomimetic strategies (Hruby, 1993; Spatola, 1983; Sawyer, 1997).\",\n \"Once a lead is identified, the developmental emphasis is directed toward optimizing potency, enhancing receptor or target specificity, and ensuring bioavailability with growing emphasis on nuclear-based therapies.\",\n \"It is clear that entry into the nucleus through membrane barriers becomes a formal obstacle.\",\n \"Various workers have reported success with pro-drugs, other bioconjugates (Niidome, 1999; Prouillat, 1998), and tactics designed to mask difficulties such as desolvation penalties by using, for example, N-methyl amides to enhance the solubility of polyamide species such as peptides (Conradi, 1992).\",\n \"More recently, there has been renewed interest in stabilized glycopeptides as drug conjugates.\",\n \"Currently, polyethylene glycol (PEG) is used as an effective bioconjugate because of its global solubility, enhanced stability through the ester linkage, and an optimum blend of non-reactivity and non-immunogenicity (Greenwald, 2000, Zalipsky, 1995).\",\n \"This knowledge and the benefits of glycoconjugation has led the present inventors to research C-sugars as bioconjugates and determine their effect when coupled to peptides and drugs.\",\n \"Through their research, the present inventors believe that the alkylated C-sugars of the present invention are particularly effective as bioconjugates.\",\n \"The presently claimed alkylated C-sugars possess some of the beneficial attributes of PEG and additionally provides stereochemical versatility via five stereocenters.\",\n \"The alkylated C-sugars can be easily and covalently linked to either a backbone or side chain peptide or pseudopeptide functionality or a drug.\",\n \"Thus, the invention has been completed based on this understanding and research.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is directed to alkylated C-sugars of the following formula: wherein n=0, 1, 2, 3 or 4, R x =aryl, alkyl, or halogen-substituted aryl or alkyl, The present invention is also directed to compositions comprising a drug and the alkylated C-sugars, as well as methods of making and using said alkylated C-sugars and compositions.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"This application is a U.S. non-provisional application which claims the benefit of U.S. provisional application Ser.\",\n \"No.\",\n \"60/274,860 filed Mar.\",\n \"9, 2001, the teachings of which are hereby incorporated by reference.\",\n \"BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to alkylated C-sugars which are new carbohydrate derivatives based on the “C-Sugar” platform.\",\n \"These alkylated C-sugars are converted from hydrophilic, hydrogen-bonded saccharide derivatives and are very stable, highly soluble and relatively low molecular weight structures.\",\n \"These alkylated C-sugars can serve as effective bioconjugates and pharmaceutical carriers.\",\n \"2.Description of the Related Art Advances in the synthetic chemistry of oligosaccharides, the most structurally diverse biopolymers, have been driven principally by the growing understanding of their role as encoders of biological information.\",\n \"Glycoconjugates on cell surfaces have a major role in biological phenomena such as the immune response, intercellular recognition, cellular adhesion, intracellular targeting, cell growth regulation, metastasis, and inflammation (Dwek, 1996; Kahane, 1996; Garegg, 1993).\",\n \"Glycosylation of proteins affects a variety of properties such as folding, packing, proteolytic resistance, conformational stability, quaternary structure, and water structure (Dwek, 1996; St. Hilaire, 1998).\",\n \"For these reasons, interest is strong in the creation and biological evaluation of oligosaccharide and glycopeptide libraries (Armstrong, 1996; Seeberger, 1996; Sofia, 1998).\",\n \"While cell surface carbohydrates are, typically, oligosaccharides, recognition by a protein typically involves a monosaccharide or disaccharide unit of the oligosaccharide.\",\n \"Consequently, monoglycosylated amino acids (and analogs) and a few glycosyl-glycosylated amino acids have been the building blocks of glycopeptide libraries.\",\n \"More interest has focused on the glycopeptide libraries, principally because cell surface carbohydrates, while they bind to proteins with high specificity, typically have weak binding constants (and limited bioavailability due to their biological instability).\",\n \"With glycopeptide libraries, the principal aim has been to create glycomimetics targeted for lectins, enzymes, and other cell surface receptors which bind carbohydrates (St. Hilaire et al., 1998; Sutherlin et al., 1996).\",\n \"For example, considerable interest has focused on E-selectin and its ligand, the sialyl Lewis X antigen because of their role in inflammation.\",\n \"Glycomimetics of sialyl Lewis X, a tetrasaccharide, have been found with binding potencies higher than the natural compound (Bertozzi, 1992) and glycosylation of peptides is known to enhance binding to this receptor (St. Hilaire, 1998).\",\n \"Various methods are available for developing small molecule therapeutics ranging from structure-based combinatorial techniques (Ellman, 1996; Gallop, 1994), computer screening approaches (Li et al., 1997), and various peptidomimetic strategies (Hruby, 1993; Spatola, 1983; Sawyer, 1997).\",\n \"Once a lead is identified, the developmental emphasis is directed toward optimizing potency, enhancing receptor or target specificity, and ensuring bioavailability with growing emphasis on nuclear-based therapies.\",\n \"It is clear that entry into the nucleus through membrane barriers becomes a formal obstacle.\",\n \"Various workers have reported success with pro-drugs, other bioconjugates (Niidome, 1999; Prouillat, 1998), and tactics designed to mask difficulties such as desolvation penalties by using, for example, N-methyl amides to enhance the solubility of polyamide species such as peptides (Conradi, 1992).\",\n \"More recently, there has been renewed interest in stabilized glycopeptides as drug conjugates.\",\n \"Currently, polyethylene glycol (PEG) is used as an effective bioconjugate because of its global solubility, enhanced stability through the ester linkage, and an optimum blend of non-reactivity and non-immunogenicity (Greenwald, 2000, Zalipsky, 1995).\",\n \"This knowledge and the benefits of glycoconjugation has led the present inventors to research C-sugars as bioconjugates and determine their effect when coupled to peptides and drugs.\",\n \"Through their research, the present inventors believe that the alkylated C-sugars of the present invention are particularly effective as bioconjugates.\",\n \"The presently claimed alkylated C-sugars possess some of the beneficial attributes of PEG and additionally provides stereochemical versatility via five stereocenters.\",\n \"The alkylated C-sugars can be easily and covalently linked to either a backbone or side chain peptide or pseudopeptide functionality or a drug.\",\n \"Thus, the invention has been completed based on this understanding and research.\",\n \"SUMMARY OF THE INVENTION The present invention is directed to alkylated C-sugars of the following formula: wherein n=0, 1, 2, 3 or 4, Rx=aryl, alkyl, or halogen-substituted aryl or alkyl, The present invention is also directed to compositions comprising a drug and the alkylated C-sugars, as well as methods of making and using said alkylated C-sugars and compositions.\",\n \"DESCRIPTION OF THE PREFERRED EMBODIMENT C-sugars are derivatives of carbohydrates in which the anomeric carbon is replaced by a methylene unit.\",\n \"The presently claimed alkylated C-sugars utilizes C-sugars instead of carbohydrates or polymers as the core conjugate.\",\n \"Through alkylation, the inventors have eliminated hydroxyl groups in favor of ether linkages.\",\n \"As background, the methods for making the alkylated C-sugars of the present invention starts with the basic approaches used in the prior art.\",\n \"As an example, the stereoselective synthesis of alpha and beta C-glucosides in Scheme 1 (see below) is representative of an approach used in the prior art (Lewis, 1982).\",\n \"The approach to Sa and 6a of Scheme 2 works equally well for the synthesis of alpha-mannoside 7a, and this is readily converted to 7b in high yield (Tang, 1998).\",\n \"The beta-linked C-glycoside 8a (Scheme 2) was obtained by Bertozzi employing free radical chemistry and the subtle manipulation of phthalimido N-protecting groups (Roe, 1996), while 8b was obtain by Hollingsworth using direct SN2 alkylation of a halo sugar (Kim, 1994).\",\n \"These methods should be adaptable to the synthesis of 9a and 9b (Scheme 2) as well.\",\n \"Compounds 7, 8, and 9 can be the starting points for the construction of an initial set of C-glycosylated amino acid building blocks which is an embodiment of the present invention.\",\n \"The key step in the synthesis of beta-GlcNAc—CH2CO—NH-Lys (Scheme 2) is the carbodiimide-mediated coupling of protected GlcNAc derivative 10 with lysine derivative 11 (P=Boc or Fmoc).\",\n \"While Hollingsworth and Bertozzi used acetyl protection of OH groups for their synthesis of 8, either route is adaptable employing benzyl ether protection.\",\n \"Benzyl ether protection has been employed in the synthesis of the analogous GlcNAc—C-glycoside 12 (Liang, 1996).\",\n \"Benzyl ether protection is preferable to acetyl in this case because it offers orthogonality of conditions for deprotection and enhanced stability for solid phase synthesis of oligopeptides.\",\n \"Further, if HF deprotection conditions result in the Friedel Crafts alkylation of Tyr residues that might be present in the glycopeptides, then 2,6-dichloro-benzyl ether protection could be employed.\",\n \"For the synthesis of alpha-Man-CH2CO—NH-Lys, alpha-Man-CH2CO—NH-Lys can be prepared by an analogous coupling of benzyl ether-protected 7b (R═H) (Tang, 1998).\",\n \"Both D and L amino acids would be used affording both diastereoisomers for use in glycopeptide assembly.\",\n \"The basic approach for the synthesis of the alpha and beta-linked C-serine analogs can be illustrated by reference to the case of alpha-Man-CH2Ser.\",\n \"Scheme 2 outlines the synthesis of alpha-Man-CH2Ser.\",\n \"Those skilled in the art can follow the teachings of Scheme 2 below to make any desired C-Sugar.\",\n \"The ester group of 7b (or the aldehyde group from the ozonization of 7a) will be reduced and the resulting alcohol converted to a sulfonate or some other leaving group.\",\n \"A glycine anion 13 (Luxen, 1993) can be used for amino acid synthesis and it can be employed as shown in Scheme 2, Reaction (1) above.\",\n \"The reaction will give a mixture of L and D diastereoisomers which is what is desired since both will be employed in glycopeptide formation.\",\n \"The diastereoisomers can be separated by chromatography.\",\n \"In the case of the GlcNAc— and GalNAc—CH2Ser derivatives, phthalimido protection may have to be employed for N in the event of H abstraction by the nucleophile or base.\",\n \"An alternative approach to C-serine derivatives has been developed by Bertozzi (Bertozzi, 1992).\",\n \"This approach is summarized as Reaction (2) of Scheme 2 above.\",\n \"One skilled in the art should be familiar with the synthesis of Cl aldehyde derivatives such as 14.For example, the alpha and beta mannose derivative 15 has been the starting point for a number of C-mannosides (Zhang, 1998).\",\n \"An advantage of the approach of reaction pathway (2) is that it allows for the selection of the amino acid stereoisomer, either L or D. The disadvantage is that isomerization of an aldehyde a isomer to the more thermodynamically stable b isomer is facile, often occurring under conditions of reaction at the aldehyde center.\",\n \"Consequently, this pathway appears to be limited to the synthesis of only b-linked C-glycosyl derivatives.\",\n \"Nonetheless, it remains an alternative approach that has been demonstrated to work.\",\n \"Scheme 3 The following scheme teaches the synthesis of an alkylated mannosylacetic acid which is a specific embodiment of the present invention: Synthesis of Permethylated Mannosylacetic Acid a. Allylation A. Hosami, Y. Sakata, H. Sakurai, “Highly Stereoselective C-allylation of Glycopyranosides with Allylsilanes Catalyzed by Silyltriflate or Iodosilane,” Tetrahedron Lett., 1984, 25, 2383.b.\",\n \"Oxidation P. A. Aristoff, P. D. Johnson, and A. W. Harrison, “Total Synthesis of a Novel Antiulcer Agent via a Modification of the Intramolecular Wadsworth-Emmons-Wittig Reaction,” JACS, 1985, 107, 7967-7974.As described earlier, the C-sugars are typically prepared in an O-benzyl protected form.\",\n \"The derivatives can then be coupled to a peptide-free amine using standard condensation procedures and the remaining peptide and carbohydrate protecting groups are then removed, usually using catalytic hydrogenation.\",\n \"This leaves a hydrophilic compound that can increase aqueous solubility and balance excessive peptide hydrophobic character.\",\n \"Analogously, the alkylated C-sugars of the present invention can thus be readily prepared as, for example, an O-methyl rather than O-benzyl derivative.\",\n \"But following peptide condensation, the O-methylation will be retained.\",\n \"These ether compounds should now have solubility properties comparable to polyethylene glycol (PEG), the well-known bioconjugate.\",\n \"After synthesis of a protected C-glycosyl residue or its desired amino acid derivative, conditions for its use in solid phase peptide construction will be optimized.\",\n \"This will be a necessary step, one which can be performed based on the teachings of others (Bertozzi, 1992; St. Hilaire, 1998; Kutterer, 1999).\",\n \"Once the alkylated C-sugars are prepared, appropriate coupling reagents should be selected.\",\n \"The alkylated C-sugars, with, for example, a free carboxylic acid residue, will be condensed to the N-terminus of the appropriate linear peptides or a pseudopeptide using traditional peptide condensing agents.\",\n \"Deprotection and cleavage from the solid support will be accomplished with anhydrous hydrogen fluoride (HF) (Sakakibara, 1971) singly or in groups (Houghten, 1986).\",\n \"Alternatively, if HF proves unusually harsh for the carbohydrate component, inventors advise utilizing a two step cleavage and deprotection approach using two methods previously developed: phase transfer catalysis for peptide-solid phase resin cleavage (Anwer and Spatola, 1992) or by ammonium formate catalytic transfer hydrogenolysis (Anwer et al., 1983).\",\n \"Alternatively, traditional hydrogenation at elevated temperature and pressure can be used if any of the hydroxylbenzyl functions prove refractory to the standard deprotection methods.\",\n \"As an additional example of a functionalized alkylated C-sugar, a simplified method of synthesis for an embodiment of the present invention can be set forth as follows in Scheme 4: Thus, the alkylated C-sugars of the present invention can be structured to comprise a free carboxylic acid, amine group or any other appropriate functional group which facilitates the coupling of the C-sugar bioconjugate to a drug or peptide.\",\n \"Also, the C-sugars may thus be coupled to a wide variety of drugs and/or peptides for the purpose of improving their solubility, membrane transport, and overall bioavailability.\",\n \"Such variability in the types of coupling partners is shown in Scheme 5 below: Among the other variables for the claimed alkylated C-sugars in addition to a free functional group are the ring size, nature, and stereochemistry of the parent carbohydrates (such as mannose, glucose, and galactose) and the nature of the O-alkyl derivatives (O-methyl: Rx═CH3; O-ethyl: Rx═CH2CH3; trifluoromethyl, R═CF3; D-2,2,2-trifluoroethyl, R═CH2CF3, as well as other alkyl, substituted alky, aryl and other heterocyclic variants).\",\n \"Moreover, both linear and cyclic peptide drug candidates may be enhanced by the attachment of the bioconjugates of the present invention.\",\n \"Beneficial properties of cyclic peptides, are also set forth in our co-pending provisional application Ser.\",\n \"No.\",\n \"60/274,846 entitled STABILIZED L-Xxx-Yyy-L-L PEPTIDOMIMETICS are further enhanced by the stabilization of the carbohydrate moiety, as is described herein.\",\n \"It is important to note that although the above disclosure primarily teaches the synthesis of N-terminal and side chain modified glycopeptides, the alkylated C-sugars taught herein can be linked to many desired molecules and as such, are considered to be novel bioconjugates.\",\n \"One skilled in the art can easily link the claimed alkylated C-sugars to many desired molecules based on the knowledge readily possessed and available to one skilled in the art.\",\n \"The desired molecules can be any substance (i.e.\",\n \"drug) which is intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, and have an effect in the structure or function of the body so as to achieve its intended use.\",\n \"Thus, the present invention should not be limited to the synthesis of N-terminal, C-terminal and side chain modified glycopeptides disclosed above.\",\n \"Instead, the present invention should be interpreted to encompass any composition comprising a drug and the presently claimed alkylated C-Sugars.\",\n \"As stated above, addition of a permethylated C-sugar to peptides can be conveniently performed through standard amide coupling reactions to the N-terminus, the C-terminus, or even to side chain functional groups.\",\n \"In the latter case, an acid functionalized C-sugar is attached to lysine while an amine substituted C-sugar may be coupled to side chain carboxylic acids such as those found in aspartic acid or glutamic acid.\",\n \"By selecting other functional group combinations, additional linkages to either linear or cyclic peptides may be effected using techniques well known to those skilled in the art.\",\n \"In the present example, a C-sugar with a carboxylic acid group is added to the N-terminus of a cyclic peptide that has been prepared by solid phase methods of synthesis.\",\n \"The final product retains the major structural characteristics required for its biological action but now is modified by a bioconjugate that provides important solubility advantages.\",\n \"In a similar fashion, other drug candidates may be appropriately modified by one or more alkylated C-sugars to assist in the transport and bioavailability of the derivatized compound.\",\n \"The present invention is further specifically described according to the following examples.\",\n \"However, the Examples are merely illustrative in nature and should not be construed to limit the scope of the claims.\",\n \"EXPERIMENTAL Synthetic and Analytical Conditions: KMnO4, NaIO4, a methyl D mannopyranose, CH3I, NaH, allyltrimethylsilane, and TMSOTf were purchased from Aldrich, and Silica gel (60 Å 70-230/mesh) was purchased from Whatman.\",\n \"Thin layer chromatography (TLC) was performed on Merck-254 silica plates in the following systems (V/V): A. Ethyl acetate:hexane (1:1) B. Ethyl acetate:hexane (1:2) The Rf values are expressed with appropriate parenthetical abbreviations.\",\n \"The plates were sprayed with a mixture of H2SO4/HCl and heated to be visualized.\",\n \"Purification was performed by column chromatography using silica gel.\",\n \"Molecular weight determinations were made on a Matrix-Assisted-Laser Desorption Ionization-Time of flight (MALDI-TOF) Mass Spectrometer Model, DE-Pro, made by Perceptive Biosystems (now Applied Biosystems) as well as on a QUATTRO-LCZ, Electron-Spray Ionization Quadripole Mass Analyzer, made by Micromass.\",\n \"Example 1 Synthesis of a Methylated D-Mannosylacetic Acid and Coupling to a Peptide A. Synthesis of the Permethylated Mannoside The 5.84 g (0.03 mole) of a methyl D mannopyranoside was dissolved in freshly distilled DMF in a 3 neck round bottom flask equipped with a condenser under a flow of argon.\",\n \"A two fold excess NaH (1.45 g, 0.06 moles) was added and the temperature was increased to 75° C. After two hours, the mixture was cooled and a two fold excess of CH3I (3.76 ml, 0.06 mole) was added.\",\n \"After an hour, the process was repeated three to four times depending on the TLC of the mixture.\",\n \"The mixture was then quenched by the dropwise addition of methanol and extracted with EtOAc and brine.\",\n \"The organic layer was dried over Na2CO3 and the solvent was removed by rotary evaporation.\",\n \"The oil was dried overnight under high vacuum and 5.21 g (69%) was obtained.\",\n \"The compound was purified on a silica column with a solvent mixture of EtOAc:hexane (1:2) then (1:1).\",\n \"3.2 g (43%) of pure product was obtained.\",\n \"The compound was characterized by TLC, 1H and 13C NMR.\",\n \"Rf=0.4 in A.\",\n \"B. Allylation of the Permethylated Mannoside The 2.82 g (0.011 mole) of permethylated mannoside was dissolved in CH3CN at 0° C. under argon.\",\n \"The allyltrimethylsilane 3.5 ml (0.022 moles) was added at once and then 2.03 ml (0.011 mole) TMSOTf was slowly added.\",\n \"Completion of the reaction was checked after two hours.\",\n \"The reaction was quenched by the dropwise addition of methanol and extracted with EtOAc and brine.\",\n \"The organic layer was dried over Na2CO3 and the solvent was removed by rotary evaporation.\",\n \"The oil was dried overnight under high vacuum and a yellowish oil was obtained.\",\n \"The compound was purified by column chromatography with a mixture of EtOAc:hexane (1:3), then (1:2).\",\n \"2.86 g (97%) of pure compound was obtained.\",\n \"The alkene was characterized by TLC, 1H and 13C NMR.\",\n \"Rf=0.14 in system B. C. Oxidation of the Alkene Aristoff's procedure to transform an alkene into an acid was applied.\",\n \"A solution of 20 g (93 mmol) of sodium metaperiodate in 300 ml of distilled water was treated with 0.33 g (2 mmol) of potassium permanganate, stirred 30 min at 25° C., and treated with 1.6 g (12 mmol) of anhydrous potassium carbonate, and then 2.8 g (11 mmol) of the alkene in 80 ml of tert-butanol was added.\",\n \"The resulting reddish-purple suspension was stirred for 2 hours at 25° C., heated with 2 ml (30 mmol) of ethylene glycol, stirred for 2 hours, acidified to pH=2 with 1M aqueous hydrochloric acid, and extracted with chloroform.\",\n \"The combined organic extracts were washed with brine and dried (Na2SO4).\",\n \"The solvents were removed in vacuum to give 1.9 g of acid as a yellowish oil (64%).\",\n \"The compound was characterized by 1H, 13C NMR, IR, MALDI-TOF and ionization mass spectrometry.\",\n \"C12H22O7 mass calculated: 278.29 mass found [M-1]: 277.3 D. Synthesis of an Alkylated C-Sugar Linked at the Amino Terminus of a Cyclic Peptide.\",\n \"The compound: was assembled by classical methods using solid phase synthesis.\",\n \"0.1 mmol of peptide loaded on the resin was treated with 0.2 mmol of permethylated mannosylacetic acid in freshly distilled DMF with TFFH (0.4 mmol) as a coupling agent and diisopropylethylamine as base.\",\n \"After 2 hours the coupling was complete according to a Kaiser (ninhydrin) test.\",\n \"The resin was washed with CH2Cl2/EtOH/CH2Cl2 and treated with anhydrous HF for 1 hour under standard conditions.\",\n \"The compound obtained (88% yield) was checked by mass spectrometry and the expected structure was confirmed.\",\n \"The disulfide bridge was formed quantitatively overnight by mixing the peptide with DMSO (excess).\",\n \"The final mass of the compound was checked by mass spectrometry and confirmed.\",\n \"Calculated for M+H: 1347.7 Found: 1347.7 Example 2 Synthesis of an Amine Funtionalized Methylated Mannose C-Sugar Following the methods disclosed above and from knowledge commonly available and possessed by one skilled in the art as reflected in the incorporated references cited herein, an aminomethyl version of an alkylated C-sugar can be synthesized based on the scheme shown below: Example 3 Synthesis of Alkalated C-Sugar Derivatives of Penicillin and Cephalasporin As stated above, the synthesis of N-terminal, C-terminal and side chain modified glycopeptides is only one of many embodiments of the present invention.\",\n \"One skilled in the art can also easily link the presently claimed alkylated C-sugars to many desired molecules such as a drug based on the knowledge readily possessed and available to one skilled in the art as reflected in the incorporated references cited herein.\",\n \"As an example, following the methods disclosed above and from knowledge commonly available and possessed by one skilled in the art, a functionalized alkylated C-sugar can be attached to an amino-penicillinic and amino-cephalasporic acid based on the scheme shown below.\",\n \"Example 4 Peptide and Pseudopeptide Derivatives of Mannosylacetic Acid Following the methods disclosed above and in our co-pending U.S. provisional application Ser.\",\n \"No.\",\n \"60/274,846 entitled STABILIZED L-Xxx-Yyy-L-L PEPTIDOMIMETICS by Amo F. Spatola and Anne-Marie Leduc, the teaching of which is hereby incorporated by reference, the following C-sugar glycopeptides have been synthesized: 1.mannosylacetyl-Leu-Glu-Gln-Leu-Leu-OH.\",\n \"2.mannosylacetyl-Leu-2-Nal φ[CH2NH]Gln-Leu-Leu-OH.\",\n \"Those skilled in the art will recognize that many modifications, substitutions and extensions of the present invention disclosed herein may be readily made without departing from the teachings and scope of the appended claims.\",\n \"Therefore, the scope of the claims is not limited to the embodiments herein described in detail, but covers all such modifications, substitutions and extensions of said embodiments.\",\n \"All references cited herein are hereby incorporated by reference.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471328"}}},{"rowIdx":590,"cells":{"text":{"kind":"list like","value":[["Circuit arrangement for switching a current of and off without the occurrence of overcurrent","There is provided a circuit arrangement for switching a current on and off without overcurrent.","The circuit arrangement includes a current source, a load associated with the current source, a switching transistor for switching the current source on and off, where the switching transistor has a parasitic capacitance between a control electrode and a first output electrode, and a shorting device between the control electrode and the first output electrode of the switching transistor for switching off the current source.","The current can be used, for example, to drive a laser diode."],["1.A circuit arrangements for switching a current on and off without overcurrent, comprising: a current source; a load associated with the current source; a switching transistor for switching the current source on and off, wherein the switching transistor has a parasitic capacitance between a control electrode and a first output electrode, which is not at constant potential; and a shorting device between the control electrode and the first output electrode of the switching transistor for switching off the current source.","2.The circuit arrangement as claimed in claim 1, wherein the shorting device comprises a transistor having: a control electrode for having a switch-on/switch-off signal applied to it; an output electrode connected to the first output electrode; and an output electrode connected to the control electrode of the switching transistor.","3.The circuit arrangement as claimed in claim 1, wherein the shorting device has an associated pull-up device.","4.The circuit arrangement as claimed in claim 1, wherein one or more of the current source, the switching transistor, the shorting device and the pull-up device are in a form selected from the group consisting of a bipolar transistor and a field-effect transistor.","5.The circuit arrangement as claimed in claim 1, wherein the load is a laser diode.","6.The circuit arrangement as claimed in claim 1, wherein the current is switched off by the switching transistor if a constant voltage applied to a control electrode of the current source is lower than a sum of threshold voltages of the current source and the switching transistor."],["<SOH> BACKGROUND OF THE INVENTION <EOH>Circuit arrangements for switching a current driving a load on and off are known.","FIG.","1 shows such a circuit arrangement, which uses a transistor 20 ′ as a current source or current sink.","The control electrode of the transistor 20 ′ is connected to a DC voltage source 80 ′.","The current source 20 ′ is connected in series with a load 40 ′, for example with a laser diode.","A switching transistor 50 ′, which is likewise connected in series with the current source 20 ′, is used as a switch for the current source.","The switching transistor 50 ′, which is in the form of a field-effect transistor, for example, is connected by means of its gate electrode 51 ′ to a driving device which comprises the transistors 75 ′ and 70 ′, for example.","The input 1 of the driving device 70 ′, 75 ′ has a switch-on/switch-off signal P applied to it, as illustrated in FIG.","2 , for example.","Between the gate 51 ′ and the drain electrode 52 ′ of the switching transistor 50 ′ there is a manufacture-dependent parasitic capacitance 60 ′, which is also known as the Miller capacitance.","As will be explained later, this parasitic capacitance is the cause of overcurrents that are brought about when the current source 20 ′ is switched off.","The gate/source voltage GS of the switching transistor 50 ′ controls the state of the switching transistor.","If this gate/source voltage GS is lower than the threshold voltage of the switching transistor 50 ′, the current IL is interrupted by the switching transistor 50 ′.","The gate/source voltage GS is S controlled by means of the driving device 70 ′, 75 ′.","However, when the switching transistor 50 ′ and hence the current source 20 ′ are disconnected, an overcurrent, also called a current spike, is produced which has its origins in the parasitic capacitance 60 ′ of the switching transistor 50 ′.","This is because a falling edge of the gate/source voltage GS on the switching transistor 50 ′ (the profile of said gate/source voltage being shown in FIG.","4 ) causes the parasitic capacitance 60 ′ to transfer the sudden voltage change on the control electrode 51 ′ to the output electrode 52 ′ of the switching transistor 50 ′ and to drive the transistor 20 ′ at a higher level.","This causes an overcurrent through the load 40 ′, as shown in FIG.","3 at the time t a .","The overcurrent flows until the parasitic capacitance's charge has been reversed.","The time t a characterizes the time at which the switch-off signal is applied to the input 1 of the driving device 70 ′, 75 ′.","In the case of certain applications, such current spikes can result in the load 40 ′ being destroyed, as can be the case with a laser diode, for example."],["<SOH> SUMMARY OF THE INVENTION <EOH>The invention is now based on the object of improving the initially described circuit arrangement for switching a current on and off such that overcurrents can be avoided when a current source is switched off.","The invention solves this technical problem with the features of claim 1 .","Accordingly, a circuit arrangement for switching a current on and off without overcurrent is provided.","The circuit arrangement has a current source, a load associated with the current source and a switching transistor for switching the current source on and off.","Depending on manufacture, the switching transistor contains a parasitic capacitance between the control electrode and a first output electrode, which causes charge-reversal currents when the current source is switched off.","A shorting device, connected between the control electrode and the first output electrode of the switching device, for switching off the current source can be used to prevent the charge-reversal currents from resulting in overcurrents in the load.","Expediently, the shorting device is in the form of a transistor whose control electrode can have a switch-on/switch-off signal applied to it.","One output electrode of the shorting device is connected to the first output electrode of the switching transistor, and the other output electrode of the shorting device is connected to the control electrode of the switching transistor.","In this way, the shorting device shorts the switching transistor and hence switchs off the current source.","In order to be able to shorten the input and output cycles, a pull-up device is connected in parallel with the shorting device and ensures that the potential on the switching transistor's control electrode can be raised at the switch-on time.","The current source, the switching transistor, the shorting device and/or the pull-up device can be in the form of bipolar transistors, field-effect transistors, particularly MOS transistors, or combinations of these technologies.","The circuit arrangement is suitable particularly for driving a laser diode as a load."],["FIELD OF THE INVENTION The invention relates to a circuit arrangement for switching a current on and off without overcurrent, that current being used to drive a laser diode, for example.","BACKGROUND OF THE INVENTION Circuit arrangements for switching a current driving a load on and off are known.","FIG.","1 shows such a circuit arrangement, which uses a transistor 20′ as a current source or current sink.","The control electrode of the transistor 20′ is connected to a DC voltage source 80′.","The current source 20′ is connected in series with a load 40′, for example with a laser diode.","A switching transistor 50′, which is likewise connected in series with the current source 20′, is used as a switch for the current source.","The switching transistor 50′, which is in the form of a field-effect transistor, for example, is connected by means of its gate electrode 51′ to a driving device which comprises the transistors 75′ and 70′, for example.","The input 1 of the driving device 70′, 75′ has a switch-on/switch-off signal P applied to it, as illustrated in FIG.","2, for example.","Between the gate 51′ and the drain electrode 52′ of the switching transistor 50′ there is a manufacture-dependent parasitic capacitance 60′, which is also known as the Miller capacitance.","As will be explained later, this parasitic capacitance is the cause of overcurrents that are brought about when the current source 20′ is switched off.","The gate/source voltage GS of the switching transistor 50′ controls the state of the switching transistor.","If this gate/source voltage GS is lower than the threshold voltage of the switching transistor 50′, the current IL is interrupted by the switching transistor 50′.","The gate/source voltage GS is S controlled by means of the driving device 70′, 75′.","However, when the switching transistor 50′ and hence the current source 20′ are disconnected, an overcurrent, also called a current spike, is produced which has its origins in the parasitic capacitance 60′ of the switching transistor 50′.","This is because a falling edge of the gate/source voltage GS on the switching transistor 50′ (the profile of said gate/source voltage being shown in FIG.","4) causes the parasitic capacitance 60′ to transfer the sudden voltage change on the control electrode 51′ to the output electrode 52′ of the switching transistor 50′ and to drive the transistor 20′ at a higher level.","This causes an overcurrent through the load 40′, as shown in FIG.","3 at the time ta.","The overcurrent flows until the parasitic capacitance's charge has been reversed.","The time ta characterizes the time at which the switch-off signal is applied to the input 1 of the driving device 70′, 75′.","In the case of certain applications, such current spikes can result in the load 40′ being destroyed, as can be the case with a laser diode, for example.","SUMMARY OF THE INVENTION The invention is now based on the object of improving the initially described circuit arrangement for switching a current on and off such that overcurrents can be avoided when a current source is switched off.","The invention solves this technical problem with the features of claim 1.Accordingly, a circuit arrangement for switching a current on and off without overcurrent is provided.","The circuit arrangement has a current source, a load associated with the current source and a switching transistor for switching the current source on and off.","Depending on manufacture, the switching transistor contains a parasitic capacitance between the control electrode and a first output electrode, which causes charge-reversal currents when the current source is switched off.","A shorting device, connected between the control electrode and the first output electrode of the switching device, for switching off the current source can be used to prevent the charge-reversal currents from resulting in overcurrents in the load.","Expediently, the shorting device is in the form of a transistor whose control electrode can have a switch-on/switch-off signal applied to it.","One output electrode of the shorting device is connected to the first output electrode of the switching transistor, and the other output electrode of the shorting device is connected to the control electrode of the switching transistor.","In this way, the shorting device shorts the switching transistor and hence switchs off the current source.","In order to be able to shorten the input and output cycles, a pull-up device is connected in parallel with the shorting device and ensures that the potential on the switching transistor's control electrode can be raised at the switch-on time.","The current source, the switching transistor, the shorting device and/or the pull-up device can be in the form of bipolar transistors, field-effect transistors, particularly MOS transistors, or combinations of these technologies.","The circuit arrangement is suitable particularly for driving a laser diode as a load.","BRIEF DESCRIPTION OF THE DRAWINGS The invention is explained in more detail below with reference to an exemplary embodiment in conjunction with the appended drawings, in which: FIG.","1 shows a circuit arrangement for switching a current on/off in accordance with the prior art, FIG.","2 shows the time-dependency diagram for a switch-on/switch-off signal, FIG.","3 shows the load current IL through the load 40′, FIG.","4 shows the time-dependency diagram of the potential profile on the control electrode of the switching transistor shown in FIG.","1, FIG.","5 shows a circuit arrangement in accordance with the invention, FIG.","6 shows the time-dependency diagram for a switch-on/switch-off signal in accordance with FIG.","2, FIG.","7 shows the time-dependency diagram of potential profile on the control electrode of the switching transistor shown in FIG.","5, FIG.","8 shows the time-dependency diagram of current through the parasitic capacitance shown in FIG.","5, and FIG.","9 shows the time-dependency diagram for a load current without overcurrent.","DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT FIG.","5 shows a circuit arrangement 10 that contains components of the circuit arrangement shown in FIG.","1.A load, for example a laser diode 40, is connected to the drain electrode of an exemplary n-channel field-effect transistor 20 operated as a current source.","The gate of the current source 20 has a constant voltage applied to it that is delivered by the DC voltage source 80.The source electrode of the current source 20 is connected to the drain electrode 52 of an exemplary n-channel field-effect transistor acting as a switching transistor 50, so that the load 40, the current source 20 and the switching transistor 50 form a series circuit.","The source electrode 53 of the switching transistor 50 is connected to ground.","In addition, there is a shorting transistor 30, for example in the form of an n-channel field-effect transistor, whose drain electrode 33 is connected to the gate 51 of the switching transistor 50, whereas the source electrode 32 of the shorting transistor 30 is connected directly to the drain electrode 52 of the switching transistor.","It is important to point out that the shorting transistor 30, unlike the circuit arrangement shown in FIG.","1, connects the gate 51 of the switching transistor 50 in the conducting state not to ground but to the drain electrode 52 of the switching transistor 50.In this way, as will be explained in detail later, the manufacture-dependent parasitic capacitance 60 between the gate 51 and the drain electrode 52 of the switching transistor 50 can be shorted at the time at which the switching transistor 50 is switched off, which simultaneously connects the switching transistor 50 to a two-terminal network across which the threshold voltage drops.","The gate 31 of the shorting transistor 30 is connected to an input connection 1 to which the switch-on/switch-off signal P shown in FIG.","6 can be applied.","The shorting transistor 30 can have a pull-up element 70 connected in parallel with it, for example a pull-up transistor in the form of a p-channel field-effect transistor whose gate is likewise connected to the input 1 of the circuit arrangement.","Unlike the shorting transistor 30, the pull-up transistor 70 is in the form of a p-channel field-effect transistor.","The source electrode of the pull-up transistor 70 is connected to the operating voltage VDD, whereas the drain electrode is connected to the gate 51 of the switching transistor 50.In this way, the gate 51 of the switching transistor 50 can be pulled more quickly at the switch-on time to a potential which is above the threshold voltage of the switching transistor 50, which means that the switching transistor can switch on more quickly and hence can switch on the current source 20 more quickly.","A detailed explanation will now be given, in conjunction with FIG.","6 to 9, of the way in which the circuit arrangement 10 works when the current is switched off.","Consideration will first be given to the phase during which the switching transistor 50 and hence the current source 20 are on.","During this time, the switch-on signal P shown in FIG.","6 is applied to the input 1 and adopts a low level during this time in the elucidated example.","On account of the low level, the shorting transistor 30 switchs off and the potential on the gate 51 of the switching transistor 50 is held at operating voltage by means of the pull-up transistor 70, which is on during this period, as shown in FIG.","7.Since the operating voltage is chosen to be higher than the threshold voltage of the switching transistor 50, a current consequently flows through the switching transistor 50 and the load current IL flows through the load 40 and the current source 20, as shown in FIG.","9.In other words, the current source 20 is on.","It will now be assumed that a switch-off signal P is applied to the input 1 of the circuit arrangement at the time t1, so that the potential at the input 1 changes from low to high.","This time t1 is shown in FIG.","6.At this time, the shorting transistor 30 starts to conduct and connects the gate 51 of the switching transistor 50 to the drain electrode 52.Consequently, the charge-reversal current I2 in the parasitic capacitance 60 flows via the shorting transistor 30, as shown in FIG.","8.This charge-reversal current thus no longer flows as a current spike through the load 40.The current I1 flowing via the pull-up transistor 70 drains to ground essentially through the shorting transistor 30 and then via the switching transistor 50.This phase lasts until the time t2, at which the voltage on the gate 51 of the switching transistor 50 has reached the latter's threshold voltage Vth.","This phase is shown in FIG.","7 by the falling edge of the gate/source voltage between the times t1 and t2.As soon as the voltage on the drain electrode 52 of the switching transistor 50 has become high enough to achieve a dip below the threshold voltage Vth of the current source 20, the current source 20 is switched off.","Since the shorting transistor 30 shorts the gate 51 and the drain electrode 52, the same potential is applied to both points.","Consequently, as mentioned, the charge-reversal current in the parasitic capacitance 60 can flow via the shorting transistor 30.The load current IL, which is free from overcurrent, shown in FIG.","9 thus falls to almost zero between the times t1 and t2.The condition for the load current IL to be disconnected by the switching transistor 50 is that the constant voltage applied to the gate of the current source 20 is lower than the sum of the threshold voltages of the current source 20 and the switching transistor 50.To be able to speed up the input and output switching processes further or to be able to extend the operating range, the potentials on the gate of the current source or on the gate of the switching transistor 50 can be lowered when switching off or raised when switching on by further permanently connected or dynamically connectable current sinks.","List of References 1 Input 10 Circuit arrangement 20, 20° Current source 30 Shorting device 31 Gate 32 Source electrode 33 Drain electrode 40, 40′ Load, laser diode 50, 50′ Switching transistor 51, 51° Control electrode, gate 52, 52′ Drain electrode 53, 53′ Source electrode 60, 60′ Parasitic capacitance 70′, 75′ Driving device 70 Pull-up transistor 80, 80′ DC voltage source VDD Operating voltage IL Load current GS Gate/source voltage P Switch-on/switch-off signal"]],"string":"[\n [\n \"Circuit arrangement for switching a current of and off without the occurrence of overcurrent\",\n \"There is provided a circuit arrangement for switching a current on and off without overcurrent.\",\n \"The circuit arrangement includes a current source, a load associated with the current source, a switching transistor for switching the current source on and off, where the switching transistor has a parasitic capacitance between a control electrode and a first output electrode, and a shorting device between the control electrode and the first output electrode of the switching transistor for switching off the current source.\",\n \"The current can be used, for example, to drive a laser diode.\"\n ],\n [\n \"1.A circuit arrangements for switching a current on and off without overcurrent, comprising: a current source; a load associated with the current source; a switching transistor for switching the current source on and off, wherein the switching transistor has a parasitic capacitance between a control electrode and a first output electrode, which is not at constant potential; and a shorting device between the control electrode and the first output electrode of the switching transistor for switching off the current source.\",\n \"2.The circuit arrangement as claimed in claim 1, wherein the shorting device comprises a transistor having: a control electrode for having a switch-on/switch-off signal applied to it; an output electrode connected to the first output electrode; and an output electrode connected to the control electrode of the switching transistor.\",\n \"3.The circuit arrangement as claimed in claim 1, wherein the shorting device has an associated pull-up device.\",\n \"4.The circuit arrangement as claimed in claim 1, wherein one or more of the current source, the switching transistor, the shorting device and the pull-up device are in a form selected from the group consisting of a bipolar transistor and a field-effect transistor.\",\n \"5.The circuit arrangement as claimed in claim 1, wherein the load is a laser diode.\",\n \"6.The circuit arrangement as claimed in claim 1, wherein the current is switched off by the switching transistor if a constant voltage applied to a control electrode of the current source is lower than a sum of threshold voltages of the current source and the switching transistor.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>Circuit arrangements for switching a current driving a load on and off are known.\",\n \"FIG.\",\n \"1 shows such a circuit arrangement, which uses a transistor 20 ′ as a current source or current sink.\",\n \"The control electrode of the transistor 20 ′ is connected to a DC voltage source 80 ′.\",\n \"The current source 20 ′ is connected in series with a load 40 ′, for example with a laser diode.\",\n \"A switching transistor 50 ′, which is likewise connected in series with the current source 20 ′, is used as a switch for the current source.\",\n \"The switching transistor 50 ′, which is in the form of a field-effect transistor, for example, is connected by means of its gate electrode 51 ′ to a driving device which comprises the transistors 75 ′ and 70 ′, for example.\",\n \"The input 1 of the driving device 70 ′, 75 ′ has a switch-on/switch-off signal P applied to it, as illustrated in FIG.\",\n \"2 , for example.\",\n \"Between the gate 51 ′ and the drain electrode 52 ′ of the switching transistor 50 ′ there is a manufacture-dependent parasitic capacitance 60 ′, which is also known as the Miller capacitance.\",\n \"As will be explained later, this parasitic capacitance is the cause of overcurrents that are brought about when the current source 20 ′ is switched off.\",\n \"The gate/source voltage GS of the switching transistor 50 ′ controls the state of the switching transistor.\",\n \"If this gate/source voltage GS is lower than the threshold voltage of the switching transistor 50 ′, the current IL is interrupted by the switching transistor 50 ′.\",\n \"The gate/source voltage GS is S controlled by means of the driving device 70 ′, 75 ′.\",\n \"However, when the switching transistor 50 ′ and hence the current source 20 ′ are disconnected, an overcurrent, also called a current spike, is produced which has its origins in the parasitic capacitance 60 ′ of the switching transistor 50 ′.\",\n \"This is because a falling edge of the gate/source voltage GS on the switching transistor 50 ′ (the profile of said gate/source voltage being shown in FIG.\",\n \"4 ) causes the parasitic capacitance 60 ′ to transfer the sudden voltage change on the control electrode 51 ′ to the output electrode 52 ′ of the switching transistor 50 ′ and to drive the transistor 20 ′ at a higher level.\",\n \"This causes an overcurrent through the load 40 ′, as shown in FIG.\",\n \"3 at the time t a .\",\n \"The overcurrent flows until the parasitic capacitance's charge has been reversed.\",\n \"The time t a characterizes the time at which the switch-off signal is applied to the input 1 of the driving device 70 ′, 75 ′.\",\n \"In the case of certain applications, such current spikes can result in the load 40 ′ being destroyed, as can be the case with a laser diode, for example.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The invention is now based on the object of improving the initially described circuit arrangement for switching a current on and off such that overcurrents can be avoided when a current source is switched off.\",\n \"The invention solves this technical problem with the features of claim 1 .\",\n \"Accordingly, a circuit arrangement for switching a current on and off without overcurrent is provided.\",\n \"The circuit arrangement has a current source, a load associated with the current source and a switching transistor for switching the current source on and off.\",\n \"Depending on manufacture, the switching transistor contains a parasitic capacitance between the control electrode and a first output electrode, which causes charge-reversal currents when the current source is switched off.\",\n \"A shorting device, connected between the control electrode and the first output electrode of the switching device, for switching off the current source can be used to prevent the charge-reversal currents from resulting in overcurrents in the load.\",\n \"Expediently, the shorting device is in the form of a transistor whose control electrode can have a switch-on/switch-off signal applied to it.\",\n \"One output electrode of the shorting device is connected to the first output electrode of the switching transistor, and the other output electrode of the shorting device is connected to the control electrode of the switching transistor.\",\n \"In this way, the shorting device shorts the switching transistor and hence switchs off the current source.\",\n \"In order to be able to shorten the input and output cycles, a pull-up device is connected in parallel with the shorting device and ensures that the potential on the switching transistor's control electrode can be raised at the switch-on time.\",\n \"The current source, the switching transistor, the shorting device and/or the pull-up device can be in the form of bipolar transistors, field-effect transistors, particularly MOS transistors, or combinations of these technologies.\",\n \"The circuit arrangement is suitable particularly for driving a laser diode as a load.\"\n ],\n [\n \"FIELD OF THE INVENTION The invention relates to a circuit arrangement for switching a current on and off without overcurrent, that current being used to drive a laser diode, for example.\",\n \"BACKGROUND OF THE INVENTION Circuit arrangements for switching a current driving a load on and off are known.\",\n \"FIG.\",\n \"1 shows such a circuit arrangement, which uses a transistor 20′ as a current source or current sink.\",\n \"The control electrode of the transistor 20′ is connected to a DC voltage source 80′.\",\n \"The current source 20′ is connected in series with a load 40′, for example with a laser diode.\",\n \"A switching transistor 50′, which is likewise connected in series with the current source 20′, is used as a switch for the current source.\",\n \"The switching transistor 50′, which is in the form of a field-effect transistor, for example, is connected by means of its gate electrode 51′ to a driving device which comprises the transistors 75′ and 70′, for example.\",\n \"The input 1 of the driving device 70′, 75′ has a switch-on/switch-off signal P applied to it, as illustrated in FIG.\",\n \"2, for example.\",\n \"Between the gate 51′ and the drain electrode 52′ of the switching transistor 50′ there is a manufacture-dependent parasitic capacitance 60′, which is also known as the Miller capacitance.\",\n \"As will be explained later, this parasitic capacitance is the cause of overcurrents that are brought about when the current source 20′ is switched off.\",\n \"The gate/source voltage GS of the switching transistor 50′ controls the state of the switching transistor.\",\n \"If this gate/source voltage GS is lower than the threshold voltage of the switching transistor 50′, the current IL is interrupted by the switching transistor 50′.\",\n \"The gate/source voltage GS is S controlled by means of the driving device 70′, 75′.\",\n \"However, when the switching transistor 50′ and hence the current source 20′ are disconnected, an overcurrent, also called a current spike, is produced which has its origins in the parasitic capacitance 60′ of the switching transistor 50′.\",\n \"This is because a falling edge of the gate/source voltage GS on the switching transistor 50′ (the profile of said gate/source voltage being shown in FIG.\",\n \"4) causes the parasitic capacitance 60′ to transfer the sudden voltage change on the control electrode 51′ to the output electrode 52′ of the switching transistor 50′ and to drive the transistor 20′ at a higher level.\",\n \"This causes an overcurrent through the load 40′, as shown in FIG.\",\n \"3 at the time ta.\",\n \"The overcurrent flows until the parasitic capacitance's charge has been reversed.\",\n \"The time ta characterizes the time at which the switch-off signal is applied to the input 1 of the driving device 70′, 75′.\",\n \"In the case of certain applications, such current spikes can result in the load 40′ being destroyed, as can be the case with a laser diode, for example.\",\n \"SUMMARY OF THE INVENTION The invention is now based on the object of improving the initially described circuit arrangement for switching a current on and off such that overcurrents can be avoided when a current source is switched off.\",\n \"The invention solves this technical problem with the features of claim 1.Accordingly, a circuit arrangement for switching a current on and off without overcurrent is provided.\",\n \"The circuit arrangement has a current source, a load associated with the current source and a switching transistor for switching the current source on and off.\",\n \"Depending on manufacture, the switching transistor contains a parasitic capacitance between the control electrode and a first output electrode, which causes charge-reversal currents when the current source is switched off.\",\n \"A shorting device, connected between the control electrode and the first output electrode of the switching device, for switching off the current source can be used to prevent the charge-reversal currents from resulting in overcurrents in the load.\",\n \"Expediently, the shorting device is in the form of a transistor whose control electrode can have a switch-on/switch-off signal applied to it.\",\n \"One output electrode of the shorting device is connected to the first output electrode of the switching transistor, and the other output electrode of the shorting device is connected to the control electrode of the switching transistor.\",\n \"In this way, the shorting device shorts the switching transistor and hence switchs off the current source.\",\n \"In order to be able to shorten the input and output cycles, a pull-up device is connected in parallel with the shorting device and ensures that the potential on the switching transistor's control electrode can be raised at the switch-on time.\",\n \"The current source, the switching transistor, the shorting device and/or the pull-up device can be in the form of bipolar transistors, field-effect transistors, particularly MOS transistors, or combinations of these technologies.\",\n \"The circuit arrangement is suitable particularly for driving a laser diode as a load.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS The invention is explained in more detail below with reference to an exemplary embodiment in conjunction with the appended drawings, in which: FIG.\",\n \"1 shows a circuit arrangement for switching a current on/off in accordance with the prior art, FIG.\",\n \"2 shows the time-dependency diagram for a switch-on/switch-off signal, FIG.\",\n \"3 shows the load current IL through the load 40′, FIG.\",\n \"4 shows the time-dependency diagram of the potential profile on the control electrode of the switching transistor shown in FIG.\",\n \"1, FIG.\",\n \"5 shows a circuit arrangement in accordance with the invention, FIG.\",\n \"6 shows the time-dependency diagram for a switch-on/switch-off signal in accordance with FIG.\",\n \"2, FIG.\",\n \"7 shows the time-dependency diagram of potential profile on the control electrode of the switching transistor shown in FIG.\",\n \"5, FIG.\",\n \"8 shows the time-dependency diagram of current through the parasitic capacitance shown in FIG.\",\n \"5, and FIG.\",\n \"9 shows the time-dependency diagram for a load current without overcurrent.\",\n \"DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT FIG.\",\n \"5 shows a circuit arrangement 10 that contains components of the circuit arrangement shown in FIG.\",\n \"1.A load, for example a laser diode 40, is connected to the drain electrode of an exemplary n-channel field-effect transistor 20 operated as a current source.\",\n \"The gate of the current source 20 has a constant voltage applied to it that is delivered by the DC voltage source 80.The source electrode of the current source 20 is connected to the drain electrode 52 of an exemplary n-channel field-effect transistor acting as a switching transistor 50, so that the load 40, the current source 20 and the switching transistor 50 form a series circuit.\",\n \"The source electrode 53 of the switching transistor 50 is connected to ground.\",\n \"In addition, there is a shorting transistor 30, for example in the form of an n-channel field-effect transistor, whose drain electrode 33 is connected to the gate 51 of the switching transistor 50, whereas the source electrode 32 of the shorting transistor 30 is connected directly to the drain electrode 52 of the switching transistor.\",\n \"It is important to point out that the shorting transistor 30, unlike the circuit arrangement shown in FIG.\",\n \"1, connects the gate 51 of the switching transistor 50 in the conducting state not to ground but to the drain electrode 52 of the switching transistor 50.In this way, as will be explained in detail later, the manufacture-dependent parasitic capacitance 60 between the gate 51 and the drain electrode 52 of the switching transistor 50 can be shorted at the time at which the switching transistor 50 is switched off, which simultaneously connects the switching transistor 50 to a two-terminal network across which the threshold voltage drops.\",\n \"The gate 31 of the shorting transistor 30 is connected to an input connection 1 to which the switch-on/switch-off signal P shown in FIG.\",\n \"6 can be applied.\",\n \"The shorting transistor 30 can have a pull-up element 70 connected in parallel with it, for example a pull-up transistor in the form of a p-channel field-effect transistor whose gate is likewise connected to the input 1 of the circuit arrangement.\",\n \"Unlike the shorting transistor 30, the pull-up transistor 70 is in the form of a p-channel field-effect transistor.\",\n \"The source electrode of the pull-up transistor 70 is connected to the operating voltage VDD, whereas the drain electrode is connected to the gate 51 of the switching transistor 50.In this way, the gate 51 of the switching transistor 50 can be pulled more quickly at the switch-on time to a potential which is above the threshold voltage of the switching transistor 50, which means that the switching transistor can switch on more quickly and hence can switch on the current source 20 more quickly.\",\n \"A detailed explanation will now be given, in conjunction with FIG.\",\n \"6 to 9, of the way in which the circuit arrangement 10 works when the current is switched off.\",\n \"Consideration will first be given to the phase during which the switching transistor 50 and hence the current source 20 are on.\",\n \"During this time, the switch-on signal P shown in FIG.\",\n \"6 is applied to the input 1 and adopts a low level during this time in the elucidated example.\",\n \"On account of the low level, the shorting transistor 30 switchs off and the potential on the gate 51 of the switching transistor 50 is held at operating voltage by means of the pull-up transistor 70, which is on during this period, as shown in FIG.\",\n \"7.Since the operating voltage is chosen to be higher than the threshold voltage of the switching transistor 50, a current consequently flows through the switching transistor 50 and the load current IL flows through the load 40 and the current source 20, as shown in FIG.\",\n \"9.In other words, the current source 20 is on.\",\n \"It will now be assumed that a switch-off signal P is applied to the input 1 of the circuit arrangement at the time t1, so that the potential at the input 1 changes from low to high.\",\n \"This time t1 is shown in FIG.\",\n \"6.At this time, the shorting transistor 30 starts to conduct and connects the gate 51 of the switching transistor 50 to the drain electrode 52.Consequently, the charge-reversal current I2 in the parasitic capacitance 60 flows via the shorting transistor 30, as shown in FIG.\",\n \"8.This charge-reversal current thus no longer flows as a current spike through the load 40.The current I1 flowing via the pull-up transistor 70 drains to ground essentially through the shorting transistor 30 and then via the switching transistor 50.This phase lasts until the time t2, at which the voltage on the gate 51 of the switching transistor 50 has reached the latter's threshold voltage Vth.\",\n \"This phase is shown in FIG.\",\n \"7 by the falling edge of the gate/source voltage between the times t1 and t2.As soon as the voltage on the drain electrode 52 of the switching transistor 50 has become high enough to achieve a dip below the threshold voltage Vth of the current source 20, the current source 20 is switched off.\",\n \"Since the shorting transistor 30 shorts the gate 51 and the drain electrode 52, the same potential is applied to both points.\",\n \"Consequently, as mentioned, the charge-reversal current in the parasitic capacitance 60 can flow via the shorting transistor 30.The load current IL, which is free from overcurrent, shown in FIG.\",\n \"9 thus falls to almost zero between the times t1 and t2.The condition for the load current IL to be disconnected by the switching transistor 50 is that the constant voltage applied to the gate of the current source 20 is lower than the sum of the threshold voltages of the current source 20 and the switching transistor 50.To be able to speed up the input and output switching processes further or to be able to extend the operating range, the potentials on the gate of the current source or on the gate of the switching transistor 50 can be lowered when switching off or raised when switching on by further permanently connected or dynamically connectable current sinks.\",\n \"List of References 1 Input 10 Circuit arrangement 20, 20° Current source 30 Shorting device 31 Gate 32 Source electrode 33 Drain electrode 40, 40′ Load, laser diode 50, 50′ Switching transistor 51, 51° Control electrode, gate 52, 52′ Drain electrode 53, 53′ Source electrode 60, 60′ Parasitic capacitance 70′, 75′ Driving device 70 Pull-up transistor 80, 80′ DC voltage source VDD Operating voltage IL Load current GS Gate/source voltage P Switch-on/switch-off signal\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471342"}}},{"rowIdx":591,"cells":{"text":{"kind":"list like","value":[["Novel genes for conditional cell ablation","A Novel deacetylase genes for use in conditional cell ablation are described.","These genes are particularly useful for the production of transgenic plants with plant parts which can be destroyed upon treatment with N-acetyl-PPT."],["1.A DNA molecule encoding a protein or polypepetide having deacetylase activity, wherein said protein has at least 70% to 75% sequence indentity with the sequence of SEQ ID No.","1.2.The DNA molecule of claim 1, wherein said protein or polypeptide comprises the sequence of SEQ ID No.","1.3.The DNA molecule of claim 1, comprising a nucleotide sequence which has at least 70% to 75% sequence identity to the sequence of SEQ ID No.","2.4.The DNA molecule of claim 3, comprising the nucleotide sequence of SEQ ID No.","2.5.The DNA molecule of claim 1, which can be isolated from Comamonas acidovorans, deposited as DSM 11070.6.A chimeric gene comprising: a) the DNA molecule of any one of claims 1 to 5, b) a plant-expressible promoter; said DNA molecule being in the same transcriptional unit and under the control of said plant-expressible promoter.","7.A plant having plant parts which can be destroyed upon induction, said plant comprising, stably integrated in its genome, a chimeric gene, comprising a) The DNA molecule of any one of claims 1 to 5, b) a plant-expressible promoter; said DNA molecule being in the same transcriptional unit and under the control of said tissue-specific, plant-expressible promoter; wherein said plant is characterized by the fact that, upon treatment with N-acetyl-PPT, plant parts are destroyed.","8.The plant of claim 7, wherein said plant-expressible promoter is a tissue-specific promoter.","9.The plant of claim 8, wherein said tissue-specific promoter is a stamen selective promoter; 10.A plant which is inducibly male-sterile, said plant comprising, stably integrated into its genome, a foreign DNA comprising a chimeric gene comprising: a) the DNA molecule of any one of claims 1 to 5; and b) a promoter directing stamen-selective expression; said DNA being in the same transcriptional unit and under the control of said stamen-selective promoter.","11.The plant of claim 10, wherein said stamen-selective promoter is selected from the group of: the TA29 promoter, the T72 promoter, the El promoter, or the CA55 promoter.","12.The plant of claim 10, wherein said foreign DNA further comprises: a herbicide resistance gene comprising a DNA encoding an RNA, protein or polypeptide that confers herbicide resistance on said plant.","13.A process for producing a plant with selectively destroyable plant tissues or parts, said process comprising: i) introducing into a plant cell or tissue a foreign DNA comprising a chimeric gene comprising: a) The DNA molecule of any one of claims 1 to 5, b) plant-expressible promoter; said DNA molecule being in the same transcriptional unit and under the control of said plant-expressible promoter.","ii) regenerating said plant from said plant cell.","14.The process of claim 13, wherein said plant-expressible promoter is a tissue-specific promoter.","15.The process of claim 14, wherein said tissue-specific promoter is a male or female organ selective promoter and said plants are inducibly male or female sterile."],["<SOH> BACKGROUND OF THE INVENTION <EOH>Deacetylases are capable of deacetylating acetylated toxins, such as N-acetyl-phosphinothricin (N-Ac-PTC or N-Ac-PPT), intracellularly, whereupon the cytotoxic activity of the toxins is restored (formation of PTC or PTC).","U.S. Pat.","No.","5,650,310, U.S. Pat.","No.","5,668,297, U.S. Pat.","No.","5,767,370, and U.S. Pat.","No.","5,767,371, describe deacetylase genes, for the production of phosphinothricin or phosphinothricyl-alanyl-alanine, as well as processes for their isolation and use.","Furthermore deacetylase genes are disclosed which are isolated from Streptomyces viridochromogenes (dea) and from E. coli (ArgE).","The use of such genes for the production of plants with selectively destroyable plant parts is described.","More specifically, a method is disclosed for the production of conditionally male-sterile plants.","This is achieved by introducing a deacetylase gene, under the control of a tapetum-specific promoter into the genome of a plant.","Upon application of N-Ac-PPT to the plant, in the tapetum cells, where the deacetylase is expressed, N-Ac-PPT is converted into PPT, which is toxic to the cells.","The cytotoxic activity in the tapetum cells inhibits the development of microspores and renders the plant male-sterile.","WO 98/27201 describes novel genes encoding amino acid deacetylases isolated from Stenotrophomonas sp.","and from Comamonas acidovorans and their use in the production of tranagenic plants.","WO 98/13504 describes the use of deacetylase genes for the production of female-sterile plants.","WO 98/39462 describes a method of hybrid seed production using conditional female sterility, whereby a plant is made conditionally female-sterile by transformation with the ArgE gene linked to a female-preferential promoter.","EP 98116492 describes the use of the deac system for the modification of plant development.","Deacetylase genes placed under control of a meristem specific promoter can be used for inducible modification of developmental characteristics of the plant such as flowering and bolting to improve yield, or influence general plant architecture.","EP 98116493 describes the use of the deac system to obtain pathogen control in plants.","Deacetylase genes placed under control of a pathogen-inducible promoter can be used to obtain plants which, upon treatment with N-acetyl-PPT become tolerant to pathogens, through selective destruction of pathogen-infected tissue.","However, the foregoing documents fail to teach or suggest the present invention."],["<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to DNA molecules encoding a protein having the biological activity of a deacetylase.","More specifically the protein having the biological activity of a deacetylase is a protein having an amino acid sequence with at least 70% to 79%, more preferably 80% to 85%, especially preferably 86% to 90%, most preferably 91% to 95%, sequence identity, more specifically which is 96% to 100% identical to the sequence of SEQ ID No.","1 or a biologically active fragment or variant thereof.","Especially preferably the DNA molecule of the invention encodes a protein having the amino acid sequence of SEQ ID No.","1 or a biologically active fragment thereof Alternately or additionally, the DNA molecule of the present invention has a nucleotide sequence which has a sequence identity of at least 70% to 79%, more preferably 80% to 85%, especially preferably 86% to 90%, most preferably 91% to 95%, sequence identity, more specifically which is 96% to 100% identical to the sequence of SEQ ID No.","2.Especially preferably the DNA molecule of the invention comprises the nucleotide sequence of SEQ ID No.","2.Additionally or alternatively, the invention relates to a DNA molecule encoding a deacetylase which can be isolated from Comamonas acidovorans , deposited as DSM 11070, which comprises the sequence of SEQ ID No.","2.The invention further relates to a protein or polypeptide having the biological activity of a deacetylase, which is encoded by a DNA molecule, such as those described above.","The invention further relates to a chimeric gene comprising the DNA molecule of the present invention placed in the same transcriptional unit and under the control of a plant expressible promoter, preferably a tissue-specific promoter.","The invention further relates to a plant having inducibly destroyable plant parts, which plant comprises, stably integrated in its genome, a chimeric gene comprising the DNA molecule of the invention, placed under control of a plant-expressible promoter, preferably a tissue-specific promoter, the plant of the invention being characterized by the fact that, upon treatment with N-acetyl-PPT, the plant tissues in which the promoter directs expression, are destroyed.","The invention further relates to an inducibly male or female-sterile plant, which plant comprises, stably integrated in its genome, the DNA molecule of the invention, placed under control of a male- or female reproductive organ selective promoter, the inducibly male- or female-sterile plant of the invention being characterized by the fact that, upon treatment with N-acetyl-PPT, specific plant tissues of the male- or female reproductive organ are destroyed.","The invention further relates to a process for producing a plant with inducibly destroyable plant tissues or parts, which process comprises: introducing into a plant cell or tissue a foreign DNA comprising a chimeric gene comprising a) the DNA molecule of the invention, encoding a protein or polypeptide having the biological activity of a deacetylase, under the control of b) a plant-expressible promoter, which is preferably tissue-specific; regenerating the plant cell or tissue into a plant optionally, inducing the destruction of the plant tissue or parts by treatment of the plant with N-acetyl-PPT.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["FIELD OF THE INVENTION The invention relates to novel DNA molecules encoding a protein having the biological activity of a deacetylase.","Preferably these DNA molecules encode a protein having a sequence similarity with a deacetylase as described herein isolated from Comamonas acidovorans.","The invention further relates to transgenic plants with plant parts which can be destroyed upon induction, comprising the DNA molecules of the invention under control of tissue-specific promoter.","All documents cited herein are incorporated herein by reference.","BACKGROUND OF THE INVENTION Deacetylases are capable of deacetylating acetylated toxins, such as N-acetyl-phosphinothricin (N-Ac-PTC or N-Ac-PPT), intracellularly, whereupon the cytotoxic activity of the toxins is restored (formation of PTC or PTC).","U.S. Pat.","No.","5,650,310, U.S. Pat.","No.","5,668,297, U.S. Pat.","No.","5,767,370, and U.S. Pat.","No.","5,767,371, describe deacetylase genes, for the production of phosphinothricin or phosphinothricyl-alanyl-alanine, as well as processes for their isolation and use.","Furthermore deacetylase genes are disclosed which are isolated from Streptomyces viridochromogenes (dea) and from E. coli (ArgE).","The use of such genes for the production of plants with selectively destroyable plant parts is described.","More specifically, a method is disclosed for the production of conditionally male-sterile plants.","This is achieved by introducing a deacetylase gene, under the control of a tapetum-specific promoter into the genome of a plant.","Upon application of N-Ac-PPT to the plant, in the tapetum cells, where the deacetylase is expressed, N-Ac-PPT is converted into PPT, which is toxic to the cells.","The cytotoxic activity in the tapetum cells inhibits the development of microspores and renders the plant male-sterile.","WO 98/27201 describes novel genes encoding amino acid deacetylases isolated from Stenotrophomonas sp.","and from Comamonas acidovorans and their use in the production of tranagenic plants.","WO 98/13504 describes the use of deacetylase genes for the production of female-sterile plants.","WO 98/39462 describes a method of hybrid seed production using conditional female sterility, whereby a plant is made conditionally female-sterile by transformation with the ArgE gene linked to a female-preferential promoter.","EP 98116492 describes the use of the deac system for the modification of plant development.","Deacetylase genes placed under control of a meristem specific promoter can be used for inducible modification of developmental characteristics of the plant such as flowering and bolting to improve yield, or influence general plant architecture.","EP 98116493 describes the use of the deac system to obtain pathogen control in plants.","Deacetylase genes placed under control of a pathogen-inducible promoter can be used to obtain plants which, upon treatment with N-acetyl-PPT become tolerant to pathogens, through selective destruction of pathogen-infected tissue.","However, the foregoing documents fail to teach or suggest the present invention.","SUMMARY OF THE INVENTION The present invention relates to DNA molecules encoding a protein having the biological activity of a deacetylase.","More specifically the protein having the biological activity of a deacetylase is a protein having an amino acid sequence with at least 70% to 79%, more preferably 80% to 85%, especially preferably 86% to 90%, most preferably 91% to 95%, sequence identity, more specifically which is 96% to 100% identical to the sequence of SEQ ID No.","1 or a biologically active fragment or variant thereof.","Especially preferably the DNA molecule of the invention encodes a protein having the amino acid sequence of SEQ ID No.","1 or a biologically active fragment thereof Alternately or additionally, the DNA molecule of the present invention has a nucleotide sequence which has a sequence identity of at least 70% to 79%, more preferably 80% to 85%, especially preferably 86% to 90%, most preferably 91% to 95%, sequence identity, more specifically which is 96% to 100% identical to the sequence of SEQ ID No.","2.Especially preferably the DNA molecule of the invention comprises the nucleotide sequence of SEQ ID No.","2.Additionally or alternatively, the invention relates to a DNA molecule encoding a deacetylase which can be isolated from Comamonas acidovorans, deposited as DSM 11070, which comprises the sequence of SEQ ID No.","2.The invention further relates to a protein or polypeptide having the biological activity of a deacetylase, which is encoded by a DNA molecule, such as those described above.","The invention further relates to a chimeric gene comprising the DNA molecule of the present invention placed in the same transcriptional unit and under the control of a plant expressible promoter, preferably a tissue-specific promoter.","The invention further relates to a plant having inducibly destroyable plant parts, which plant comprises, stably integrated in its genome, a chimeric gene comprising the DNA molecule of the invention, placed under control of a plant-expressible promoter, preferably a tissue-specific promoter, the plant of the invention being characterized by the fact that, upon treatment with N-acetyl-PPT, the plant tissues in which the promoter directs expression, are destroyed.","The invention further relates to an inducibly male or female-sterile plant, which plant comprises, stably integrated in its genome, the DNA molecule of the invention, placed under control of a male- or female reproductive organ selective promoter, the inducibly male- or female-sterile plant of the invention being characterized by the fact that, upon treatment with N-acetyl-PPT, specific plant tissues of the male- or female reproductive organ are destroyed.","The invention further relates to a process for producing a plant with inducibly destroyable plant tissues or parts, which process comprises: introducing into a plant cell or tissue a foreign DNA comprising a chimeric gene comprising a) the DNA molecule of the invention, encoding a protein or polypeptide having the biological activity of a deacetylase, under the control of b) a plant-expressible promoter, which is preferably tissue-specific; regenerating the plant cell or tissue into a plant optionally, inducing the destruction of the plant tissue or parts by treatment of the plant with N-acetyl-PPT.","DETAILED DESCRIPTION The term “gene” as used herein refers to any DNA sequence comprising several operably linked DNA fragments such as a promoter region, a 5′ untranslated region (the 5′UTR), a coding region (which may or may not code for a protein), and an untranslated 3′ region (3′UTR) comprising a polyadenylation site.","Typically in plant cells, the 5′UTR, the coding region and the 3′UTR are transcribed into an RNA of which, in the case of a protein encoding gene, the coding region is translated into a protein.","A gene may include additional DNA fragments such as, for example, introns.","The term “chimeric” when referring to a gene or DNA sequence is used to refer to the fact that the gene or DNA sequence comprises at least two functionally relevant DNA fragments (such as promoter, 5′UTR, coding region, 3′UTR intron) that are not naturally associated with each other and/or originate, for example, from different sources.","“Foreign” referring to a gene or DNA sequence with respect to a plant species is used to indicate that the gene or DNA sequence is not naturally found in that plant species, or is not naturally found in that genetic locus in that plant species.","The term “foreign DNA” will be used herein to refer to a DNA sequence as it has incorporated into the genome of a plant as a result of transformation.","A genome of a plant, plant tissue or plant cell, as used herein, refers to any genetic material in the plant, plant tissue or plant cell, and includes both the nuclear and the plastid (chloroplast and mitochondrial) genome.","A “fragment” of a DNA molecule or protein sequence as used herein refers to a truncated sequence of the original sequence (nucleic acid or amino acid) referred to, which can vary in length but of which the minimum size is sufficient to ensure the (encoded) protein to be biologically active, the maximum size not being critical.","A “variant” of a sequence is used herein to indicate a DNA molecule or protein of which the sequence (nucleic or amino acid) is essentially identical to the sequence to which the term refers.","Sequences which are “essentially identical” means that when two sequences are aligned, the percent sequence identity, i.e.","the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the sequences, is higher than 70%-80%, preferably 81-85%, more preferably 86-90%, especially preferably 91-95%, most preferably 96-100%, more specifically is 100%.","The alignment of two nucleotide sequences is performed by the algorithm as described by Wilbur and Lipmann (1983, Proc.","Natl.","Acad.","Sci.","U.S.A. 80:726) using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4.Preferably the DNA molecule of the invention is a sequence which is essentially identical to a coding sequence of a deacetylase from Comamonas acidovorans, which has been adapted for preferred codon usage in plants.","The present invention relates to a DNA molecule or coding sequence encoding a protein having the activity of a deacetylase.","A “deacetylase” or a protein with deacetylase activity as used herein refers to a molecule which is capable of deacetylating N-acetyl PPT (which in itself—i.e.","in the absence of a deacetylase- is not toxic to plants or plant cells or tissues), to produce PPT which is cytotoxic to plants.","Deacetylase activity can be measured by radioactive or non-radioactive assays as described in WO 98/27201 and U.S. Pat.","No.","5,668,297.Presence of deacetylase activity in plant tissues can be determined by treatment of the tissue with the substrate N-acetyl-PPT, and evaluating production of PPT, which should result in cell death.","A protein or polypeptide which is “biologically active” as used herein refers to the fact that it has deacetylase activity.","In a preferred embodiment of the invention, the DNA molecule encoding a protein having the biological activity of a deacetylase, is a DNA which can be isolated from the deposit with International deposit No.","DSM 11070 (as described in WO 98/27201 and U.S. Pat.","No.","6,177,616) deposited at the.","Deutsche Sammlung für Microorganismen und Zelikulturen GmbH, Masenroder Weg 1b, D-38124.Braunsenweig.","More specifically such a DNA molecule is a DNA-molecule encoding a deacetylase having a sequence which is essentially identical to the sequence of SEQ ID No.","1, or a biologically active fragment thereof.","Additionally or alternatively such a DNA molecule comprises a nucleotide sequence which is essentially identical to the sequence of SEQ ID No.","2, or corresponds to a DNA sequence which hybridizes with the sequence of SEQ ID No.","2 under standard stringent conditions.","A preferred embodiment of the DNA molecule of the present invention is a DNA molecule encoding a deacetylase comprising a sequence which is essentially identical to SEQ ID No.","1, isolated from Comamonas acidovorans.","Based on the homology of the DNA sequence isolated from Comamonas acidovorans to N-acetyl-Ornithine transferase genes, the deacetylases of present invention will be referred to as “ArgJ” deacetylases and the genes comprising a DNA molecule encoding such an ArgJ deacetylase (or a biologically active fragment thereof) will be referred to as an “ArgJ gene”.","The isolation of sequences encompassed by the present invention which are essentially identical to the sequence of SEQ ID No.1 isolated from Comamomas acidovorans, is possible using standard strategies and techniques that are well known in the art.","For example, using all or a portion of the amino acid sequence of the Comamonas ArgJ protein, one may readily design specific oligonucleotide probes, including degenerate oligonucleotide probes (i.e., a mixture of all possible coding sequences for a given amino acid sequence).","The oligonucleotides may be based upon the sequence of either DNA strand and any appropriate portion of the ArgJ coding sequence.","General methods for designing and preparing such probes are provided, for example, in Ausubel et al.","Current Protocols in Molecular Biology, Wiley Interscience, New York and Berger and Kimmel, Guide to Molecular Cloning techniques, 1987, Academic Press, New York.","These oligonucleotides are useful for ArgJ gene isolation, either through their use as probes capable of hybridizing to ArgJ complementary sequences or as primers for various amplification techniques, for example PCR cloning strategies.","For detection or isolation of closely related ArgJ sequences having greater than 80% sequence identity, high stringency conditions are preferably used; such Stringent conditions as used herein refers to the hybridizing conditions described by Sambrook et al.","(1989) (Molecular Cloning: A.","Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory Press, NY).","As used herein N-acetyl-PPT refers to N-acetyl-phosphinothricin or a composite structure thereof, which in itself is not toxic to plant cells but is converted to a toxin (phosphinothricin, PPT) by a deacetylase.","Thus, the term N-acetyl-PPT refers to any formulation of N-acetyl-phosphinothricin, or derivatives or related compounds, such as acetylated forms of desmethylphosphinothricine (2-amino-4-hydroxyphosphinobutyric acid), bialaphos (phosphinothricyl-alanyl-alanine), desmethylbialaphos and the like, their salts, racemic mixtures and active enantiomers, which can be used in the context of the present invention.","According to the present invention, plant-expressible promoter is a promoter which directs expression in plants.","Preferably, the plant-expressible promoter of the invention is a tissue-specific promoter, which is a promoter directing expression selectively in a specific cell or tissue of the plant.","A broad range of tissue-specific promoters are described in the literature, such as, but not limited to, meristem-specific promoters (such as the promoter of the AGL8 MADS box gene; Yanofsky, 1995, The plant cell 7:1763-1771), female-organ specific promoters (such as the PSTMG-type promoters described in U.S. Pat.","No.","4,633,441), leaf-tissue specific promoters (such as the leaf-specific Ca/b promoter from Arabidopsis described by Cannon et al., 1990, Plant Mol Biol 15(1)39-47 or the tomato RbcS-3C promoter described by Kyozuka et al., 1993, Plant Physiol 102(3):991-1000).","In a preferred embodiment of the invention, the tissue-specific promoter is male or female organ selective promoter, directing expression selectively in male or female organs of the plant.","A preferred male organ selective promoter is a stamen-selective promoter which directs expression in selective stamen cells (including anther cells and pollen), preferably directing expression essentially only in stamen cells.","More specifically, the stamen-selective promoter is a tapetum-specific promoter directing expression specifically in tapetum cells.","A number of stamen-selective promoters are known in the art, such as, but not limited to, the TA29 promoter from tobacco (U.S. Pat.","No.","5,652,354), the CA55 promoter isolated form corn (WO 92/00275, U.S. Pat.","No.","5,589,610), the T72 and E1 promoters isolated from rice (U.S. Pat.","No.","5,639,948), SGBG (U.S. Pat.","No.","5,837,850), and 5126 (U.S. Pat.","No.","5,689,049).","Tissue-specific promoters as used herein includes promoters which are also subject to other kinds of regulation (such as, but not limited to, time-dependent, stress-dependent, environment-dependent, chemically inducible, etc.).","Alternatively, the plant-expressible promoters of the present invention are not tissue-specific but are subject to other kinds of regulation (such as, but not limited to, time-dependent, stress-dependent, environment-dependent, chemically inducible, etc.).","A plant with conditionally or inducibly destroyable parts refers to a plant in which, under specific conditions, specific plant tissues or parts can be destroyed.","“Plant part” is meant to refer to any plant organ (such as, but not limited to male or female reproductive organs, leaves, vegetative meristem, roots) or tissue (such as, but not limited to reproductive tissues, embryogenic tissues), including the complete plant.","More specifically, these conditions are understood to refer to the treatment of the plant with an acetylated toxin, which normally is not toxic to the plant or plant cells, but in the presence of a deacetylase in the plant cell is converted into a substance that is cytotoxic.","A conditionally male-sterile plant refers to a plant which, under normal growing conditions, is male fertile, i.e.","produces fertile pollen, and which can be made male-sterile under specific conditions.","Treatment of the plant can refer to administering the substance in any way, such as, but not limited to manual treatment, or spraying of the plant, plant part (or of the field or greenhouse wherein the plant is grown), or through other forms of irrigation.","Treatment of a plant with N-acetyl PPT will result in uptake of N-acetyl PPT by the leaf (or other) cells and transportation to different plant organs.","Thus, treatment of a plant which comprises a chimeric gene comprising a deacetylase coding sequence expressed in specific cells of the plant, will result in the conversion of N-acetyl PPT to PPT in those cells, causing these cells to die.","In a specific embodiment of the invention, the transgenic plant, comprising in its genome the DNA molecule of the invention encoding a protein having the biological activity of a deacetylase, additionally comprises, a chimeric gene conferring on said plants resistance a herbicide to PPT, such as, but not limited to the herbicide resistance genes, pat or bar described in EP-A-0 242 236, EP-A-0 242 246, EP-A-0 257 542.These genes encode enzymes which are capable of converting PPT into N-acetyl-PPT.","This N-acetyl-PPT can be transported throughout the plant and act as the substrate for a deacetylase specifically expressed in certain plant cells or tissues.","It is understood that according to this specific embodiment, induction of tissue-specific cytotoxicity will also occur upon treatment of the plant with PPT (EP-A-0 531 716, U.S. Pat.","No.","5,668,297).","Preferably, the deacetylase of the present invention and the enzyme conferring herbicide resistance gene will be expressed or transported to different cellular compartments (i.e.","cytoplasm, chloroplast) to avoid re-conversion of the PPT to N-acetyl PPT, where this is not intended.","A chimeric marker gene as used herein is a gene comprising a marker DNA under the control of a plant-expressible promoter.","The marker DNA encodes an RNA, protein or polypeptide, which, when expressed in a plant or plant cell, allows such plants or plant cells to be distinguished and separated from plants or plant cells not expressing the marker DNA.","Examples of marker genes are genes that provide a specific color to the cells (such as the A1 gene, Meyer et al.","1987, Nature 330:667) or genes that encode a particular resistance to the plant cells or the plant such as antibiotic resistance (e.g.","the aac(6′) gene, encoding resistance to gentamycin, WO94/01560).","Preferred marker genes are herbicide resistance genes.","A herbicide resistance gene as used herein is a gene conferring to the plant or plant cell resistance to a herbicide.","Examples of herbicide resistance genes are the genes encoding resistance to phenmedipham (such as the pmph gene, U.S. Pat.","No.","5,347,047; U.S. Pat.","No.","5,543,306), the genes encoding resistance to glyphosate (such as the EPSPS genes, U.S. Pat.","No.","551047), genes encoding bromoxynyl resistance (such as described in U.S. Pat.","No.","4,810,648) genes encoding resistance to sulfonylurea (such as described in EPA 0 360 750), genes encoding resistance to the herbicide dalapon (such as described in WO 99/27116), and genes encoding resistance to cyanamide (such as described in WO 98/48023 and WO 98/56238).","The cytotoxic effect of certain toxins may be enhanced by targeting to the specific parts of the cell, such as the chloroplast or mitochondria.","Phosphinothricin is a glutamine synthase inhibitor.","In plants, glutamine synthase exitst in multiple isozymic forms that can be localized within the cell in the cytosol and plastids.","Thus, it can be contemplated that targeting of the deacetylase to the chloroplast, whereby N-acetyl-PPT is converted to PPT in the chloroplast, increases cytotoxic efficiency.","Thereto, the foreign DNA comprising the deacetylase coding sequence can comprise an additional foreign DNA encoding a transit peptide.","The additional DNA is preferably located between the first promoter and the deacetylase coding sequence.","Alternatively, it can be desired that the marker protein or polypeptide is transported from the cytoplasm into chloroplasts or mitochondria of the transformed plant cells.","Thereto, an additional DNA encoding a transit peptide is located between the marker DNA and its promoter.","By transit peptide is meant a polypeptide fragment which is normally associated with a chloroplast or mitochondrial protein or subunit of the protein and is produced in a cell as a precursor protein encoded by the DNA of the cell.","The transit peptide, is responsible for the translocation process of the nuclearencoded chloroplast or mitochondrial protein or subunit into the chloroplasts or mitochondria, and during such a process, the transit peptide is separated or proteolytically removed from the chloroplast or mitochondrial protein or subunit.","One or more of such additional DNA's can be provided in the foreign DNAs of this invention as generally described and exemplified in European patent publication 0,189,707 and European patent EP 0 344 029.Transformation of the plants of the invention can occur in a number of ways described in the art.","Such methods include, but are not limited to Agrobacterium-mediated transformation (BP 0,116,718, DeBlaere: et al., 1987, Meth.","Enzymol.","153:277-293), microinjection (Crossway et al., 1986, Mol.","Gen. Genet.","102:179), direct gene transfer (Pazowski et al., 1984, EMBO J.","3:2717-2722), electrapomtion (Riggs et al., 1986, Proc.","Natl.","Acad.","Sci.","USA 83:5602-5606), or by particle gun bombardment (WO92/09696; Potrykus et al.","1991, Annu.","Rev.","Plant Physiol.","Plant Mol.","Biol.","42:205-225); see also Christou P. 1994, Agro-Food Industry Hi-Tech 2:17; Langridge et al.","1992, Plant J.","2:631; Vasil, 1994, Plant Mol.","Biol.","25:925; Chen D., 1998, Methods Mol.","Biol.","81:373.Alternatively, the foreign DNA comprising the chimeric genes of the invention can be inserted into the plastid genome of the cell or cells of a plant.","Gene transfer can be carried out, for example, by particle gun bombardment (as described by Svab et al., 1990, Proc.","Natl.","Acad.","Sci.","USA 87:8526-8530) or by Agrobacterium-mediated transformation (De Block et al., 1985, EMBO J.","4:1367).","As used herein “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof.","Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein.","A chimeric gene comprising a DNA sequences which is functionally or structurally defined, may comprise additional DNA sequences, etc.","Unless otherwise stated, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al.","(1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al.","(1994) Current Protocols in Molecular Biology, Current Protocols, USA.","Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D.","Croy published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.","In the description and examples, reference is made to the following sequences: SEQ ID No.","1: Amino acid sequence of the ArgJ deacetylase from Comamonas acidovorans SEQ ID No.","2: Nucleotide sequence encoding the Arg J deacetylase EXAMPLES Example 1 Isolation of Bacterial Deacetylase Genes The isolation of micro-organisms having N-acetyl-PPT deacetylase activity was performed as described in WO98/27201.Briefly, different soil samples were grown on Minimal Essential Medium containing only chitin as a carbon source.","As chitin can only be broken down after deacetylation, microorganisms having deacetylase activity will preferentially grow on this medium.","After subsequent replating and isolation of single colony bacteria, different colonies could be isolated that were capable of converting radioactive N-acetyl-PPT to PPT.","One of the isolated bacteria was identified as Comamonas acidovorans and deposited at the Deutsche Sammlung fur Microorganismen und Zellkulturen under accession number DSM 11070.In a next step the genes encoding for N-acetyl-PPT deacetylase activity were isolated.","A specific selection method was developed to be able to isolate the deacetylase genes in a “Shot-gun” cloning method.","The method is based on the principle that a gene product capable of deacetylating N-acetyl-PPT should also be able to process a related compound N-acetyl-Ornithine as a substrate.","The deacetylation of N-acetyl-ornithine is a step in the arginine biosynthesis and mutants that are incapable of performing this step are auxotrophic for arginine.","One such mutant, the E. coli nullmutant (XS1D2), which has a deletion in its ArgE gene, was described by Mountain et al.","(1984, Mol.","Gen. Genet.","197:82-89).","Total DNA from Comamonas acidovorans was isolated, digested with EcoRI and ligated within the EcoRI site of the vector pACYC184 (Cang and Cohen, 1987, J Bacteriol.","134:1141-1156).","The ligation mixture was then introduced by electroporation into the cells of the mutant E. coli XS1D2.Of 16.000 tested shot-gun clones, 14 were able to complement the arginine auxotrophy of the E. coli mutant.","One of the complementing clones (E4.2) comprised a 4,2 kb EcoRI fragment.","Hybridisation experiments confirmed that this fragment originated from the C. acidovorans genome and did not show any cross-hybridization with any of the other fragments in other clones.","Through subsequent subdloning, the minimal sequence for retaining complementation of the arginine auxotrophy was determined.","This sequence was then sequenced as a double strand and the open reading frame was identified.","Based on a homology with databank sequence, the sequence was termed as the ArgJ-gene.","Example 2 The ArgJ Coding Sequence of C. Acidovorans Through subsequent subdloning, the minimal sequence for retaining complementation of the arginine auxotrophy was determined as a 2.2 kb HindIII/PstI fragment (SEQ ID No.","2).","An open reading frame of 1230 bp was identified, encoding a protein of 410 amino acids (SEQ ID No.","1).","A potential ribosomal binding site is located at about 10 bp before the start-codon.","Comparison of the amino acid sequence of the open reading frame with known sequences, strong homology was found with a number of N-acetyl-ornithin-transacetylases (Table 1).","The strongest homology was found with N-acetyl-ornithin-transacetylase encoded by the ArgJ-gene from Neisseria gonorrhoeae.","Therefore the complete open reading frame of the C. acidovorans fragment was named the ArgJ coding sequence.","Upstream of the ArgJ coding sequence another, be it incomplete, open reading frame can be found.","Comparison with databank sequences showed that this is part of the secA-gene of C. acidovorans.","This sequence however, does not seem of importance for the complementation of the E. coli ArgE-mutant, since a PCR fragment comprising only the ArgJ-gene can restore arginin auxotrophy in the mutant.","TABLE 1 Protein sequences found to be homologous to the sequence encoded by the C. acidovorans gene Homologous gene Origin Encoded protein % Identical AA ArgJ Neisseria gonorrhea N-acetyl-ornithin- 54.5% transferase ArgJ Bacillus subtilis N-acetyl-ornithin- 37.4% transferase ArgJ Bacillus N-acetyl-ornithin- 37.5% stearothermophilus transferase ArgJ Saccharomyces N-acetyl-ornithin- 36.1% cervisiae transferase Example 3 Expression of the ArgJ Coding Sequence in E. coli Through the selection procedure, it was established that the protein encoded by the ArgJ coding sequence from C. acidovorans was capable of complementing the E. coli ArgE mutant by its deacetylase activity.","To establish whether this protein would also deacetylate N-acetyl-PPT, both coding sequences were over-expressed in E. coli, and the supernatant of the culture was analyzed for its ability to deacetylate N-acetyl-PPT.","The coding sequence was cloned into a vector with high copy number under control of the lacZ promoter.","The incorporation of a stop codon made overexpression of the deacetylase sequence possible without interference of open reading frames of the lacZ gene.","Compared to the isolated fragments, these construct showed significantly stronger complementation of the E. coli ArgE mutant.","Example 4 Expression of the ArgJ Coding Region from C. acidovorans in Tobacco The ArgJ coding sequence was recloned into the binary vector pROK1 under control of the Cauliflower Mosaic Virus 35S promoter sequence.","The resulting plasmid p35SARG comprising the 35S promoter, ArgJ coding sequence and 35S terminator was transformed in Agrobacterium tumefaciens (strain ATHV) using standard methods.","Tobacco (Nicotiana tabacum) leaf disks were transformed with the recombinant Agrobacteria according to the method of Horsch et al.","(1985, Science 227:1229-1231) and selected on kanamycin medium.","Seven independent transformants were regenerated.","In a drop experiment, the leaves of the transgenic plants (and a number of control plants) were treated with 5 μl each of the following concentrations of N-acetyl- D, L-PPT: 4 mg/ml (=15 mM), 1 mg/ml (=3.75 mM), 0.4 mg/ml (=1.5 mM), 0.1 mg/ml (=0.38 mM).","The treatment sites were examined for lightening or necrosis formation.","Four plants showed clearly visible leaf damage, apparent 4 to 5 days after treatment with a dose of 4 mg/ml or 1 mg/ml.","Example 5 Tapetum-Specific Expression of the ArgJ Gene in Tobacco The ArgJ coding sequence was recloned into the binary vector pROK1 under control of the TA29 promoter of tobacco.","The resulting plasmid pTA29ARG comprising the TA29 promoter, ArgJ coding sequence and 35S terminator was transformed in Agrobacterium tumefaciens (strain ATHV) using standard methods.","Tobacco (Nicotiana tabacurn) leaf disks were transformed with the recombinant Agrobacteria according to the method of Horsch et al.","(1985, above) and selected on kanamycin medium.","To investigate tapetum-specific expression of the ArgJ deacetylase, flower buds of the transgenic lines were treated with N-acetyl-PPT to induce male-sterile flowers.","In each case only one side of the inflorescence (the total area of the developing flower bud) was treated with N-acetyl PPT, in order to eliminate possible contamination with natural male-sterility phenomena.","Treatment was carried out on flower buds of 2 to 4 mm large.","Transgenic and control plants were treated with 10 μl of 5 mg/ml N-acetyl PPT solution (0.1% Genapol as wetting agent).","After the blooming of the buds (about 9-11 days after conclusion of the treatment) the plants were investigated for the occurrence of male-sterile flowers.","As expected, no reaction was seen in either the control or the wild-type tobacco flowers.","In three out of seven transgenic plants, male-sterile flowers developed from the drop-treated flowerbuds.","The anthers of these flowers did not contain any pollen and their filaments were markedly shorter than those of treated wild-type flowers.","Anther dehiscence occurs in these flowers, but is delayed compared to controls.","If cross-pollination is avoided, the male-sterile flowers develop an empty seedpod, as pollen for self-pollination is lacking.","Otherwise these flowers can not be distinguished from untreated flowers."]],"string":"[\n [\n \"Novel genes for conditional cell ablation\",\n \"A Novel deacetylase genes for use in conditional cell ablation are described.\",\n \"These genes are particularly useful for the production of transgenic plants with plant parts which can be destroyed upon treatment with N-acetyl-PPT.\"\n ],\n [\n \"1.A DNA molecule encoding a protein or polypepetide having deacetylase activity, wherein said protein has at least 70% to 75% sequence indentity with the sequence of SEQ ID No.\",\n \"1.2.The DNA molecule of claim 1, wherein said protein or polypeptide comprises the sequence of SEQ ID No.\",\n \"1.3.The DNA molecule of claim 1, comprising a nucleotide sequence which has at least 70% to 75% sequence identity to the sequence of SEQ ID No.\",\n \"2.4.The DNA molecule of claim 3, comprising the nucleotide sequence of SEQ ID No.\",\n \"2.5.The DNA molecule of claim 1, which can be isolated from Comamonas acidovorans, deposited as DSM 11070.6.A chimeric gene comprising: a) the DNA molecule of any one of claims 1 to 5, b) a plant-expressible promoter; said DNA molecule being in the same transcriptional unit and under the control of said plant-expressible promoter.\",\n \"7.A plant having plant parts which can be destroyed upon induction, said plant comprising, stably integrated in its genome, a chimeric gene, comprising a) The DNA molecule of any one of claims 1 to 5, b) a plant-expressible promoter; said DNA molecule being in the same transcriptional unit and under the control of said tissue-specific, plant-expressible promoter; wherein said plant is characterized by the fact that, upon treatment with N-acetyl-PPT, plant parts are destroyed.\",\n \"8.The plant of claim 7, wherein said plant-expressible promoter is a tissue-specific promoter.\",\n \"9.The plant of claim 8, wherein said tissue-specific promoter is a stamen selective promoter; 10.A plant which is inducibly male-sterile, said plant comprising, stably integrated into its genome, a foreign DNA comprising a chimeric gene comprising: a) the DNA molecule of any one of claims 1 to 5; and b) a promoter directing stamen-selective expression; said DNA being in the same transcriptional unit and under the control of said stamen-selective promoter.\",\n \"11.The plant of claim 10, wherein said stamen-selective promoter is selected from the group of: the TA29 promoter, the T72 promoter, the El promoter, or the CA55 promoter.\",\n \"12.The plant of claim 10, wherein said foreign DNA further comprises: a herbicide resistance gene comprising a DNA encoding an RNA, protein or polypeptide that confers herbicide resistance on said plant.\",\n \"13.A process for producing a plant with selectively destroyable plant tissues or parts, said process comprising: i) introducing into a plant cell or tissue a foreign DNA comprising a chimeric gene comprising: a) The DNA molecule of any one of claims 1 to 5, b) plant-expressible promoter; said DNA molecule being in the same transcriptional unit and under the control of said plant-expressible promoter.\",\n \"ii) regenerating said plant from said plant cell.\",\n \"14.The process of claim 13, wherein said plant-expressible promoter is a tissue-specific promoter.\",\n \"15.The process of claim 14, wherein said tissue-specific promoter is a male or female organ selective promoter and said plants are inducibly male or female sterile.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>Deacetylases are capable of deacetylating acetylated toxins, such as N-acetyl-phosphinothricin (N-Ac-PTC or N-Ac-PPT), intracellularly, whereupon the cytotoxic activity of the toxins is restored (formation of PTC or PTC).\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,650,310, U.S. Pat.\",\n \"No.\",\n \"5,668,297, U.S. Pat.\",\n \"No.\",\n \"5,767,370, and U.S. Pat.\",\n \"No.\",\n \"5,767,371, describe deacetylase genes, for the production of phosphinothricin or phosphinothricyl-alanyl-alanine, as well as processes for their isolation and use.\",\n \"Furthermore deacetylase genes are disclosed which are isolated from Streptomyces viridochromogenes (dea) and from E. coli (ArgE).\",\n \"The use of such genes for the production of plants with selectively destroyable plant parts is described.\",\n \"More specifically, a method is disclosed for the production of conditionally male-sterile plants.\",\n \"This is achieved by introducing a deacetylase gene, under the control of a tapetum-specific promoter into the genome of a plant.\",\n \"Upon application of N-Ac-PPT to the plant, in the tapetum cells, where the deacetylase is expressed, N-Ac-PPT is converted into PPT, which is toxic to the cells.\",\n \"The cytotoxic activity in the tapetum cells inhibits the development of microspores and renders the plant male-sterile.\",\n \"WO 98/27201 describes novel genes encoding amino acid deacetylases isolated from Stenotrophomonas sp.\",\n \"and from Comamonas acidovorans and their use in the production of tranagenic plants.\",\n \"WO 98/13504 describes the use of deacetylase genes for the production of female-sterile plants.\",\n \"WO 98/39462 describes a method of hybrid seed production using conditional female sterility, whereby a plant is made conditionally female-sterile by transformation with the ArgE gene linked to a female-preferential promoter.\",\n \"EP 98116492 describes the use of the deac system for the modification of plant development.\",\n \"Deacetylase genes placed under control of a meristem specific promoter can be used for inducible modification of developmental characteristics of the plant such as flowering and bolting to improve yield, or influence general plant architecture.\",\n \"EP 98116493 describes the use of the deac system to obtain pathogen control in plants.\",\n \"Deacetylase genes placed under control of a pathogen-inducible promoter can be used to obtain plants which, upon treatment with N-acetyl-PPT become tolerant to pathogens, through selective destruction of pathogen-infected tissue.\",\n \"However, the foregoing documents fail to teach or suggest the present invention.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to DNA molecules encoding a protein having the biological activity of a deacetylase.\",\n \"More specifically the protein having the biological activity of a deacetylase is a protein having an amino acid sequence with at least 70% to 79%, more preferably 80% to 85%, especially preferably 86% to 90%, most preferably 91% to 95%, sequence identity, more specifically which is 96% to 100% identical to the sequence of SEQ ID No.\",\n \"1 or a biologically active fragment or variant thereof.\",\n \"Especially preferably the DNA molecule of the invention encodes a protein having the amino acid sequence of SEQ ID No.\",\n \"1 or a biologically active fragment thereof Alternately or additionally, the DNA molecule of the present invention has a nucleotide sequence which has a sequence identity of at least 70% to 79%, more preferably 80% to 85%, especially preferably 86% to 90%, most preferably 91% to 95%, sequence identity, more specifically which is 96% to 100% identical to the sequence of SEQ ID No.\",\n \"2.Especially preferably the DNA molecule of the invention comprises the nucleotide sequence of SEQ ID No.\",\n \"2.Additionally or alternatively, the invention relates to a DNA molecule encoding a deacetylase which can be isolated from Comamonas acidovorans , deposited as DSM 11070, which comprises the sequence of SEQ ID No.\",\n \"2.The invention further relates to a protein or polypeptide having the biological activity of a deacetylase, which is encoded by a DNA molecule, such as those described above.\",\n \"The invention further relates to a chimeric gene comprising the DNA molecule of the present invention placed in the same transcriptional unit and under the control of a plant expressible promoter, preferably a tissue-specific promoter.\",\n \"The invention further relates to a plant having inducibly destroyable plant parts, which plant comprises, stably integrated in its genome, a chimeric gene comprising the DNA molecule of the invention, placed under control of a plant-expressible promoter, preferably a tissue-specific promoter, the plant of the invention being characterized by the fact that, upon treatment with N-acetyl-PPT, the plant tissues in which the promoter directs expression, are destroyed.\",\n \"The invention further relates to an inducibly male or female-sterile plant, which plant comprises, stably integrated in its genome, the DNA molecule of the invention, placed under control of a male- or female reproductive organ selective promoter, the inducibly male- or female-sterile plant of the invention being characterized by the fact that, upon treatment with N-acetyl-PPT, specific plant tissues of the male- or female reproductive organ are destroyed.\",\n \"The invention further relates to a process for producing a plant with inducibly destroyable plant tissues or parts, which process comprises: introducing into a plant cell or tissue a foreign DNA comprising a chimeric gene comprising a) the DNA molecule of the invention, encoding a protein or polypeptide having the biological activity of a deacetylase, under the control of b) a plant-expressible promoter, which is preferably tissue-specific; regenerating the plant cell or tissue into a plant optionally, inducing the destruction of the plant tissue or parts by treatment of the plant with N-acetyl-PPT.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"FIELD OF THE INVENTION The invention relates to novel DNA molecules encoding a protein having the biological activity of a deacetylase.\",\n \"Preferably these DNA molecules encode a protein having a sequence similarity with a deacetylase as described herein isolated from Comamonas acidovorans.\",\n \"The invention further relates to transgenic plants with plant parts which can be destroyed upon induction, comprising the DNA molecules of the invention under control of tissue-specific promoter.\",\n \"All documents cited herein are incorporated herein by reference.\",\n \"BACKGROUND OF THE INVENTION Deacetylases are capable of deacetylating acetylated toxins, such as N-acetyl-phosphinothricin (N-Ac-PTC or N-Ac-PPT), intracellularly, whereupon the cytotoxic activity of the toxins is restored (formation of PTC or PTC).\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,650,310, U.S. Pat.\",\n \"No.\",\n \"5,668,297, U.S. Pat.\",\n \"No.\",\n \"5,767,370, and U.S. Pat.\",\n \"No.\",\n \"5,767,371, describe deacetylase genes, for the production of phosphinothricin or phosphinothricyl-alanyl-alanine, as well as processes for their isolation and use.\",\n \"Furthermore deacetylase genes are disclosed which are isolated from Streptomyces viridochromogenes (dea) and from E. coli (ArgE).\",\n \"The use of such genes for the production of plants with selectively destroyable plant parts is described.\",\n \"More specifically, a method is disclosed for the production of conditionally male-sterile plants.\",\n \"This is achieved by introducing a deacetylase gene, under the control of a tapetum-specific promoter into the genome of a plant.\",\n \"Upon application of N-Ac-PPT to the plant, in the tapetum cells, where the deacetylase is expressed, N-Ac-PPT is converted into PPT, which is toxic to the cells.\",\n \"The cytotoxic activity in the tapetum cells inhibits the development of microspores and renders the plant male-sterile.\",\n \"WO 98/27201 describes novel genes encoding amino acid deacetylases isolated from Stenotrophomonas sp.\",\n \"and from Comamonas acidovorans and their use in the production of tranagenic plants.\",\n \"WO 98/13504 describes the use of deacetylase genes for the production of female-sterile plants.\",\n \"WO 98/39462 describes a method of hybrid seed production using conditional female sterility, whereby a plant is made conditionally female-sterile by transformation with the ArgE gene linked to a female-preferential promoter.\",\n \"EP 98116492 describes the use of the deac system for the modification of plant development.\",\n \"Deacetylase genes placed under control of a meristem specific promoter can be used for inducible modification of developmental characteristics of the plant such as flowering and bolting to improve yield, or influence general plant architecture.\",\n \"EP 98116493 describes the use of the deac system to obtain pathogen control in plants.\",\n \"Deacetylase genes placed under control of a pathogen-inducible promoter can be used to obtain plants which, upon treatment with N-acetyl-PPT become tolerant to pathogens, through selective destruction of pathogen-infected tissue.\",\n \"However, the foregoing documents fail to teach or suggest the present invention.\",\n \"SUMMARY OF THE INVENTION The present invention relates to DNA molecules encoding a protein having the biological activity of a deacetylase.\",\n \"More specifically the protein having the biological activity of a deacetylase is a protein having an amino acid sequence with at least 70% to 79%, more preferably 80% to 85%, especially preferably 86% to 90%, most preferably 91% to 95%, sequence identity, more specifically which is 96% to 100% identical to the sequence of SEQ ID No.\",\n \"1 or a biologically active fragment or variant thereof.\",\n \"Especially preferably the DNA molecule of the invention encodes a protein having the amino acid sequence of SEQ ID No.\",\n \"1 or a biologically active fragment thereof Alternately or additionally, the DNA molecule of the present invention has a nucleotide sequence which has a sequence identity of at least 70% to 79%, more preferably 80% to 85%, especially preferably 86% to 90%, most preferably 91% to 95%, sequence identity, more specifically which is 96% to 100% identical to the sequence of SEQ ID No.\",\n \"2.Especially preferably the DNA molecule of the invention comprises the nucleotide sequence of SEQ ID No.\",\n \"2.Additionally or alternatively, the invention relates to a DNA molecule encoding a deacetylase which can be isolated from Comamonas acidovorans, deposited as DSM 11070, which comprises the sequence of SEQ ID No.\",\n \"2.The invention further relates to a protein or polypeptide having the biological activity of a deacetylase, which is encoded by a DNA molecule, such as those described above.\",\n \"The invention further relates to a chimeric gene comprising the DNA molecule of the present invention placed in the same transcriptional unit and under the control of a plant expressible promoter, preferably a tissue-specific promoter.\",\n \"The invention further relates to a plant having inducibly destroyable plant parts, which plant comprises, stably integrated in its genome, a chimeric gene comprising the DNA molecule of the invention, placed under control of a plant-expressible promoter, preferably a tissue-specific promoter, the plant of the invention being characterized by the fact that, upon treatment with N-acetyl-PPT, the plant tissues in which the promoter directs expression, are destroyed.\",\n \"The invention further relates to an inducibly male or female-sterile plant, which plant comprises, stably integrated in its genome, the DNA molecule of the invention, placed under control of a male- or female reproductive organ selective promoter, the inducibly male- or female-sterile plant of the invention being characterized by the fact that, upon treatment with N-acetyl-PPT, specific plant tissues of the male- or female reproductive organ are destroyed.\",\n \"The invention further relates to a process for producing a plant with inducibly destroyable plant tissues or parts, which process comprises: introducing into a plant cell or tissue a foreign DNA comprising a chimeric gene comprising a) the DNA molecule of the invention, encoding a protein or polypeptide having the biological activity of a deacetylase, under the control of b) a plant-expressible promoter, which is preferably tissue-specific; regenerating the plant cell or tissue into a plant optionally, inducing the destruction of the plant tissue or parts by treatment of the plant with N-acetyl-PPT.\",\n \"DETAILED DESCRIPTION The term “gene” as used herein refers to any DNA sequence comprising several operably linked DNA fragments such as a promoter region, a 5′ untranslated region (the 5′UTR), a coding region (which may or may not code for a protein), and an untranslated 3′ region (3′UTR) comprising a polyadenylation site.\",\n \"Typically in plant cells, the 5′UTR, the coding region and the 3′UTR are transcribed into an RNA of which, in the case of a protein encoding gene, the coding region is translated into a protein.\",\n \"A gene may include additional DNA fragments such as, for example, introns.\",\n \"The term “chimeric” when referring to a gene or DNA sequence is used to refer to the fact that the gene or DNA sequence comprises at least two functionally relevant DNA fragments (such as promoter, 5′UTR, coding region, 3′UTR intron) that are not naturally associated with each other and/or originate, for example, from different sources.\",\n \"“Foreign” referring to a gene or DNA sequence with respect to a plant species is used to indicate that the gene or DNA sequence is not naturally found in that plant species, or is not naturally found in that genetic locus in that plant species.\",\n \"The term “foreign DNA” will be used herein to refer to a DNA sequence as it has incorporated into the genome of a plant as a result of transformation.\",\n \"A genome of a plant, plant tissue or plant cell, as used herein, refers to any genetic material in the plant, plant tissue or plant cell, and includes both the nuclear and the plastid (chloroplast and mitochondrial) genome.\",\n \"A “fragment” of a DNA molecule or protein sequence as used herein refers to a truncated sequence of the original sequence (nucleic acid or amino acid) referred to, which can vary in length but of which the minimum size is sufficient to ensure the (encoded) protein to be biologically active, the maximum size not being critical.\",\n \"A “variant” of a sequence is used herein to indicate a DNA molecule or protein of which the sequence (nucleic or amino acid) is essentially identical to the sequence to which the term refers.\",\n \"Sequences which are “essentially identical” means that when two sequences are aligned, the percent sequence identity, i.e.\",\n \"the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the sequences, is higher than 70%-80%, preferably 81-85%, more preferably 86-90%, especially preferably 91-95%, most preferably 96-100%, more specifically is 100%.\",\n \"The alignment of two nucleotide sequences is performed by the algorithm as described by Wilbur and Lipmann (1983, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"U.S.A. 80:726) using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4.Preferably the DNA molecule of the invention is a sequence which is essentially identical to a coding sequence of a deacetylase from Comamonas acidovorans, which has been adapted for preferred codon usage in plants.\",\n \"The present invention relates to a DNA molecule or coding sequence encoding a protein having the activity of a deacetylase.\",\n \"A “deacetylase” or a protein with deacetylase activity as used herein refers to a molecule which is capable of deacetylating N-acetyl PPT (which in itself—i.e.\",\n \"in the absence of a deacetylase- is not toxic to plants or plant cells or tissues), to produce PPT which is cytotoxic to plants.\",\n \"Deacetylase activity can be measured by radioactive or non-radioactive assays as described in WO 98/27201 and U.S. Pat.\",\n \"No.\",\n \"5,668,297.Presence of deacetylase activity in plant tissues can be determined by treatment of the tissue with the substrate N-acetyl-PPT, and evaluating production of PPT, which should result in cell death.\",\n \"A protein or polypeptide which is “biologically active” as used herein refers to the fact that it has deacetylase activity.\",\n \"In a preferred embodiment of the invention, the DNA molecule encoding a protein having the biological activity of a deacetylase, is a DNA which can be isolated from the deposit with International deposit No.\",\n \"DSM 11070 (as described in WO 98/27201 and U.S. Pat.\",\n \"No.\",\n \"6,177,616) deposited at the.\",\n \"Deutsche Sammlung für Microorganismen und Zelikulturen GmbH, Masenroder Weg 1b, D-38124.Braunsenweig.\",\n \"More specifically such a DNA molecule is a DNA-molecule encoding a deacetylase having a sequence which is essentially identical to the sequence of SEQ ID No.\",\n \"1, or a biologically active fragment thereof.\",\n \"Additionally or alternatively such a DNA molecule comprises a nucleotide sequence which is essentially identical to the sequence of SEQ ID No.\",\n \"2, or corresponds to a DNA sequence which hybridizes with the sequence of SEQ ID No.\",\n \"2 under standard stringent conditions.\",\n \"A preferred embodiment of the DNA molecule of the present invention is a DNA molecule encoding a deacetylase comprising a sequence which is essentially identical to SEQ ID No.\",\n \"1, isolated from Comamonas acidovorans.\",\n \"Based on the homology of the DNA sequence isolated from Comamonas acidovorans to N-acetyl-Ornithine transferase genes, the deacetylases of present invention will be referred to as “ArgJ” deacetylases and the genes comprising a DNA molecule encoding such an ArgJ deacetylase (or a biologically active fragment thereof) will be referred to as an “ArgJ gene”.\",\n \"The isolation of sequences encompassed by the present invention which are essentially identical to the sequence of SEQ ID No.1 isolated from Comamomas acidovorans, is possible using standard strategies and techniques that are well known in the art.\",\n \"For example, using all or a portion of the amino acid sequence of the Comamonas ArgJ protein, one may readily design specific oligonucleotide probes, including degenerate oligonucleotide probes (i.e., a mixture of all possible coding sequences for a given amino acid sequence).\",\n \"The oligonucleotides may be based upon the sequence of either DNA strand and any appropriate portion of the ArgJ coding sequence.\",\n \"General methods for designing and preparing such probes are provided, for example, in Ausubel et al.\",\n \"Current Protocols in Molecular Biology, Wiley Interscience, New York and Berger and Kimmel, Guide to Molecular Cloning techniques, 1987, Academic Press, New York.\",\n \"These oligonucleotides are useful for ArgJ gene isolation, either through their use as probes capable of hybridizing to ArgJ complementary sequences or as primers for various amplification techniques, for example PCR cloning strategies.\",\n \"For detection or isolation of closely related ArgJ sequences having greater than 80% sequence identity, high stringency conditions are preferably used; such Stringent conditions as used herein refers to the hybridizing conditions described by Sambrook et al.\",\n \"(1989) (Molecular Cloning: A.\",\n \"Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory Press, NY).\",\n \"As used herein N-acetyl-PPT refers to N-acetyl-phosphinothricin or a composite structure thereof, which in itself is not toxic to plant cells but is converted to a toxin (phosphinothricin, PPT) by a deacetylase.\",\n \"Thus, the term N-acetyl-PPT refers to any formulation of N-acetyl-phosphinothricin, or derivatives or related compounds, such as acetylated forms of desmethylphosphinothricine (2-amino-4-hydroxyphosphinobutyric acid), bialaphos (phosphinothricyl-alanyl-alanine), desmethylbialaphos and the like, their salts, racemic mixtures and active enantiomers, which can be used in the context of the present invention.\",\n \"According to the present invention, plant-expressible promoter is a promoter which directs expression in plants.\",\n \"Preferably, the plant-expressible promoter of the invention is a tissue-specific promoter, which is a promoter directing expression selectively in a specific cell or tissue of the plant.\",\n \"A broad range of tissue-specific promoters are described in the literature, such as, but not limited to, meristem-specific promoters (such as the promoter of the AGL8 MADS box gene; Yanofsky, 1995, The plant cell 7:1763-1771), female-organ specific promoters (such as the PSTMG-type promoters described in U.S. Pat.\",\n \"No.\",\n \"4,633,441), leaf-tissue specific promoters (such as the leaf-specific Ca/b promoter from Arabidopsis described by Cannon et al., 1990, Plant Mol Biol 15(1)39-47 or the tomato RbcS-3C promoter described by Kyozuka et al., 1993, Plant Physiol 102(3):991-1000).\",\n \"In a preferred embodiment of the invention, the tissue-specific promoter is male or female organ selective promoter, directing expression selectively in male or female organs of the plant.\",\n \"A preferred male organ selective promoter is a stamen-selective promoter which directs expression in selective stamen cells (including anther cells and pollen), preferably directing expression essentially only in stamen cells.\",\n \"More specifically, the stamen-selective promoter is a tapetum-specific promoter directing expression specifically in tapetum cells.\",\n \"A number of stamen-selective promoters are known in the art, such as, but not limited to, the TA29 promoter from tobacco (U.S. Pat.\",\n \"No.\",\n \"5,652,354), the CA55 promoter isolated form corn (WO 92/00275, U.S. Pat.\",\n \"No.\",\n \"5,589,610), the T72 and E1 promoters isolated from rice (U.S. Pat.\",\n \"No.\",\n \"5,639,948), SGBG (U.S. Pat.\",\n \"No.\",\n \"5,837,850), and 5126 (U.S. Pat.\",\n \"No.\",\n \"5,689,049).\",\n \"Tissue-specific promoters as used herein includes promoters which are also subject to other kinds of regulation (such as, but not limited to, time-dependent, stress-dependent, environment-dependent, chemically inducible, etc.).\",\n \"Alternatively, the plant-expressible promoters of the present invention are not tissue-specific but are subject to other kinds of regulation (such as, but not limited to, time-dependent, stress-dependent, environment-dependent, chemically inducible, etc.).\",\n \"A plant with conditionally or inducibly destroyable parts refers to a plant in which, under specific conditions, specific plant tissues or parts can be destroyed.\",\n \"“Plant part” is meant to refer to any plant organ (such as, but not limited to male or female reproductive organs, leaves, vegetative meristem, roots) or tissue (such as, but not limited to reproductive tissues, embryogenic tissues), including the complete plant.\",\n \"More specifically, these conditions are understood to refer to the treatment of the plant with an acetylated toxin, which normally is not toxic to the plant or plant cells, but in the presence of a deacetylase in the plant cell is converted into a substance that is cytotoxic.\",\n \"A conditionally male-sterile plant refers to a plant which, under normal growing conditions, is male fertile, i.e.\",\n \"produces fertile pollen, and which can be made male-sterile under specific conditions.\",\n \"Treatment of the plant can refer to administering the substance in any way, such as, but not limited to manual treatment, or spraying of the plant, plant part (or of the field or greenhouse wherein the plant is grown), or through other forms of irrigation.\",\n \"Treatment of a plant with N-acetyl PPT will result in uptake of N-acetyl PPT by the leaf (or other) cells and transportation to different plant organs.\",\n \"Thus, treatment of a plant which comprises a chimeric gene comprising a deacetylase coding sequence expressed in specific cells of the plant, will result in the conversion of N-acetyl PPT to PPT in those cells, causing these cells to die.\",\n \"In a specific embodiment of the invention, the transgenic plant, comprising in its genome the DNA molecule of the invention encoding a protein having the biological activity of a deacetylase, additionally comprises, a chimeric gene conferring on said plants resistance a herbicide to PPT, such as, but not limited to the herbicide resistance genes, pat or bar described in EP-A-0 242 236, EP-A-0 242 246, EP-A-0 257 542.These genes encode enzymes which are capable of converting PPT into N-acetyl-PPT.\",\n \"This N-acetyl-PPT can be transported throughout the plant and act as the substrate for a deacetylase specifically expressed in certain plant cells or tissues.\",\n \"It is understood that according to this specific embodiment, induction of tissue-specific cytotoxicity will also occur upon treatment of the plant with PPT (EP-A-0 531 716, U.S. Pat.\",\n \"No.\",\n \"5,668,297).\",\n \"Preferably, the deacetylase of the present invention and the enzyme conferring herbicide resistance gene will be expressed or transported to different cellular compartments (i.e.\",\n \"cytoplasm, chloroplast) to avoid re-conversion of the PPT to N-acetyl PPT, where this is not intended.\",\n \"A chimeric marker gene as used herein is a gene comprising a marker DNA under the control of a plant-expressible promoter.\",\n \"The marker DNA encodes an RNA, protein or polypeptide, which, when expressed in a plant or plant cell, allows such plants or plant cells to be distinguished and separated from plants or plant cells not expressing the marker DNA.\",\n \"Examples of marker genes are genes that provide a specific color to the cells (such as the A1 gene, Meyer et al.\",\n \"1987, Nature 330:667) or genes that encode a particular resistance to the plant cells or the plant such as antibiotic resistance (e.g.\",\n \"the aac(6′) gene, encoding resistance to gentamycin, WO94/01560).\",\n \"Preferred marker genes are herbicide resistance genes.\",\n \"A herbicide resistance gene as used herein is a gene conferring to the plant or plant cell resistance to a herbicide.\",\n \"Examples of herbicide resistance genes are the genes encoding resistance to phenmedipham (such as the pmph gene, U.S. Pat.\",\n \"No.\",\n \"5,347,047; U.S. Pat.\",\n \"No.\",\n \"5,543,306), the genes encoding resistance to glyphosate (such as the EPSPS genes, U.S. Pat.\",\n \"No.\",\n \"551047), genes encoding bromoxynyl resistance (such as described in U.S. Pat.\",\n \"No.\",\n \"4,810,648) genes encoding resistance to sulfonylurea (such as described in EPA 0 360 750), genes encoding resistance to the herbicide dalapon (such as described in WO 99/27116), and genes encoding resistance to cyanamide (such as described in WO 98/48023 and WO 98/56238).\",\n \"The cytotoxic effect of certain toxins may be enhanced by targeting to the specific parts of the cell, such as the chloroplast or mitochondria.\",\n \"Phosphinothricin is a glutamine synthase inhibitor.\",\n \"In plants, glutamine synthase exitst in multiple isozymic forms that can be localized within the cell in the cytosol and plastids.\",\n \"Thus, it can be contemplated that targeting of the deacetylase to the chloroplast, whereby N-acetyl-PPT is converted to PPT in the chloroplast, increases cytotoxic efficiency.\",\n \"Thereto, the foreign DNA comprising the deacetylase coding sequence can comprise an additional foreign DNA encoding a transit peptide.\",\n \"The additional DNA is preferably located between the first promoter and the deacetylase coding sequence.\",\n \"Alternatively, it can be desired that the marker protein or polypeptide is transported from the cytoplasm into chloroplasts or mitochondria of the transformed plant cells.\",\n \"Thereto, an additional DNA encoding a transit peptide is located between the marker DNA and its promoter.\",\n \"By transit peptide is meant a polypeptide fragment which is normally associated with a chloroplast or mitochondrial protein or subunit of the protein and is produced in a cell as a precursor protein encoded by the DNA of the cell.\",\n \"The transit peptide, is responsible for the translocation process of the nuclearencoded chloroplast or mitochondrial protein or subunit into the chloroplasts or mitochondria, and during such a process, the transit peptide is separated or proteolytically removed from the chloroplast or mitochondrial protein or subunit.\",\n \"One or more of such additional DNA's can be provided in the foreign DNAs of this invention as generally described and exemplified in European patent publication 0,189,707 and European patent EP 0 344 029.Transformation of the plants of the invention can occur in a number of ways described in the art.\",\n \"Such methods include, but are not limited to Agrobacterium-mediated transformation (BP 0,116,718, DeBlaere: et al., 1987, Meth.\",\n \"Enzymol.\",\n \"153:277-293), microinjection (Crossway et al., 1986, Mol.\",\n \"Gen. Genet.\",\n \"102:179), direct gene transfer (Pazowski et al., 1984, EMBO J.\",\n \"3:2717-2722), electrapomtion (Riggs et al., 1986, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 83:5602-5606), or by particle gun bombardment (WO92/09696; Potrykus et al.\",\n \"1991, Annu.\",\n \"Rev.\",\n \"Plant Physiol.\",\n \"Plant Mol.\",\n \"Biol.\",\n \"42:205-225); see also Christou P. 1994, Agro-Food Industry Hi-Tech 2:17; Langridge et al.\",\n \"1992, Plant J.\",\n \"2:631; Vasil, 1994, Plant Mol.\",\n \"Biol.\",\n \"25:925; Chen D., 1998, Methods Mol.\",\n \"Biol.\",\n \"81:373.Alternatively, the foreign DNA comprising the chimeric genes of the invention can be inserted into the plastid genome of the cell or cells of a plant.\",\n \"Gene transfer can be carried out, for example, by particle gun bombardment (as described by Svab et al., 1990, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 87:8526-8530) or by Agrobacterium-mediated transformation (De Block et al., 1985, EMBO J.\",\n \"4:1367).\",\n \"As used herein “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof.\",\n \"Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein.\",\n \"A chimeric gene comprising a DNA sequences which is functionally or structurally defined, may comprise additional DNA sequences, etc.\",\n \"Unless otherwise stated, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al.\",\n \"(1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al.\",\n \"(1994) Current Protocols in Molecular Biology, Current Protocols, USA.\",\n \"Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D.\",\n \"Croy published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.\",\n \"In the description and examples, reference is made to the following sequences: SEQ ID No.\",\n \"1: Amino acid sequence of the ArgJ deacetylase from Comamonas acidovorans SEQ ID No.\",\n \"2: Nucleotide sequence encoding the Arg J deacetylase EXAMPLES Example 1 Isolation of Bacterial Deacetylase Genes The isolation of micro-organisms having N-acetyl-PPT deacetylase activity was performed as described in WO98/27201.Briefly, different soil samples were grown on Minimal Essential Medium containing only chitin as a carbon source.\",\n \"As chitin can only be broken down after deacetylation, microorganisms having deacetylase activity will preferentially grow on this medium.\",\n \"After subsequent replating and isolation of single colony bacteria, different colonies could be isolated that were capable of converting radioactive N-acetyl-PPT to PPT.\",\n \"One of the isolated bacteria was identified as Comamonas acidovorans and deposited at the Deutsche Sammlung fur Microorganismen und Zellkulturen under accession number DSM 11070.In a next step the genes encoding for N-acetyl-PPT deacetylase activity were isolated.\",\n \"A specific selection method was developed to be able to isolate the deacetylase genes in a “Shot-gun” cloning method.\",\n \"The method is based on the principle that a gene product capable of deacetylating N-acetyl-PPT should also be able to process a related compound N-acetyl-Ornithine as a substrate.\",\n \"The deacetylation of N-acetyl-ornithine is a step in the arginine biosynthesis and mutants that are incapable of performing this step are auxotrophic for arginine.\",\n \"One such mutant, the E. coli nullmutant (XS1D2), which has a deletion in its ArgE gene, was described by Mountain et al.\",\n \"(1984, Mol.\",\n \"Gen. Genet.\",\n \"197:82-89).\",\n \"Total DNA from Comamonas acidovorans was isolated, digested with EcoRI and ligated within the EcoRI site of the vector pACYC184 (Cang and Cohen, 1987, J Bacteriol.\",\n \"134:1141-1156).\",\n \"The ligation mixture was then introduced by electroporation into the cells of the mutant E. coli XS1D2.Of 16.000 tested shot-gun clones, 14 were able to complement the arginine auxotrophy of the E. coli mutant.\",\n \"One of the complementing clones (E4.2) comprised a 4,2 kb EcoRI fragment.\",\n \"Hybridisation experiments confirmed that this fragment originated from the C. acidovorans genome and did not show any cross-hybridization with any of the other fragments in other clones.\",\n \"Through subsequent subdloning, the minimal sequence for retaining complementation of the arginine auxotrophy was determined.\",\n \"This sequence was then sequenced as a double strand and the open reading frame was identified.\",\n \"Based on a homology with databank sequence, the sequence was termed as the ArgJ-gene.\",\n \"Example 2 The ArgJ Coding Sequence of C. Acidovorans Through subsequent subdloning, the minimal sequence for retaining complementation of the arginine auxotrophy was determined as a 2.2 kb HindIII/PstI fragment (SEQ ID No.\",\n \"2).\",\n \"An open reading frame of 1230 bp was identified, encoding a protein of 410 amino acids (SEQ ID No.\",\n \"1).\",\n \"A potential ribosomal binding site is located at about 10 bp before the start-codon.\",\n \"Comparison of the amino acid sequence of the open reading frame with known sequences, strong homology was found with a number of N-acetyl-ornithin-transacetylases (Table 1).\",\n \"The strongest homology was found with N-acetyl-ornithin-transacetylase encoded by the ArgJ-gene from Neisseria gonorrhoeae.\",\n \"Therefore the complete open reading frame of the C. acidovorans fragment was named the ArgJ coding sequence.\",\n \"Upstream of the ArgJ coding sequence another, be it incomplete, open reading frame can be found.\",\n \"Comparison with databank sequences showed that this is part of the secA-gene of C. acidovorans.\",\n \"This sequence however, does not seem of importance for the complementation of the E. coli ArgE-mutant, since a PCR fragment comprising only the ArgJ-gene can restore arginin auxotrophy in the mutant.\",\n \"TABLE 1 Protein sequences found to be homologous to the sequence encoded by the C. acidovorans gene Homologous gene Origin Encoded protein % Identical AA ArgJ Neisseria gonorrhea N-acetyl-ornithin- 54.5% transferase ArgJ Bacillus subtilis N-acetyl-ornithin- 37.4% transferase ArgJ Bacillus N-acetyl-ornithin- 37.5% stearothermophilus transferase ArgJ Saccharomyces N-acetyl-ornithin- 36.1% cervisiae transferase Example 3 Expression of the ArgJ Coding Sequence in E. coli Through the selection procedure, it was established that the protein encoded by the ArgJ coding sequence from C. acidovorans was capable of complementing the E. coli ArgE mutant by its deacetylase activity.\",\n \"To establish whether this protein would also deacetylate N-acetyl-PPT, both coding sequences were over-expressed in E. coli, and the supernatant of the culture was analyzed for its ability to deacetylate N-acetyl-PPT.\",\n \"The coding sequence was cloned into a vector with high copy number under control of the lacZ promoter.\",\n \"The incorporation of a stop codon made overexpression of the deacetylase sequence possible without interference of open reading frames of the lacZ gene.\",\n \"Compared to the isolated fragments, these construct showed significantly stronger complementation of the E. coli ArgE mutant.\",\n \"Example 4 Expression of the ArgJ Coding Region from C. acidovorans in Tobacco The ArgJ coding sequence was recloned into the binary vector pROK1 under control of the Cauliflower Mosaic Virus 35S promoter sequence.\",\n \"The resulting plasmid p35SARG comprising the 35S promoter, ArgJ coding sequence and 35S terminator was transformed in Agrobacterium tumefaciens (strain ATHV) using standard methods.\",\n \"Tobacco (Nicotiana tabacum) leaf disks were transformed with the recombinant Agrobacteria according to the method of Horsch et al.\",\n \"(1985, Science 227:1229-1231) and selected on kanamycin medium.\",\n \"Seven independent transformants were regenerated.\",\n \"In a drop experiment, the leaves of the transgenic plants (and a number of control plants) were treated with 5 μl each of the following concentrations of N-acetyl- D, L-PPT: 4 mg/ml (=15 mM), 1 mg/ml (=3.75 mM), 0.4 mg/ml (=1.5 mM), 0.1 mg/ml (=0.38 mM).\",\n \"The treatment sites were examined for lightening or necrosis formation.\",\n \"Four plants showed clearly visible leaf damage, apparent 4 to 5 days after treatment with a dose of 4 mg/ml or 1 mg/ml.\",\n \"Example 5 Tapetum-Specific Expression of the ArgJ Gene in Tobacco The ArgJ coding sequence was recloned into the binary vector pROK1 under control of the TA29 promoter of tobacco.\",\n \"The resulting plasmid pTA29ARG comprising the TA29 promoter, ArgJ coding sequence and 35S terminator was transformed in Agrobacterium tumefaciens (strain ATHV) using standard methods.\",\n \"Tobacco (Nicotiana tabacurn) leaf disks were transformed with the recombinant Agrobacteria according to the method of Horsch et al.\",\n \"(1985, above) and selected on kanamycin medium.\",\n \"To investigate tapetum-specific expression of the ArgJ deacetylase, flower buds of the transgenic lines were treated with N-acetyl-PPT to induce male-sterile flowers.\",\n \"In each case only one side of the inflorescence (the total area of the developing flower bud) was treated with N-acetyl PPT, in order to eliminate possible contamination with natural male-sterility phenomena.\",\n \"Treatment was carried out on flower buds of 2 to 4 mm large.\",\n \"Transgenic and control plants were treated with 10 μl of 5 mg/ml N-acetyl PPT solution (0.1% Genapol as wetting agent).\",\n \"After the blooming of the buds (about 9-11 days after conclusion of the treatment) the plants were investigated for the occurrence of male-sterile flowers.\",\n \"As expected, no reaction was seen in either the control or the wild-type tobacco flowers.\",\n \"In three out of seven transgenic plants, male-sterile flowers developed from the drop-treated flowerbuds.\",\n \"The anthers of these flowers did not contain any pollen and their filaments were markedly shorter than those of treated wild-type flowers.\",\n \"Anther dehiscence occurs in these flowers, but is delayed compared to controls.\",\n \"If cross-pollination is avoided, the male-sterile flowers develop an empty seedpod, as pollen for self-pollination is lacking.\",\n \"Otherwise these flowers can not be distinguished from untreated flowers.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471370"}}},{"rowIdx":592,"cells":{"text":{"kind":"list like","value":[["Bushing with controlled elastic absorption of radial stress","Bushing, in particular of the kind with controlled elastic absorption of radial stress, includes a cylindrical tubular element (1) out of self-lubricating material onto which a ring (2) is freely fitted out of an elastically buckling material, wherein the internal surface of the ring out of elastically buckling material is associated to an annular reinforcing fitting (3) out of a material that does not give radially and which gives the bushing the required radial stiffness and limits the longitudinal deformations of the ring (2)."],["1.A bushing, in particular of the kind with controlled elastic absorption of radial stress, comprising a cylindrical tubular element (1) out of self-lubricating material onto which a ring (2) is freely fitted out of a elastically buckling material, characterized in that the internal surface of said ring out of elastically buckling material is associated to an annular reinforcing fitting (3) out of a material that does not give radially.","2.A bushing according to claim 1, characterized in that the external surface of said ring (2) out of elastically buckling material comprises bulge ribbings (4).","3.A bushing according to claim 1, characterized in that said bulge ribbings (4) are placed at regular distances one from the other.","4.A bushing according to claim 1, characterized in that said bulge ribbings (4) develop in a longitudinal direction substantially parallel to the axis of said bushing.","5.A bushing according to claim 1, characterized in that said bulge ribbings (4) have an annular shape.","6.A bushing according to claim 1, characterized in that said ring (2) is out of rubber.","7.A bushing according to claim 1, characterized in that said annular reinforcing insert (3) is fixed to said ring (2) by means of vulcanization.","8.A bushing according to claim 1, characterized in that said element (1) and said ring (2) are respectively provided with stop collars (5, 6).","9.A bushing according to claim 1, characterized in that it comprises a plane metal washer that may be placed against said stop collar (6).","10.A vehicle characterized in that it comprises at least one bushing according to claim 1."],["The present invention concerns a bushing with elastic absorption of radial stress with a control means for the radial compressibility.","Bushings with elastic absorption of radial stress are used for the connection to an axis of a pin, subject to a shifting angular to said axis.","They are also used in many fields and among these the one of haulage vehicles.","Here they are employed, e.g., for the connection of the leaf spring eye of the back suspension to the undercarriage of the vehicle, or also for the connection of the supporting arms of the front wheels to the undercarriage as well as to the hub of said wheels, or still in the connection of connecting rods, arms etc.","Usually, such bushings are realized by means of two tubular, cylindrical and coaxial elements out of metal; between said elements, an elastic rubber ring is placed, coupled to the same by means of vulcanization.","The reciprocal angular shifting of the elements connected by the bushing is allowed by the torsional buckling of the rubber ring, which at the same time produces the effect of absorbing possible direct crashes and radial stress.","The assembling takes place by fixing, by interference, the outer tubular element to an appropriate housing, while the pin gets fixed into the inner tubular element, always by interference.","This kind of bushing has a number of inconveniences, among which the breaking of the rubber ring caused by torsional stress; the need of expensive mechanical working of the metal surfaces that must be coupled by interference; the assembling difficulty; the high weight and cost of the bushing due to the necessary mechanic working and to the vulcanization process of the rubber ring.","For solving in particular the first one of said inconveniences, i.e.","the fatigue failure of the rubber ring, a bushing is known mainly comprising an internal cylindrical tubular element, out of self-lubricating material.","On said tubular element an elastic rubber ring is freely fitted and gets fixed by means of vulcanization to an outer cylindrical tubular element, realized out of metal and coaxial to the first one.","The assembling of said bushing takes place, in this case, by fixing by interference the outer cylindrical tubular element to the assembling seat, after boring the latter, while the pin is free to rotate onto the internal surface of the element out of self-lubricating material.","Consequently, it won't transmit any torsional stress to the rubber ring, and the latter has the only task of absorbing the pushes and the stress in radial direction.","This mentioned solution eliminates the risk of torsional fatigue failure of the rubber ring and increases the reliability and the life of said bushing; furthermore, it does not request the fixing by interference of the pin to the internal cylindrical tubular element of said bushing.","However, said solution does not eliminate the inconveniences linked to the need of preparing for said bushing a seat for the interference assembling of the outer metal tubular element, with consequent high costs due to the mechanical working, to the difficulty of said assembling, to the high weight and cost of said bushing.","Furthermore, from Utility Model application no.","CR97U000005 filed on Apr.","17, 1997 of the same applicant, a particular kind of bushing is known, mainly comprising an inner cylindrical tubular element out of self-lubricating material, onto which a ring out of rubber or of any other elastically buckling material is freely fitted.","The outer cylindrical surface of said ring is provided with rises that allow the interference assembling of said rubber ring directly into the housing seat of said bushing, without particular working or preparation of the latter.","This kind of bushing shows the advantage of having a simple and saving realization: in fact, it does not need the outer cylindrical tubular element fixed by means of vulcanization to the rubber ring, and does not need special mechanical working.","The assembling of the bushing takes place by forcing the buckling rubber ring into its housing seat, is greatly simplified with respect to the reciprocal forcing of metal parts.","Furthermore, the mechanical working of said housing seat requires less limiting dimensional tolerances and therefore is less expensive.","However, this kind of bushing has the disadvantage that it is not suitable for those application that require a high radial stiffness and the capacity of bearing great radial loads.","As an example: if said bushings are used on haulage vehicles for the connection of the leaf springs of the back suspensions of the undercarriage of the vehicle, they may be applied only to vehicle with a capacity up to about 10 tons.","A further inconvenience consists in that in the presence of great radial loads, the rubber ring is subject to relevant buckling in longitudinal direction that may cause expansion and stretching of the rubber, that reduce durability and reliability of said bushing.","It is the aim of the present invention to eliminate all above mentioned inconveniences.","In particular, it is the aim of the present invention to realize a bushing with elastic absorption of radial stress that allows the control of the radial compressibility, and that may sustain radial loads of considerable entity in a reliable manner, and that the realization thereof is convenient and the assembly easy.","The aims set forth are reached by means of a bushing with elastic absorption of radial stress with a control means for the radial compressibility according to the present invention, comprising a cylindrical tubular element out of self-lubricating material onto which a ring is freely fitted out of a elastically budding material, characterized in that the internal surface of said ring out of elastically buckling material is associated to an annular reinforcing fitting out of a material that does not give radially.","For making the coupling between the bushing and the circular seat into which it gets inserted as less expensive as possible, the outer surface of said ring out of elastically buckling material comprises bulge ribbings that cooperate in interference with the internal surface of said seat.","Said ring out of elastically buckling material may be advantageously realized out of rubber, while said reinforcing annular insert is advantageously realized out of glass loaded nylon; its thickness is correlated to the entity of the radial charges to bear and is coupled by means of vulcanization to the internal surface of the rubber ring.","Said bulge ribbings on the outer surface of the ring out of buckling material, according to the cited Utility Model of the same applicant, are advantageously placed at regular distances one from the other and in a longitudinal direction, parallel to the axis of the bushing.","In an alternative, bulges with annular shape, parallel one to the other, might perform the same function.","The main advantages obtained by means of the bushing with elastic absorption of radial stress with a control means for the radial compressibility according to the present invention consist in that said bushing is able to bear high radial loads due to the control of the radial buckling assured by the reinforcing insert, and in that the reinforcing insert, connected by vulcanization to the internal surface of said rubber ring, limits and contains the longitudinal buckling of said ring.","A further advantage consists in that by suitably choosing the thickness of the internal reinforcing insert, it is possible to realize bushings with different degrees of radial stiffness, fitting to different application fields.","The present invention will be explained more in detail hereinbelow relating to the enclosed drawings in which an embodiment is shown.","FIG.","1 shows a perspective view of a bushing with elastic absorption of radial stress With a control means for the radial compressibility, according to the present invention.","FIG.","2 shows said bushing in an axial section.","FIGS.","3 and 4 show said bushing in a top and a bottom view.","Relating now to the drawings, the bushing with elastic absorption of radial stress with a control means for the radial compressibility according to the present invention mainly comprises an internal cylindrical tubular element 1 out of plastic self-lubricating material, onto which a rubber ring 2 is freely fitted.","An annular reinforcement 3 of suitable thickness is fixed on the internal cylindrical surface of said rubber ring 2, and it is out of a material that does not give radially, like e.g.","glass loaded nylon.","The coupling between said ring 2 and said reinforcement 3 is advantageously realized by means of a vulcanization process.","The external cylindrical surface of said ring 2 has a plurality of bulge ribbings 4 that locally increase the thickness of said ring 2.Furthermore, in the embodiment shown, element 1 and ring 2 have stop collars 5 and 6 that allow the bushing to bear the compression stress parallel to the axis of said bushing.","In addition, it is possible to place a plane metal washer—not shown—of suitable thickness, similar to the one of collars 5 and 6, against said collar 6: said washer allows to distribute the longitudinal force transmitted to the bushing onto a greater surface and in a uniform manner, thus increasing the resistance of said bushing to said compression stress.","It is evident that any variant of the bushing according to the present invention, of generic or simplified kind, will not be provided with said collars 5 and 6.The assembling of the bushing according to the present invention takes place by pushing the external surface of said rubber ring 2 into the housing seat of said bushing.","During such operation, the bulge ribbings 4 get compressed and elastically buckled, while the space between one ribbing and the other allows a free lateral extension of the same.","Thus the bushing will be fixed in its own seat by the friction between the buckled rubber bulges and the internal surface of the housing seat.","The pin in freely rotating on the internal surface of said tubular element 1.During normal working, said rubber ring 2 absorbs crashes and radial stress transmitted by the pin to the bushing, while the annular reinforcement 3 of suitable thickness gives the bushing the required radial stiffness, limits its squeezing and the longitudinal deformation of the rubber ring 2."]],"string":"[\n [\n \"Bushing with controlled elastic absorption of radial stress\",\n \"Bushing, in particular of the kind with controlled elastic absorption of radial stress, includes a cylindrical tubular element (1) out of self-lubricating material onto which a ring (2) is freely fitted out of an elastically buckling material, wherein the internal surface of the ring out of elastically buckling material is associated to an annular reinforcing fitting (3) out of a material that does not give radially and which gives the bushing the required radial stiffness and limits the longitudinal deformations of the ring (2).\"\n ],\n [\n \"1.A bushing, in particular of the kind with controlled elastic absorption of radial stress, comprising a cylindrical tubular element (1) out of self-lubricating material onto which a ring (2) is freely fitted out of a elastically buckling material, characterized in that the internal surface of said ring out of elastically buckling material is associated to an annular reinforcing fitting (3) out of a material that does not give radially.\",\n \"2.A bushing according to claim 1, characterized in that the external surface of said ring (2) out of elastically buckling material comprises bulge ribbings (4).\",\n \"3.A bushing according to claim 1, characterized in that said bulge ribbings (4) are placed at regular distances one from the other.\",\n \"4.A bushing according to claim 1, characterized in that said bulge ribbings (4) develop in a longitudinal direction substantially parallel to the axis of said bushing.\",\n \"5.A bushing according to claim 1, characterized in that said bulge ribbings (4) have an annular shape.\",\n \"6.A bushing according to claim 1, characterized in that said ring (2) is out of rubber.\",\n \"7.A bushing according to claim 1, characterized in that said annular reinforcing insert (3) is fixed to said ring (2) by means of vulcanization.\",\n \"8.A bushing according to claim 1, characterized in that said element (1) and said ring (2) are respectively provided with stop collars (5, 6).\",\n \"9.A bushing according to claim 1, characterized in that it comprises a plane metal washer that may be placed against said stop collar (6).\",\n \"10.A vehicle characterized in that it comprises at least one bushing according to claim 1.\"\n ],\n [\n \"The present invention concerns a bushing with elastic absorption of radial stress with a control means for the radial compressibility.\",\n \"Bushings with elastic absorption of radial stress are used for the connection to an axis of a pin, subject to a shifting angular to said axis.\",\n \"They are also used in many fields and among these the one of haulage vehicles.\",\n \"Here they are employed, e.g., for the connection of the leaf spring eye of the back suspension to the undercarriage of the vehicle, or also for the connection of the supporting arms of the front wheels to the undercarriage as well as to the hub of said wheels, or still in the connection of connecting rods, arms etc.\",\n \"Usually, such bushings are realized by means of two tubular, cylindrical and coaxial elements out of metal; between said elements, an elastic rubber ring is placed, coupled to the same by means of vulcanization.\",\n \"The reciprocal angular shifting of the elements connected by the bushing is allowed by the torsional buckling of the rubber ring, which at the same time produces the effect of absorbing possible direct crashes and radial stress.\",\n \"The assembling takes place by fixing, by interference, the outer tubular element to an appropriate housing, while the pin gets fixed into the inner tubular element, always by interference.\",\n \"This kind of bushing has a number of inconveniences, among which the breaking of the rubber ring caused by torsional stress; the need of expensive mechanical working of the metal surfaces that must be coupled by interference; the assembling difficulty; the high weight and cost of the bushing due to the necessary mechanic working and to the vulcanization process of the rubber ring.\",\n \"For solving in particular the first one of said inconveniences, i.e.\",\n \"the fatigue failure of the rubber ring, a bushing is known mainly comprising an internal cylindrical tubular element, out of self-lubricating material.\",\n \"On said tubular element an elastic rubber ring is freely fitted and gets fixed by means of vulcanization to an outer cylindrical tubular element, realized out of metal and coaxial to the first one.\",\n \"The assembling of said bushing takes place, in this case, by fixing by interference the outer cylindrical tubular element to the assembling seat, after boring the latter, while the pin is free to rotate onto the internal surface of the element out of self-lubricating material.\",\n \"Consequently, it won't transmit any torsional stress to the rubber ring, and the latter has the only task of absorbing the pushes and the stress in radial direction.\",\n \"This mentioned solution eliminates the risk of torsional fatigue failure of the rubber ring and increases the reliability and the life of said bushing; furthermore, it does not request the fixing by interference of the pin to the internal cylindrical tubular element of said bushing.\",\n \"However, said solution does not eliminate the inconveniences linked to the need of preparing for said bushing a seat for the interference assembling of the outer metal tubular element, with consequent high costs due to the mechanical working, to the difficulty of said assembling, to the high weight and cost of said bushing.\",\n \"Furthermore, from Utility Model application no.\",\n \"CR97U000005 filed on Apr.\",\n \"17, 1997 of the same applicant, a particular kind of bushing is known, mainly comprising an inner cylindrical tubular element out of self-lubricating material, onto which a ring out of rubber or of any other elastically buckling material is freely fitted.\",\n \"The outer cylindrical surface of said ring is provided with rises that allow the interference assembling of said rubber ring directly into the housing seat of said bushing, without particular working or preparation of the latter.\",\n \"This kind of bushing shows the advantage of having a simple and saving realization: in fact, it does not need the outer cylindrical tubular element fixed by means of vulcanization to the rubber ring, and does not need special mechanical working.\",\n \"The assembling of the bushing takes place by forcing the buckling rubber ring into its housing seat, is greatly simplified with respect to the reciprocal forcing of metal parts.\",\n \"Furthermore, the mechanical working of said housing seat requires less limiting dimensional tolerances and therefore is less expensive.\",\n \"However, this kind of bushing has the disadvantage that it is not suitable for those application that require a high radial stiffness and the capacity of bearing great radial loads.\",\n \"As an example: if said bushings are used on haulage vehicles for the connection of the leaf springs of the back suspensions of the undercarriage of the vehicle, they may be applied only to vehicle with a capacity up to about 10 tons.\",\n \"A further inconvenience consists in that in the presence of great radial loads, the rubber ring is subject to relevant buckling in longitudinal direction that may cause expansion and stretching of the rubber, that reduce durability and reliability of said bushing.\",\n \"It is the aim of the present invention to eliminate all above mentioned inconveniences.\",\n \"In particular, it is the aim of the present invention to realize a bushing with elastic absorption of radial stress that allows the control of the radial compressibility, and that may sustain radial loads of considerable entity in a reliable manner, and that the realization thereof is convenient and the assembly easy.\",\n \"The aims set forth are reached by means of a bushing with elastic absorption of radial stress with a control means for the radial compressibility according to the present invention, comprising a cylindrical tubular element out of self-lubricating material onto which a ring is freely fitted out of a elastically budding material, characterized in that the internal surface of said ring out of elastically buckling material is associated to an annular reinforcing fitting out of a material that does not give radially.\",\n \"For making the coupling between the bushing and the circular seat into which it gets inserted as less expensive as possible, the outer surface of said ring out of elastically buckling material comprises bulge ribbings that cooperate in interference with the internal surface of said seat.\",\n \"Said ring out of elastically buckling material may be advantageously realized out of rubber, while said reinforcing annular insert is advantageously realized out of glass loaded nylon; its thickness is correlated to the entity of the radial charges to bear and is coupled by means of vulcanization to the internal surface of the rubber ring.\",\n \"Said bulge ribbings on the outer surface of the ring out of buckling material, according to the cited Utility Model of the same applicant, are advantageously placed at regular distances one from the other and in a longitudinal direction, parallel to the axis of the bushing.\",\n \"In an alternative, bulges with annular shape, parallel one to the other, might perform the same function.\",\n \"The main advantages obtained by means of the bushing with elastic absorption of radial stress with a control means for the radial compressibility according to the present invention consist in that said bushing is able to bear high radial loads due to the control of the radial buckling assured by the reinforcing insert, and in that the reinforcing insert, connected by vulcanization to the internal surface of said rubber ring, limits and contains the longitudinal buckling of said ring.\",\n \"A further advantage consists in that by suitably choosing the thickness of the internal reinforcing insert, it is possible to realize bushings with different degrees of radial stiffness, fitting to different application fields.\",\n \"The present invention will be explained more in detail hereinbelow relating to the enclosed drawings in which an embodiment is shown.\",\n \"FIG.\",\n \"1 shows a perspective view of a bushing with elastic absorption of radial stress With a control means for the radial compressibility, according to the present invention.\",\n \"FIG.\",\n \"2 shows said bushing in an axial section.\",\n \"FIGS.\",\n \"3 and 4 show said bushing in a top and a bottom view.\",\n \"Relating now to the drawings, the bushing with elastic absorption of radial stress with a control means for the radial compressibility according to the present invention mainly comprises an internal cylindrical tubular element 1 out of plastic self-lubricating material, onto which a rubber ring 2 is freely fitted.\",\n \"An annular reinforcement 3 of suitable thickness is fixed on the internal cylindrical surface of said rubber ring 2, and it is out of a material that does not give radially, like e.g.\",\n \"glass loaded nylon.\",\n \"The coupling between said ring 2 and said reinforcement 3 is advantageously realized by means of a vulcanization process.\",\n \"The external cylindrical surface of said ring 2 has a plurality of bulge ribbings 4 that locally increase the thickness of said ring 2.Furthermore, in the embodiment shown, element 1 and ring 2 have stop collars 5 and 6 that allow the bushing to bear the compression stress parallel to the axis of said bushing.\",\n \"In addition, it is possible to place a plane metal washer—not shown—of suitable thickness, similar to the one of collars 5 and 6, against said collar 6: said washer allows to distribute the longitudinal force transmitted to the bushing onto a greater surface and in a uniform manner, thus increasing the resistance of said bushing to said compression stress.\",\n \"It is evident that any variant of the bushing according to the present invention, of generic or simplified kind, will not be provided with said collars 5 and 6.The assembling of the bushing according to the present invention takes place by pushing the external surface of said rubber ring 2 into the housing seat of said bushing.\",\n \"During such operation, the bulge ribbings 4 get compressed and elastically buckled, while the space between one ribbing and the other allows a free lateral extension of the same.\",\n \"Thus the bushing will be fixed in its own seat by the friction between the buckled rubber bulges and the internal surface of the housing seat.\",\n \"The pin in freely rotating on the internal surface of said tubular element 1.During normal working, said rubber ring 2 absorbs crashes and radial stress transmitted by the pin to the bushing, while the annular reinforcement 3 of suitable thickness gives the bushing the required radial stiffness, limits its squeezing and the longitudinal deformation of the rubber ring 2.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471426"}}},{"rowIdx":593,"cells":{"text":{"kind":"list like","value":[["Method and compositions for evaluating risk of developing type 2 diabetes in people of chinese descent","Methods and compositions for identifying mutations and polymorphisms in mutant genes encoding gene product involved in insulin secretion, for example, hepatocyte nuclear factor-1∝, glucokinase, amylin and mitochondrial DNA are disclosed.","Specifically, a microchip comprising a combination of at least two different mutant genes wherein each gene comprises at least one mutation indicative of a predisposition for type-2 diabetes in a member of a Chinese population is disclosed.","A kit comprising the microchip, an isolated nucleic acid, primers and probes which are specifically used to screen or identify the mutations in genes of hepatocyte nuclear factor-1∝, glucokinase, amylin and mitochondrial DNA are also disclosed."],["1.A microchip comprising: a combination of at least two different mutant nucleic acid sequences of a wild-type nucleic acid sequence, wherein each wild-type nucleic acid sequence encodes a protein involved in insulin secretion, wherein said gene comprises at least one mutation indicative of a predisposition for type 2 diabetes in a member of a Chinese population.","2.The microchip according to claim 1, wherein said nucleic acid sequences comprise nucleic acid selected from the group consisting of genomic DNA, complementary DNA and messenger RNA.","3.The microchip according to claim 1, wherein said type 2 diabetes is maturity onset diabetes of the young.","4.The microchip according to claim 1, wherein said microchip further comprises a genetic marker that uniquely identifies a member of a Chinese population.","5.A microchip comprising: a combination of at least two different nucleic acid sequences, wherein each nucleic acid sequence encodes a gene product involved in insulin secretion wherein said gene comprises at least one mutation indicative of a predisposition for type 2 diabetes in a human subject of a Chinese population, wherein said gene product is selected from the group consisting of a glucokinase, a hepatocyte nuclear factor 1α, an amylin and a mitochondrial tRNA (Leu) (UUR).","6.A microchip comprising: at least one each of a combination of different nucleic acid sequences, wherein each nucleic acid sequence encodes a protein selected from the group consisting of glucokinase, hepatocyte nuclear factor la, amylin and mitochondrial tRNA(Leu)(UTR), wherein said glucokinase gene comprises at least one mutation selected from the group consisting of V101M, I110T, A119D, Q239R, and G385V, and said hepatocyte nuclear factor la gene comprises at least one mutation selected from the group consisting of G20R, A116V, IVS2nt-G→A, R203H, S432C, and I618M, and said amylin gene comprises the mutation S20G, and said mitochondrial tRNA(Leu)(UUR) gene comprises the mutation A3243G.","7.A microchip comprising at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:10.8.A microassay system comprising a microchip according to claim 1.9.A kit comprising a microchip according to claim 1.10.A nucleic acid primer comprised of SEQ ID NO: 34.11.A nucleic acid primer comprised of SEQ ID NO: 35.12.A nucleic acid primer comprised of SEQ ID NO: 36.13.A nucleic acid primer comprised of SEQ ID NO: 37.14.A nucleic acid probe that specifically anneals to a nucleic acid encoding a mutant gene of a wild-type gene involved in insulin secretion, wherein said mutant gene comprises at least one mutation indicative of increased risk for type 2 diabetes in a human subject of a Chinese population, and wherein said nucleic acid probe does not bind to said wild-type gene.","15.An isolated nucleic acid encoding a mutant gene of a wild-type gene that encodes a protein involved in the secretion of insulin, wherein said mutant gene comprises at least one mutation associated with increased risk for type 2 diabetes in a subject of a Chinese population.","16.The isolated nucleic acid according to claim 15, wherein said mutation is a single nucleotide polymorphism.","17.The isolated nucleic acid according to claim 15, wherein said mutation is selected from the group consisting of a missense, a nonsense, an insertion and a deletion mutation.","18.The isolated nucleic acid according to claim 15, wherein said wild-type gene encodes hepatocyte nuclear factor la, and said mutation is A116V.","19.The isolated nucleic acid according to claim 15, wherein said wild-type gene encodes glucokinase, and said mutation is selected from the group consisting of V101M and Q239R.","20.An isolated nucleic acid encoding a mutant gene of a wild-type gene that encodes a protein involved in the secretion of insulin, wherein said mutant gene is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 10.21.An isolated amino acid sequence encoded by a mutant gene of a wild-type gene encoding a protein involved in the secretion of insulin, wherein said mutant gene comprises at least on mutation associated with increased risk for type 2 diabetes in a member of a Chinese population.","22.An antibody that specifically binds a protein encoded by a mutant gene of a wild type gene encoding a protein involved in the secretion of insulin, wherein said mutant gene comprises at least on mutation associated with increased risk for type 2 diabetes in a member of a Chinese population, and wherein said antibody does not bind to a protein encoded by said wild-type gene.","23.A method of determining a genetic predisposition of a member of a Chinese population to develop type 2 diabetes, said method comprising the step of: contacting a sample comprising nucleic acid from said member with a combination of at least two nucleic acid sequences, wherein each nucleic acid sequence encodes a mutant gene of a wild-type gene encoding a protein involved in insulin secretion, wherein each mutant gene comprises at least one mutation indicative of a predisposition of a member of a Chinese population to develop type 2 diabetes, whereby identification of at least one of said mutations in said sample is indicative of a genetic predisposition for type 2 diabetes in said member of a Chinese population.","24.A method for detecting an increased risk of an individual of a Chinese population with decreased insulin secretory function to develop type 2 diabetes, said method comprising the step of: contacting a sample comprising nucleic acid from said individual with a combination of at least two different nucleic acid sequences, wherein each nucleic acid sequence encodes a mutant gene of a wild-type gene encoding a protein involved in insulin secretion, wherein each mutant gene comprises at least one mutation indicative of a predisposition of a member of a Chinese population to develop type 2 diabetes, wherein identification of at least one of said mutations in said sample is indicative of an increased risk for type 2 diabetes in said individual of a Chinese population.","25.The method according to claim 23, wherein said combination of at least two different nucleic acid sequences are attached to a microchip.","26.The method according to claim 23, wherein said nucleic acid sample is obtained from bodily fluid or tissue.","27.The method according to claim 23, wherein said wild-type gene encodes a gene product selected from the group consisting of hepatocyte nuclear factor 1α, glucokinase, amylin and mitochondrial tRNA(Leu)(UUR).","28.A method of determining a genetic predisposition of a member of a Chinese population to develop type 2 diabetes, said method comprising the step of: contacting a sample comprising nucleic acid from said member with a combination of at least two different nucleic acid sequences selected from the group consisting of G20R, A116V, IVS2nt-G→A, R203H, S432C, and I618M of hepatocyte nuclear factor 1α; V101M, I110T, A119D, Q239R, and G385V of glucokinase; S20G of amylin, and A3243G of mitochondrial tRNA(Leu)(UUR), wherein each nucleic acid sequence encodes a mutant gene of a wild-type gene encoding a protein involved in insulin secretion, wherein each mutant gene comprises at least one mutation indicative of a predisposition of a member of a Chinese population to develop type 2 diabetes, and wherein said identification of one of said mutations in said sample is indicative of a genetic predisposition for type 2 diabetes in said member of a Chinese population.","29.A method for detecting an increased risk of an individual of a Chinese population with decreased insulin secretory function to develop type 2 diabetes, said method comprising the step of: contacting a sample from said individual with a combination of at least two different nucleic acid sequences selected from the group consisting of G20R, A116V, IVS2nt-G→A, R203H, S432C, and I618M of hepatocyte nuclear factor 1α;V101M, I110T, A119D, Q239R, and G385V of glucokinase; S20G of amylin, and A3243G of mitochondrial tRNA(Leu)(UUR), wherein each nucleic acid sequence encodes a mutant gene of a wild-type gene encoding a protein involved in insulin secretion, wherein each mutant gene comprises at least one mutation indicative of a predisposition of an individual of a Chinese population to develop type 2 diabetes, and wherein the identification of at least one of said mutations in said sample is indicative of an increased risk for type 2 diabetes in said individual of a Chinese population.","30.A method for screening for genetic mutations in an individual of a Chinese population diagnosed with type 2 diabetes, said method comprising the steps of: contacting a sample from said individual with a combination of at least two different nucleic acid sequences, wherein each nucleic acid sequence encodes a mutant gene of a wild-type gene encoding a protein involved in insulin secretion, wherein each mutant gene comprises at least one mutation indicative of a predisposition of a member of a Chinese population to develop type 2 diabetes, and wherein identification of at least one of said mutations in said sample is indicative of an etiology of said type 2 diabetes in said individual of a Chinese population.","31.The method according to claim 30, wherein said individual has been diagnosed with maturity onset diabetes of the young.","32.The method according to claim 30, wherein said individual has at least one primary family member that has been diagnosed with maturity onset diabetes of the young.","33.The method according to claim 30, wherein said mutation is selected from the group consisting of a missense, a nonsense, an insertion and a deletion mutation.","34.A method for screening for genetic mutations indicative of increased risk of an individual of a Chinese population to develop type 2 diabetes, said method comprising the steps of: contacting a sample from said individual with a combination of at least two different nucleic acid sequences selected from the group consisting of G20R, A116V, IVS2nt-G→A, R203H, S432C, and I618M of hepatocyte nuclear factor 1α;V101M, I110T, A119D, Q239R, and G385V of glucokinase; S20G of amylin, and A3243G of mitochondrial tRNA(Leu)(UTR), wherein each nucleic acid sequence encodes a mutant gene of a wild-type gene encoding a protein involved in insulin secretion, wherein each mutant gene comprises at least one mutation indicative of a predisposition of an individual of a Chinese population to develop type 2 diabetes.","35.A method for screening for a genetic predisposition to develop type 2 diabetes in an individual of a Chinese population having at least one primary family member that has been diagnosed with type 2 diabetes, said method comprising the steps of: contacting a sample comprising nucleic acid from said individual with a combination of at least two different nucleic acid sequences, wherein each nucleic acid sequence encodes a mutant gene of a wild-type gene encoding a protein involved in insulin secretion, wherein each mutant gene comprises at least one mutation indicative of a predisposition of a member of a Chinese population to develop type 2 diabetes, and wherein identification of at least one of said mutations in said sample is indicative of a genetic predisposition to develop type 2 diabetes in said individual of a Chinese population."],["<SOH> BACKGROUND <EOH>Although people of Chinese ancestry account for >20% of the world's population (Chan, et al(1997) 20: 1785), very little is known about the genetic factors that contribute to the development of diabetes in this population.","The prevalence of diabetes amongst Chinese people varies from <1% in some rural areas in mainland China to 6-12% in Hong Kong, Singapore, and Taiwan (Chan, et al (1997), supra).","Hong Kong can be regarded as a paradigm of future China.","The prevalence of diabetes mellitus is reaching epidemic proportions amongst Hong Kong Chinese, with type 2 diabetes being the predominant form in pateints with early-or late-onset of disease (Chan and Cockram (1997) Diabetes Care 20: 1785).","Type 2 diabetes mellitus is a heterogeneous disease that is caused by both genetic and environmental factors.","The age-adjusted prevalence of diabetes in the Chinese population has increased from 7.7% in 1990 (Cockram, et al (1993) Diabetes Res and Clin Practice 21: 67) to 8.9% in 1995 (Cockram and Chan (1999) In: Diabetes in the New Millennium, Pot Still Press, Sydney, pp.","11-22).","In a population-based study conducted in 1995, the crude prevalence of diabetes mellitus was 9.6%, rising from 1.7% in those aged under 40 years to 25% in those older than 60 years (Janus (1997) Clin Exp Pharmacol Physiol 24: 987).","There is a high prevalence of obesity (43%) and positive family history of diabetes (50%) in Chinese patients presenting with acute or early onset diabetes (Chan, et al (1993) Postgrad Med J 69: 204; Ko, et al (1998) 35: 761).","These findings indicate that genetic factors, in addition to environmental factors, can be an important cause of early onset diabetes in this population.","Because type 2 diabetes is an insidious disease, it is estimated that as many as half of the individuals in Hong Kong that would be considered diabetic remain undiagnosed.","Most patients are finally diagnosed only when presenting with overt symptoms that often are the consequence of advanced disease.","Clinic as well as population-based studies reveal that about 17% of diabetic patients in Hong Kong are diagnosed before age 35 years (Chan, et al (1993) Postgrad Med 69: 204; Janus (1996) The Hong Kong cardovascular risk factor prevalence study 1995-1996 Dept of Clin Biochem, Queen Mary Hospital of Hong Kong, Hong Kong, 1997).","Due to their anticipated long duration of disease, it is important to classify and characterize the nature of diabetes in these young patients to facilitate early diagnosis and appropriate treatments.","Current methods of diagnosing type 2 diabetes generally involve assessing phenotypic parameters, such as measuring fasting serum glucose levels by administering an oral glucose tolerance test (OGTT) to determine impaired glucose tolerance (IGT) or impaired fasting glucose (IFG).","Phenotypic assessments of persons suspected of having type 2 diabetes are important, but they are limited in that patients generally receive a diagnosis only after presentation with overt symptoms.","Furthermore, because the common symptoms of type 2 diabetes are a consequence of a combination heterogenous genetic and environmental causes, the therapies provided are general with regard to the disease rather than targetted to the specific etiology of the individual patient.","Numerous studies have attempted to correlate the increased risk for development of type 2 diabetes with a mutation of a specific gene, but the results of these studies repeatedly demonstrate that no one mutated gene can be attributed as the major cause of type 2 diabetes, emphasizing the heterogeneous nature of this disease.","Furthermore, a mutation in a particular gene that correlates with increased risk for developing type 2 diabetes in individuals of one ethnic population is not relevant to individuals of a second ethnic population, wherein the risk for type 2 diabetes in individuals of the second ethnic population will correlate with a different mutation or a mutation in a completely different gene.","It is therefore of interest to identify additional genetic mutations and polymorphisms that are indicative of an increased risk for developing type 2 diabetes in people of Chinese ancestry, and to develop methods that can be effectively employed to prophylactically identify asymptomatic Chinese individuals with a genetic predisposition for type 2 diabetes.","Relevant Literature Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes characterized by autosomal dominant inheritance, early onset (usually before 25 years of age) and a primary defect in pancreatic β-cell function (Fajans (1990) Diabetes Care 13: 49; Chan, et al (1990) Diabetic Med 7: 211; Byrne, et al (1996) Diabetes 45: 1503).","This form of diabetes can result from mutations in at least five different genes including those encoding the glycolytic enzyme glucokinase (Froguel, et al (1993) New Engl J Med 328: 697), the liver-enriched transcription factors expressed in the pancreatic β-cell, which are hepatocyte nuclear factors HNF-1α (Yamagata, et al (1996) Nature 384: 455), HNF-1β (Horikawa, et al (1997) Nature Genet 17: 384), and HNF-4α (Yamagata, et al (1996) Nature 384: 458), and insulin promoter factor-1 (IPF-1) (Stoffers, et al (1997) Nature Genet 17: 138).","Some mutations and polymorphisms in the glucokinase and HNF-1α genes that are associated with the genetic predisposition of a Chinese individual to develop type 2 diabetes mellitus have been initially identified in Ng, et al ( Diabetic Medicine 1999, 16: 956, herein incorporated by reference), but this manuscript does not disclose how these mutations and polymorphisms might be used to identify Chinese individuals with increased risk of developing type 2 diabetes.","U.S. Pat.","No.","5,541,060 discloses the results of screening a cohort of sixteen French families having MODY and the identification of several missense mutations in the glucokinase gene, however none of the mutations identified are relevant to individuals of Chinese descent.","U.S. Pat.","No.","5,800,998 discloses a point mutation at nucleotide 414 of human HNF 1α, but this single point mutation is not associated with a genetic predisposition of a Chinese individual to develop type 2 diabetes.","Major susceptibility loci for non-insulin dependent diabetes have been identified through genome scans of individuals in Mexican-American (Hanis, et al (1996) Nature Genet 13: 161) and Finnish (Mahtani, et al (1996) Nature Genet 14: 90) populations, but not in individuals of a Chinese population.","Specific microsatellite regions of genomic DNA can be correlated with major susceptibility loci that closely associate with the increased risk of a Chinese subject to develop type 2 diabetes.","For instance, Le Stunff, et al ( Nature Genet.","(2000) 26: 444) have reported that particular alleles of the insulin gene variable number of tandem repeat (VNTR) locus are associated with obesity and type 2 diabetes.","Also, microsatellite polymorphisms flanking the glucokinase have been associated with type 2 diabetes in a Taiwanese population (Wu, et al (1995) Diabetes Res Clin Pract 30: 21)."],["<SOH> SUMMARY OF THE INVENTION <EOH>Compositions and methods are provided, wherein a unique combination of genetic markers indicative of a genetic predisposition for developing type 2 diabetes in members of a Chinese population is described.","The invention is exemplified by a combination of mutated gene sequences from wild-type genes that are involved in insulin secretory function, including hepatocyte nuclear factor 1α (HNF-1α), glucokinase, amylin and mitochondrial DNA.","The combination of representative mutations include G20R, A116V, IVS2nt→GA, R203H, S432C and 1618M of HNF-1α; V101M, 1110T, A119D, Q239R and G385V of glucokinase; S20G of amylin; and A3243G of mitochondrial tRNA Leu(UUR) .","The combination of the mutated genes of interest will be most efficiently used for screening individuals at increased risk by attaching them to a microchip.","Embodiments of methods for determining or detecting the genetic predisposition of a Chinese individual to develop type 2 diabetes include obtaining a sample containing genomic nucleic acid from a Chinese patient, such as a tissue biopsy or a blood sample, and contacting that sample with a representative combination of at least two mutated genes of interest, then subjecting the sample DNA together with the patient's DNA to hybridization conditions stringent enough to detect nucleotide differences of at least one base pair.","Alternatively, particular genes of interest from the genomic DNA of a Chinese individual at risk are screened using PCR primer pairs and PCR-RFLP techniques to identify the presence or absence of a mutation known to be associated with type 2 diabetes.","The methods further encompass screening the genomic DNA of Chinese individuals who have been diagnosed with type 2 diabetes or who have a primary family member with type 2 diabetes for additional associative mutations in identified genes or for mutations correlative with the predisposition of a member of a Chinese population to develop type 2 diabetes in additional candidate genes, such as those associated with diabetic kidney disease and obesity.","The invention further provides for nucleic acid primers and probes that are specifically used to identify mutations, for instance by PCR or hybridization, of wild-type genes involved in insulin secretion that are associated with an increased risk of a Chinese subject to develop type 2 diabetes.","Additionally, proteins translated from genes carrying at least one mutation associated with increased risk of a Chinese individual to develop type 2 diabetes find use in functional diagnostic assays and in the production of diagnostic antibodies that bind to the mutant but not the wild-type protein.","The prophylactic detection of mutations and polymorphisms that are indicative of a genetic predisposition of a Chinese individual to develop type 2 diabetes finds application in providing clinicians with information that allows for early detection and therapy initiation before the onset of overt symptoms or complications, and that enables clinicians to administer specifically targetted therapies that address the etiology of an individual's disease."],["FIELD OF THE INVENTION This subject invention relates to the identification and use of mutations and polymorphisms in mutant genes of wild-type genes involved in insulin secretory function that are associated with the increased risk of a Chinese individual to develop type 2 diabetes.","The invention is exemplified by a combination of mutations, uniquely identified in Chinese individuals with a positive family history of type 2 diabetes, in the genes encoding hepatocyte nuclear factor-1∝, glucokinase, amylin and mitochondrial DNA.","The combination of mutated genes finds use in screening Chinese individuals at risk of developing type 2 diabetes and in providing physicians with information to enable them to apply patient tailored therapies.","BACKGROUND Although people of Chinese ancestry account for >20% of the world's population (Chan, et al(1997) 20: 1785), very little is known about the genetic factors that contribute to the development of diabetes in this population.","The prevalence of diabetes amongst Chinese people varies from <1% in some rural areas in mainland China to 6-12% in Hong Kong, Singapore, and Taiwan (Chan, et al (1997), supra).","Hong Kong can be regarded as a paradigm of future China.","The prevalence of diabetes mellitus is reaching epidemic proportions amongst Hong Kong Chinese, with type 2 diabetes being the predominant form in pateints with early-or late-onset of disease (Chan and Cockram (1997) Diabetes Care 20: 1785).","Type 2 diabetes mellitus is a heterogeneous disease that is caused by both genetic and environmental factors.","The age-adjusted prevalence of diabetes in the Chinese population has increased from 7.7% in 1990 (Cockram, et al (1993) Diabetes Res and Clin Practice 21: 67) to 8.9% in 1995 (Cockram and Chan (1999) In: Diabetes in the New Millennium, Pot Still Press, Sydney, pp.","11-22).","In a population-based study conducted in 1995, the crude prevalence of diabetes mellitus was 9.6%, rising from 1.7% in those aged under 40 years to 25% in those older than 60 years (Janus (1997) Clin Exp Pharmacol Physiol 24: 987).","There is a high prevalence of obesity (43%) and positive family history of diabetes (50%) in Chinese patients presenting with acute or early onset diabetes (Chan, et al (1993) Postgrad Med J 69: 204; Ko, et al (1998) 35: 761).","These findings indicate that genetic factors, in addition to environmental factors, can be an important cause of early onset diabetes in this population.","Because type 2 diabetes is an insidious disease, it is estimated that as many as half of the individuals in Hong Kong that would be considered diabetic remain undiagnosed.","Most patients are finally diagnosed only when presenting with overt symptoms that often are the consequence of advanced disease.","Clinic as well as population-based studies reveal that about 17% of diabetic patients in Hong Kong are diagnosed before age 35 years (Chan, et al (1993) Postgrad Med 69: 204; Janus (1996) The Hong Kong cardovascular risk factor prevalence study 1995-1996 Dept of Clin Biochem, Queen Mary Hospital of Hong Kong, Hong Kong, 1997).","Due to their anticipated long duration of disease, it is important to classify and characterize the nature of diabetes in these young patients to facilitate early diagnosis and appropriate treatments.","Current methods of diagnosing type 2 diabetes generally involve assessing phenotypic parameters, such as measuring fasting serum glucose levels by administering an oral glucose tolerance test (OGTT) to determine impaired glucose tolerance (IGT) or impaired fasting glucose (IFG).","Phenotypic assessments of persons suspected of having type 2 diabetes are important, but they are limited in that patients generally receive a diagnosis only after presentation with overt symptoms.","Furthermore, because the common symptoms of type 2 diabetes are a consequence of a combination heterogenous genetic and environmental causes, the therapies provided are general with regard to the disease rather than targetted to the specific etiology of the individual patient.","Numerous studies have attempted to correlate the increased risk for development of type 2 diabetes with a mutation of a specific gene, but the results of these studies repeatedly demonstrate that no one mutated gene can be attributed as the major cause of type 2 diabetes, emphasizing the heterogeneous nature of this disease.","Furthermore, a mutation in a particular gene that correlates with increased risk for developing type 2 diabetes in individuals of one ethnic population is not relevant to individuals of a second ethnic population, wherein the risk for type 2 diabetes in individuals of the second ethnic population will correlate with a different mutation or a mutation in a completely different gene.","It is therefore of interest to identify additional genetic mutations and polymorphisms that are indicative of an increased risk for developing type 2 diabetes in people of Chinese ancestry, and to develop methods that can be effectively employed to prophylactically identify asymptomatic Chinese individuals with a genetic predisposition for type 2 diabetes.","Relevant Literature Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes characterized by autosomal dominant inheritance, early onset (usually before 25 years of age) and a primary defect in pancreatic β-cell function (Fajans (1990) Diabetes Care 13: 49; Chan, et al (1990) Diabetic Med 7: 211; Byrne, et al (1996) Diabetes 45: 1503).","This form of diabetes can result from mutations in at least five different genes including those encoding the glycolytic enzyme glucokinase (Froguel, et al (1993) New Engl J Med 328: 697), the liver-enriched transcription factors expressed in the pancreatic β-cell, which are hepatocyte nuclear factors HNF-1α (Yamagata, et al (1996) Nature 384: 455), HNF-1β (Horikawa, et al (1997) Nature Genet 17: 384), and HNF-4α (Yamagata, et al (1996) Nature 384: 458), and insulin promoter factor-1 (IPF-1) (Stoffers, et al (1997) Nature Genet 17: 138).","Some mutations and polymorphisms in the glucokinase and HNF-1α genes that are associated with the genetic predisposition of a Chinese individual to develop type 2 diabetes mellitus have been initially identified in Ng, et al (Diabetic Medicine 1999, 16: 956, herein incorporated by reference), but this manuscript does not disclose how these mutations and polymorphisms might be used to identify Chinese individuals with increased risk of developing type 2 diabetes.","U.S. Pat.","No.","5,541,060 discloses the results of screening a cohort of sixteen French families having MODY and the identification of several missense mutations in the glucokinase gene, however none of the mutations identified are relevant to individuals of Chinese descent.","U.S. Pat.","No.","5,800,998 discloses a point mutation at nucleotide 414 of human HNF 1α, but this single point mutation is not associated with a genetic predisposition of a Chinese individual to develop type 2 diabetes.","Major susceptibility loci for non-insulin dependent diabetes have been identified through genome scans of individuals in Mexican-American (Hanis, et al (1996) Nature Genet 13: 161) and Finnish (Mahtani, et al (1996) Nature Genet 14: 90) populations, but not in individuals of a Chinese population.","Specific microsatellite regions of genomic DNA can be correlated with major susceptibility loci that closely associate with the increased risk of a Chinese subject to develop type 2 diabetes.","For instance, Le Stunff, et al (Nature Genet.","(2000) 26: 444) have reported that particular alleles of the insulin gene variable number of tandem repeat (VNTR) locus are associated with obesity and type 2 diabetes.","Also, microsatellite polymorphisms flanking the glucokinase have been associated with type 2 diabetes in a Taiwanese population (Wu, et al (1995) Diabetes Res Clin Pract 30: 21).","SUMMARY OF THE INVENTION Compositions and methods are provided, wherein a unique combination of genetic markers indicative of a genetic predisposition for developing type 2 diabetes in members of a Chinese population is described.","The invention is exemplified by a combination of mutated gene sequences from wild-type genes that are involved in insulin secretory function, including hepatocyte nuclear factor 1α (HNF-1α), glucokinase, amylin and mitochondrial DNA.","The combination of representative mutations include G20R, A116V, IVS2nt→GA, R203H, S432C and 1618M of HNF-1α; V101M, 1110T, A119D, Q239R and G385V of glucokinase; S20G of amylin; and A3243G of mitochondrial tRNALeu(UUR).","The combination of the mutated genes of interest will be most efficiently used for screening individuals at increased risk by attaching them to a microchip.","Embodiments of methods for determining or detecting the genetic predisposition of a Chinese individual to develop type 2 diabetes include obtaining a sample containing genomic nucleic acid from a Chinese patient, such as a tissue biopsy or a blood sample, and contacting that sample with a representative combination of at least two mutated genes of interest, then subjecting the sample DNA together with the patient's DNA to hybridization conditions stringent enough to detect nucleotide differences of at least one base pair.","Alternatively, particular genes of interest from the genomic DNA of a Chinese individual at risk are screened using PCR primer pairs and PCR-RFLP techniques to identify the presence or absence of a mutation known to be associated with type 2 diabetes.","The methods further encompass screening the genomic DNA of Chinese individuals who have been diagnosed with type 2 diabetes or who have a primary family member with type 2 diabetes for additional associative mutations in identified genes or for mutations correlative with the predisposition of a member of a Chinese population to develop type 2 diabetes in additional candidate genes, such as those associated with diabetic kidney disease and obesity.","The invention further provides for nucleic acid primers and probes that are specifically used to identify mutations, for instance by PCR or hybridization, of wild-type genes involved in insulin secretion that are associated with an increased risk of a Chinese subject to develop type 2 diabetes.","Additionally, proteins translated from genes carrying at least one mutation associated with increased risk of a Chinese individual to develop type 2 diabetes find use in functional diagnostic assays and in the production of diagnostic antibodies that bind to the mutant but not the wild-type protein.","The prophylactic detection of mutations and polymorphisms that are indicative of a genetic predisposition of a Chinese individual to develop type 2 diabetes finds application in providing clinicians with information that allows for early detection and therapy initiation before the onset of overt symptoms or complications, and that enables clinicians to administer specifically targetted therapies that address the etiology of an individual's disease.","BRIEF DESCRIPTION OF DRAWINGS FIG.","1A shows the nucleic acid sequence of human nuclear factor 1a (HNF-1α) exon 1 with the G20R mutation (SEQ ID NO: 1).","The wild-type sequence is GenBank number U72612.FIG.","1B shows the nucleic acid sequence HNF-1α exon 2 with the A116V mutation (SEQ ID NO: 2).","The wild-type sequence is GenBank number U72613.FIG.","2 shows the nucleic acid sequence of HNF-1α exons 3 and 4 depicting the splice acceptor site mutation IVS2nt-1G→A (SEQ ID NO: 3) and the missense mutation R203H (SEQ ID NO: 4).","The wild-type sequence is GenBank number U72614.FIG.","3A shows the nucleic acid sequence of HNF-1α exons 5 and 6 with the S432C mutation (SEQ ID NO : 5).","The wild-type sequence is GenBank number U72615.FIG.","3B shows the nucleic acid sequence of HNF-1α exon 10 with the I618M mutation (SEQ ID NO: 6).","The wild-type sequence is GenBank number U72618.FIG.","4A shows the nucleic acid sequence of human glucokinase exon 3, depicting the mutations V101M (SEQ ID NO: 7), I110T (SEQ ID NO: 8) and A119D (SEQ ID NO: 9).","The wild-type sequence is GenBank number AF041016.FIG.","4B shows the nucleic acid sequence of human glucokinase exon 7 with the Q239R mutation (SEQ ID NO: 10).","The wild-type sequence is GenBank AF041019.FIG.","4C shows the nucleic acid sequence of human glucokinase exon 9 with the G385V mutation (SEQ ID NO: 11).","The wild-type sequence is GenBank number AF041021.FIG.","5 shows the nucleic acid sequence of the human amylin gene exon 3 with the S20G mutation (SEQ ID NO: 12).","The wild-type sequence is GenBank number X52819.FIG.","6 shows the nucleic acid sequence base pairs 3001-3480 of the human mitochondrion complete genome, depicting the A3243G mutation (SEQ ID NO: 13).","The wild-type sequence is GenBank number J01415.FIG.","7 shows the pedigrees of families with mutations/polymorphisms in the glucokinase (HK84) or HNF-1α gene (HK10 and HK54).","Individuals with diabetes are noted by filled symbols; individuals with impaired fasting glucose (IFG) or impaired glucose tolerance (IGT) by grey symbols; non-diabetic individuals by open symbols and untested by hatched symbols.","The arrow indicates the proband.","Present age, age at diagnosis and genotype of glucokinase or HNF-1α of tested individuals are noted: N, normal; M, mutation/polymorphism.","FIG.","8 shows the pedigrees of families with an mt3243 mutation.","Individuals with diabetes are noted by filled symbols, IGT by grey symbols, non-diabetic individuals by open symbols, and untested individuals by hatched symbols.","The arrow indicates the proband.","Present age, age of diagnosis, audiogram and genotype are also shown.","N, normal; M, mutant allele.","FIG.","9A-9J show the pedigrees of 10 families carrying the HNF-1α (9A-9B), glucokinase (9C-9E), mt3243 (9F-9H) or amylin S20G (9I-9J) gene mutations/polymorphisms.","Subjects with diabetes are noted by black symbols, subjects with IFG or IGT by grey symbols, non-diabetic and untested subjects by open symbols.","The genotype of the family members is indicated by: N, wild-type allele; and M, mutant/variant allele.","Present age, age at diagnosis, therapy and complications are stated in this order.","The proband is indicated by an arrow.","Abbreviations: Oral, oral drugs; Ins, insulin; R, retinopathy; K, albuminuria; U, neuropathy; H, hearing impairment.","FIG.","10 shows the pedigree of a Chinese family with HNF-1α IVS2nt-1G→A mutation.","Subjects with diabetes are represented by black symbols, subjects with IGT by grey symbols and untested ones by open symbols.","The genotype of family members is indicated: N, normal allele; and M, mutant allele.","The proband is indicated by an arrow.","CP, C-peptide; GST, glucagon stimulation test; Complications: R, retinopathy; K, nephropathy; U, neuropathy.","DESCRIPTION OF THE SPECIFIC EMBODIMENTS Compositions and methods are provided, wherein a unique combination of genetic markers indicative of a genetic predisposition for developing type 2 diabetes in members of a Chinese population is described.","The invention comprises as compositions: (1) a combination of nucleic acid sequences from wild-type genes that encode proteins important for insulin secretory function, each nucleic acid sequence having a mutation uniquely associated with the genetic predisposition of a Chinese individual to develop type 2 diabetes, (2) nucleic acid sequences encoding glucokinase and HNF-1α and carrying previously unreported mutations indicative of the increased risk of a Chinese person to develop type 2 diabetes, (3) a microchip having attached to it at least two of the mutated genes of interest, and (4) nucleic acid primers used to detect the unique mutations in the genes of interest.","The methods involve: (1) obtaining genomic DNA from a Chinese subject, (2) combining the genomic DNA with either a combination of the mutated nucleic acids of interest or a combination of primers used to identify the presence or absence of a mutation in a gene of interest, and (3) detecting for the presence or absence of mutations, either by identifying mismatches between the patient's DNA and a wild-type or mutant nucleic acid sequence by hybridization techniques, or by amplifying regions of the patients DNA that contain putative mutations by PCR, and subjecting the amplicons to restriction endonucleases and/or DNA sequencing.","Advantages of the present invention include that the method of screening uses genetic markers shown to cosegregate with type 2 diabetes in persons of Chinese ancestry to assess whether a given patient is at increased risk for developing type 2 diabetes.","The mutations and polymorphisms used for screening are specifically applicable to individuals of Chinese descent.","As a further advantage, the screening can be based upon the presence or absence of a combination of at least two different mutations or polymorphisms to provide for even more accurate and reliable evaluations because the contributing factors to development of type 2 diabetes are heterogeneous.","Identification of particular mutations or polymorphisms in an individual offers the advantage that with this information physicians are able to provide more specific and appropriate therapies for individual patients, and to guide a patient in making lifestyle adjustments to ameliorate or delay symptoms of diabetes and associated complications.","Because type 2 diabetes is often an insidious disease, representative combinations of genetic markers indicative of a predisposition in a Chinese individual to develop the disease can be used to screen populations of individuals who may be at increased risk for developing type 2 diabetes so that they can be given appropriate therapy before overt diabetic symptoms or complications are realized.","Likewise, family members of an individual diagnosed with type 2 diabetes can be screened for the particular mutants/polymorphisms of the affected individual to quickly identify family members also at increased risk of developing type 2 diabetes.","By a member of a Chinese population is intended to include any individual of Chinese ancestry.","In certain cases, for instance when a mutation in a gene involved in the secretion of insulin is dominant for increasing the risk of a Chinese individual to develop type 2 diabetes, a member of a Chinese population will encompass those individuals with at least one parent of Chinese descent.","A member of a Chinese population may be more specifically identified by HLA haplotyping.","For example, HLA class I and class II frequencies among a Hong Kong Chinese population have been studied by Chang and Hawkins (Hum Immunol (1997) 56: 125).","Numerous studies have been carried out to determine HLA class I and class II alleles that are more frequently or even uniquely found in members of a Chinese population, and alleles with strong associations.","Shaw et al and Shen et al have studied HLA polymorphism and allele frequency and association of Chinese populations in Taiwan (Tissue Antigens (1997) 50: 610; Tissue Antigens (1999) 53: 51; J Formos Med Assoc (1999) 98: 11).","Allele frequency and associations found in Chinese individuals of mainland China have been reported by Trejaut et al (Eur J Immunogenet (1996) 23: 437), Shieh, et al (Transfusion (1996) 36: 818), Zhao et al (Eur J Immunogenet (1993) 20: 293), Wang, et al (Tissue Antigens (1993) 41: 223; Hum Immunol (1992) 33: 129), Lee, et al (Eur J. Immunogenet (1999) 26: 275), and Gao et al (Hum Immunol (1991) 32: 269; Tissue Antigens (1991) 38: 24; Immunogenetics (1991) 34: 401).","Additionally, a Chinese individual may be objectively defined by “DNA fingerprinting” techniques well known to those in the art, where microsatellite, short tandem repeat (STR) and variable number tandem repeat (VNTR) loci specific to individuals of Chinese descent are identified.","Numerous examples of such ethnic genotyping studies have been reported (Meng, et al (1999) J Forensic Sci 44: 1273; Yoshimoto, et al (1999) Int J Legal Med 113: 15; Wu, et al (1999) J Forensic Sci 44: 1039; Evett, et al (1996) Am J Hum Genet 58: 398; Gill and Evett (1995) Genetica 96: 69; Balazs (1993) EXS 67: 193; Lan, et al (1992) Arch Kriminol 189: 169; and Hwu, et al (1992) J Formos Med Assoc 91: 839).","All of these above references are incorporated herein by reference.","Whereas insulin resistance is a strong predictor of type 2 diabetes, it is not sufficient for manifestation of the disease (So, et al (2000) Hong Kong J. Med 6: 69-76).","A relative insulin deficiency is essential to the development of hyperglycemia, setting up a vicious cycle wherein elevated glucose levels are toxic to pancreatic β-cells, thereby inducing insulin resistance and decreased β-cell secretory function.","Based in the intrinsic interconnection between insulin secretion and action, the invention is exemplified by a combination of mutated gene sequences from wild-type genes that are involved in insulin secretory function, including hepatocyte nuclear factor 1α (HNF-1α), glucokinase, amylin and mitochondrial DNA.","By “genes involved in insulin secretory function” and “genes involved in insulin secretion” is intended genes in which a heterozygous mutation has a dominant-negative effect on normal pancreatic β-cell secretory function.","The invention is primarily concerned with a representative array of gene markers, the combination of which is uniquely indicative of the genetic predisposition of a member of a Chinese population to develop type 2 diabetes (referred to hereinafter as “genes of interest”).","The combination of representative mutations is exemplified by G20R, A116V, IVS2nt→GA, R203H, S432C and 1618M of HNF-1α; V101M, I110T, A119D, Q239R and G385V of glucokinase; S20G of amylin; and A3243G of mitochondrial tRNALeu(UUR)”.","The mutation IVS2nt→GA represents a splice acceptor site mutation that likely results in a truncated translation product.","A representative combination of the mutated genes of interest finds particular use in the prophylactic screening of (i) Chinese individuals who have been diagnosed with maturity onset diabetes of the young (MODY) to determine the etiology of their disease, (ii) Chinese individuals that have a positive family history of type 2 diabetes to determine their likelihood of developing diabetic symptoms, and (iii) and Chinese individuals deemed to be at greater risk of developing diabetic symptoms because of correlative phenotypic characteristics (i. e. obese individuals).","The combination of the mutated genes of interest will be most efficiently used for screening-individuals at increased risk by attaching them to a microchip or other solid support.","A specific kind of microchip is not critical, except that it must be able to present a representative array of at least two different nucleic acid sequences, each with a mutation or polymorphism indicative of the increased risk of a Chinese individual to develop type 2 diabetes.","Additionally, it will be useful to attach representative wild-type nucleic acid sequences to the chip as comparative controls.","The microarray will normally involve a plurality of different nucleic acid sequences, usually be at least 10, more usually at least 20, frequently at least 50, but may have as many as 100 or more.","Chips that will find use with the present invention are known in the art (for example, see U.S. Pat.","Nos.","5,741,644, 5,837,832 and 6,183,970).","Additionally, other solid substrates may be used for the covalent attachment of representative combinations of mutated nucleic acid sequences of interest, including beads and slides.","Solid supports can be made out of glass or silicon oxide or other materials that can be adapted to be covalently attached to oligonucleotide sequences by the introduction of functionalities which react with oligonucleotides.","One may use a variety of approaches to bind the nucleic acid to the solid substrate.","By using chemically reactive solid substrates, one may provide for a chemically reactive group to be present on the nucleic acid, which will react with the chemically active solid substrate surface.","For example, by using silicate esters, halides or other reactive silicon species on the surface, the nucleic acid may be modified to react with the silicon moiety.","One may form silicon esters for covalent bonding of the nucleic acid to the surface.","Instead of silicon functionalities, one may use organic addition polymers.","e. g. styrene, acrylates and methacrylates, vinyl ethers and esters, and the like, where functionalities are present which can react with a functionality present on the nucleic acid.","For example, amino groups, activated halides, carboxyl groups, mercaptan groups, epoxides, and the like, may be provided in accordance with conventional ways.","The linkages may be amides, amidines, amines, esters, ethers, thioethers, dithioethers, and the like.","Methods for forming these covalent linkages may be found in U.S. Pat.","No.","5,565,324 and U.S. Pat.","No.","6,156,501.The invention also contemplates a microassay system and a kit that comprises a solid support having attached to it a representative array of nucleic acid sequences, each with a mutation or polymorphism associated with the genetic disposition of a Chinese individual to develop type 2 diabetes.","The microassay system or kit would contain, for example, a microchip or beads to which are attached wild-type and mutant nucleic acid sequences from genes that encode proteins involved in insulin secretory function, preferentially wild-type and mutant sequences from HNF-1α, glucokinase, amylin and mitochondrial DNA.","Additionally, mutant and wild-type sequences known to hybridize and known not to hybridize under stringent conditions to those sequences immobilized on the support would be included as positive and negative controls, respectively.","A microassay system or kit with nucleic sequences immobilized on a solid support would involve screening by hybridization detection (fluorescent or radioactive signal upon duplex formation).","Alternatively, another microassay system or kit would include primer pairs that anneal to nucleic acid sequences encoding proteins involved in insulin secretion.","The primer pairs specifically anneal to flanking regions of the genes that putatively contain mutations associated with type 2 diabetes, such that PCR amplification with such primers would reveal the presence or absence of an associative mutation of interes.","Such a kit or microassay system would also contain representative mutant and wild-type sequences as controls, and screening would be carried out using PCR and sequencing or through PCR restriction fragment length polymorphism analysis (RFLP) and electrophoresis.","Type 2 diabetes is a heterogeneous disease, and no single mutation or single mutated gene can be fully attributed to the manifestation of its symptoms.","Therefore, a combination of at least two different nucleic acid sequences encoding mutations or polymorphisms of closely associated with increased risk of a person of Chinese descent to develop type 2 diabetes is attached to a microchip or is individually screened.","By “at least two different nucleic acid sequences” is intended two different nucleic acid sequences from the same wild-type gene having different mutations, or two different mutant nucleic acid sequences from two different wild-type genes.","Preferably, at least one of the mutant; sequences A116V of HNF-1α (SEQ ID NO: 2), V101M (SEQ ID NO: 7) or Q239R (SEQ D NO: 10) are attached to the microchip or solid support.","By wild-type gene is intended one that is not associated with type 2 diabetes, and this would include any allelic variant of the wild-type gene, at any frequency, and that encodes a protein that functions in its expected manner without inducing pathological symptoms.","By mutant gene is intended one whose sequence has been modified by insertions, deletions, or substitutions of at least one nucleic acid base pair, wherein the modification may result in detectable changes in the expression or function of the mutant gene product as compared to the wild-type gene product.","In the genes of interest for the invention, a mutant gene is associated with type 2 diabetes.","The nucleic acid sequences may be from genomic DNA, complementary DNA (cDNA) or from messenger RNA (mRNA).","They may be synthetic or isolated from human bodily tissue or fluid.","The mutations preferably occur, but do not need to occur, in a translated region of a nucleic acid sequence that encodes a protein that in wild-type form is involved in glucose metabolism or insulin secretion.","Within a translated nucleic acid sequence, a mutation can be a missense mutation, replacing one amino acid with another amino acid, or a nonsense mutation, replacing an amino acid with a stop codon.","Mutations can also be insertions or deletions of at least one nucleic acid in either a coding or in non-coding region, such as a region that controls the transcription of a gene, including promoters, enhancers, response elements, signal sequences and polyadenylation signals, and the like.","Single nucleotide polymorphisms (SNPs), preferably but not necessarily occurring within the translated regions of nucleic acid sequences that encode proteins involved in glucose metabolism or insulin secretion and that correlate with increased risk of type 2 diabetes are also contemplated by the present invention.","Such SNPs can be identified by correlating mutations in known genes that cosegregate with development of type 2 diabetes in members of families with a positive history of the disease.","Additionally, SNPs that occur in non-translated and translated regions can be identified through genome-wide scans and correlate linkage analyses of family pedigrees.","The use of microarray technologies also can be conveniently applied to identifying SNPs of interest.","Embodiments of methods for determining or detecting the genetic predisposition of a Chinese individual to develop type 2 diabetes include obtaining a sample containing genomic nucleic acid from a Chinese patient, such as tissue from autopsy or biopsy, or a blood sample, and contacting that sample with a representative combination of at least two mutated genes of interest, then subjecting the sample DNA together with the patient's DNA to hybridization conditions stringent enough to detect nucleotide differences of at least one base pair.","Alternatively, particular genes of interest from the genomic DNA of a Chinese individual at risk are subjected to restriction fragmentation and then screened using PCR primer pairs and PCR-RFLP techniques to identify the presence or absence of a mutation known to be associated with type 2 diabetes.","The methods further encompass screening the genomic DNA of Chinese individuals that have been diagnosed with type 2 diabetes or that have a primary family member with type 2 diabetes for additional associative mutations in identified genes or for mutations correlative with the predisposition of a member of a Chinese population to develop type 2 diabetes in additional candidate genes, such as those associated with diabetic kidney disease and obesity.","Mutations are most efficiently identified in Chinese families with a positive history of developing type 2 diabetes (i. e. families with members that develop MODY).","However, identified associative mutations are useful for identifying the increased risk in any member of a Chinese population.","In practicing a method of identifying the mutations associated with the genotype of a Chinese individual who is at increased risk for developing type 2 diabetes, Chinese subjects with (i) a confirmed diagnosis of type 2 diabetes, (ii) a positive familial history of type 2 diabetes or (iii) phenotypically determined elevated risk factors (e. g. obesity) are identified by clinical testing, pedigree analysis, and linkage analysis, using standard diagnostic criteria and interview procedures, and DNA or RNA samples are obtained from the subjects.","A sample of genomic DNA is obtained from any nucleated cell source or body fluid.","Examples of cell sources available in clinical practice include blood cells, buccal cells, cervicovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy.","Body fluids include blood, urine, cerebrospinal fluid, amniotic fluid, and tissue exudates at the site of infection or inflammation.","DNA is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art.","It will be understood that the particular method used to extract DNA will depend on the nature of the source.","A variety of techniques are then employed to identify the presence or absence of new or known mutant sequences.","First, the sequences of genes known to be involved in insulin secretory function may be subjected to direct DNA sequencing, using methods that are standard in the art.","Mutations may be detected using a PCR-RFLP, in which pairs of olignucleotides are used to prime amplification reactions and the sizes of the amplification products, cleaved or uncleaved by restriction endonucleases, are compared with those of control products.","Other useful techniques include Single-Strand Conformation Polymorphism analysis (SSCP), denaturing gradient gel electrophoresis, and two dimensional gel electrophoresis, EMC, and the like.","Detection of known mutations, such as those exemplified by the invention, may alternatively be detected using nucleic acid probes that contain mutations of interest in sufficiently stringent hybridization conditions.","Appropriate stringency conditions for identifying mutations of at least one base pair in a mutant sequence of a gene involved in insulin secretory function, for example, in 6× sodium chloride/sodium citrate (SSC) at at least 42° C., preferably at about 43,44 or 45° C., followed by a wash of 2×SSC at 50° C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y.","(1989).","To optimize conditions, both salt and temperature may be varied, or either the temperature or salt concentration may be held constant while the other variable is changed.","For example, the salt concentration in the wash step can be selected from a low stringency of about 2×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. The temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C. to high stringency conditions at about 65° C. Optimal conditions will additionally depend on the length of the nucleic acid probe used, and the scale at which the hybridization takes place.","High stringency hybridization conditions using nucleotides attached to a microchip may require lower temperatures.","One can perform a series of routine thermal equilibrium experiments to determine optimal hybridization discrimination between wild-type and mutant gene sequences of interest, by starting a low stringency temperature of about 20° C. and increasing the temperature in successive 5° C. temperature intervals.","The nucleic acid probes of the invention are nucleic acid sequences from the mutated genes of interest.","They are at least 8,12,15 or 20 base pairs in length, but can be 50,80 or 100 base pairs in length, and may even be 250 or 500 base pairs in length, and include at least one associative mutation but may include multiple mutations, and can be as long as the length of the transcribed gene.","The length of the probe chosen will be optimized based on the better base pair mismatch discrimination of shorter probes and the better duplex stability of longer probes (see U.S. Pat.","No.","6,156,601 and U.S. Pat.","No.","6,197,506, herein incorporated by reference).","The length of the probe used should enable discrimination between a mutant and wild-type gene with at least one base-pair mutation.","For detection of hybridized probes, light detectable means are preferred, although other methods of detection may be employed, such as radioactivity, atomic spectrum, and the like.","For light detectable means, one may use fluorescence, phosphoresence, absorption, chemiluminescence, or the like.","The most convenient will be fluorescence, which may take many forms.","One may use individual fluorescers or pairs of fluorescers, particularly where one wishes to have a plurality of emission wavelengths with large Stokes shifts.","Illustrative fluorescers which have found use include fluorescein, rhodamine, Texas red, cyanine dyes, phycoerythrins, thiazole orange and blue, etc.","When using pairs of dyes, one may have one dye on one molecule and the other dye on another molecule which binds to the first molecule.","For example, one may have one dye on the first or bound component and the other dye on the second or complexing component.","The important factor is that the two dyes when the two components are bound are close enough for efficient energy transfer (see U.S. Pat.","No.","5,992,617).","The identification of the presence or absence of known mutations can also conveniently be detected by PCR followed by restriction analysis and/or sequencing using techniques well known to those in the art.","PCR analysis furthermore offers an efficient technique for identifying new mutations in genes already known to contain mutations that correlate with the predisposition of a Chinese individual to develop type 2 diabetes, or in identifying associative mutations in additional candidate genes.","PCR primers should be at least 12 base pairs in length, preferably 15-18 base pairs in length, and may be as long as 25-30 base pairs in length.","They can be designed to anneal to the wild-type gene sequence in regions that flank a mutation in a gene of interest, such that extension from the primer amplifies a region that allows the detection of the presence or absence of a mutation of interest.","Primers can also be designed such that their extension results in an amplified sequence only in the presence of either a wild-type or mutant gene, as desired.","This can be accomplished by designing a primer with at least one nucleotide at the 3′end that is mismatched with the wild-type sequence, but matched to a mutant sequence.","The invention is exemplified by primer pairs used to screen HNF-1α, glucokinase, amylin and human mitochondrial DNA for mutations.","Of particular interest are nucleic acid primers that can be used to screen mutations in HNF-1α and glucokinase that have not yet been previously reported (see for example, SEQ ID Nos: 34-36).","Simultaneous sequencing of several nucleic acid samples can also be carried out on a microchip (see U.S. Pat.","No.","6,197,506) For SSCP, primers are designed that amplify DNA products of about 250-300 bp in length across non-duplicated segments of the gene of interest.","For each amplification product, one gel system and two running conditions are used.","Each amplification product is applied to a 10% polyacrylamide gel containing 10% glycerol.","Separate aliquots of each amplimer are subjected to electrophoresis at 8 W at room temperature for 16 hours and at 30 W at 4° C. for 5.4 hours.","These conditions were previously shown to identify 98% of the known mutations in the CFTR gene (Ravnik-Glavac et al, (1994) Hum Mol Genet 3: 801).","As with identification of associative mutations of interest, identification of associative SNPs that correlate with the increased risk of a Chinese individual to develop type 2 diabetes can be accomplished by nucleic acid sequencing of desired regions of genomic or complementary DNA.","Screening for SNPs is pursued most efficiently using microarray technologies where attached nucleic acid sequences attached to a solid support such as a microchip are exposed to hybridization conditions that allow the discrimination between two nucleic acid sequences that differ at one nucleotide (see for example, Wang, et al (1998) Science 280: 1077; and Hacia, et al (1998) Nature Genet.","18: 155).","Alternatively, mass spectrophotometers can be used to identify small mass differences in PCR products that have single nucleotide polymorphisms (see Kirpekar, et al (1998) Nucleic Acids Res 26: 2554).","A further means of analyzing genetic information is” dynamic allele specific hybridization” (DASH).","This technique uses labeled oligonucleotides in a multiwell format that will fluoresce when the oligonucleotide exists in a double-stranded form, but not when it is in single-stranded form.","Adding a single strand of the DNA to be tested allows the strands to hybridize.","The temperature at which the strands denature will allow identification of the base at the SNP.","The DASH technique has the advantages of being technically simple, and not requiring expensive equipment.","Additional techniques that can be used in the screening for SNPs associated with the genetic predisposition of a Chinese person to develop type 2 diabetes include exonuclease resistance, microsequencing, solution-phase or solid phase extension of ddNTPs, and oligonucleotide ligation assay (as described in U.S. Pat.","No.","5,952,174, herein incorporated by reference).","After the presence of an associative mutation or SNP is detected by any of the above techniques, the specific nucleic acid alteration comprising the mutation is identified by direct DNA sequence analysis or restriction analysis or a combination of both.","In this manner, previously unidentified mutations in genes that encode proteins involved in insulin secretion, or in genes associated with obesity or diabetic kidney disease may be defined.","For instance, new mutations could be identified with other genes known to closely correlate with familial type 2 diabetes in Chinese subjects (e.g., other MODY genes).","Examples of additional MODY genes include hepatocyte nuclear factor 4α (HNF-4α), hepatocyte nuclear factor 1β (HNF-1β), and insulin promoter factor 1 (IPF-1).","Additional candidate genes of particular interest for screening because mutations or polymorphisms of the wild type genes are positively associated with type 2 diabetes and nephropathy in Chinese individuals include those that encode angiotensin converting enzyme (ACE)/angiotensinogen (AGT) (Tomino, et al (1999) Nephron 82: 139; Hsieh, et al (2000) Nephrol Dial Transplant 15: 1008; Thomas, et al (2001) Diabetes Care 24; 356), aldose reductase (Ko, et al (1995) Diabetes 44: 727; Moczulski, et al (1999) Diabetologia 42: 94) and plasminogen activator inhibitor-1 (PAI-1) (Wong, et al (2000) Kidney Int 57: 632).","Nucleic acid sequences that encode genes involved with glucose metabolism, insulin resistance, obesity and diabetic kidney can also be screened to identify mutations in, for example, proteins that influence insulin binding to its receptor, that are involved in the insulin signaling pathway, that influence glucose uptake and cell metabolism.","Specific examples include associative mutations in the α or β chain of the insulin receptor, the insulin receptor substrate proteins (IRS-1 and IRS-2), glucose transporter proteins GLUT2 and GLUT4, and transcription factors HNF-3β and NeuroDI/Beta2 and to correlated any identified mutation an/or polymorphism with indidence of type 2 diabetes.","Examples of candidate genes where mutations or polymorphisms have been shown to be associated with type 2 diabetes and obesity in other populations include genes that encode the transporter GLUT4 (Abel, et al (2001) Nature 409: 729), the beta-3-adrenergic receptor (Oeveren van-Dybicz, et al (2001) Diabetes Obes Metab 3: 47), the hormone resistin (Steppan, et al2001) Nature 409: 307), the peroxisome proliferator-activated receptor gamma2 PPARgamma) (Hasstedt, et al (2001) J Clin Endocrinol Metab 86: 536), uncoupling protein-1 (UCP-1) (Heilbronn, et al (2000) Diabetologia 43: 242), leptin (Ohshiro, et al (2000) J Mol Med 78: 516), G protein beta 3 subunit and insulin receptor substrate-1 (Rosskopf, et al (2000) 5: 484), and the dopamine D2 receptor (Jenkinson, et al (2000) Int J Obes Relat Metab Disord 24: 1233).","Additionally, mutations or polymorphisms shown to be closely associated with type 2 diabetes and nephropathy in other populations include genes that encode the G protein beta 3 subunit (Beige, et al (2000) Nephrol Dial Transplant 15: 1384; Zychma, et al (2000) Am J Nephrol 20: 305), methylenetetrahydrofolate reductase (MTHFR) (Shpichinetsky, et al (2000) J Nutr 130: 2493, the glucose transporter GLUT1 (Grzeszczak, et al (2001) Kidney Int 59: 631), and paraoxonase (PON1) (Inoue, et al (2000) Metabolism 49: 1400).","Additionally, proteins translated from genes carrying at least one mutation associated with increased risk of a Chinese individual to develop type 2 diabetes are contemplated by the invention and find use in functional diagnostic assays and in the production of diagnostic antibodies that bind to the mutant but not the wild-type protein.","The polypeptides may be the translational products of the entire mutant gene, as well as peptides of twelve or more amino acids derived therefrom that contain at least one mutation of interest.","The polypeptide (s) may be isolated from human tissues obtained by biopsy or autopsy, or may be produced in a heterologous cell by recombinant DNA methods, well known to those in the art (as disclosed in Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor, 1989), or Current Protocols in Molecular Biology (Eds.","Aufubel, Brent, Kingston, More, Feidman, Smith and Stuhl,Greene Publ.","Assoc., Wiley-Interscience, NY, N.Y., 1992), both references herein incorporated by reference).","Peptides comprising HNF-1α-, glucokinase- or amylin specific sequences may be derived from isolated larger polypeptides described above, using proteolytic cleavages by e. g. proteases such as trypsin and chemical treatments such as cyanogen bromide that are well-known in the art.","Alternatively, peptides up to 60 residues in length can be routinely synthesized in milligram quantities using commercially available peptide synthesizers.","Recombinant translational products are expressed from vectors comprising mutant nucleic acid sequences of wild-type nucleic acid sequences that encode proteins involved in insulin secretion.","Exemplified mutant nucleic acid sequences of interest include those that encode HNF-1α, glucokinase or amylin with single amino acid residue changes, as depicted in SEQ ID Nos: 1-13, and particularly SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 10.A large number of vectors, including plasmid and fungal vectors, have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.","Vectors used for expression will also include a promoter operably linked to the mutant polypeptide encoding portion, that is preferably the cDNA sequence of the mutated gene of interest or a part thereof that encodes a polypeptide of at least 12 amino acids.","The encoded polypeptide may be expressed by using any suitable commercially available vectors, and any suitable host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.","The particular choice of vector/host is not critical to the operation of the invention.","Appropriate host cells include bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells.","Of particular interest are E.coli, B.Subtilis, Saccharomyces cerevisiae, SF9 cells, C129 cells, 293 cells, Neurospora, and CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoid cell lines.","Preferred replication systems include M13, ColEI, SV40, baculovirus, lambda, adenovirus, and the like.","A large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts.","Examples of these regions, methods of isolation, manner of manipulation, etc.","are known in the art.","Under appropriate expression conditions, host cells can be used as a source of recombinantly produced mutant polypeptides of interest.","The translational products of mutant HNF-1α, glucokinase or amylin, and/or fragments or portions thereof may be used to produce specific antibodies.","The antibodies may be polyclonal or monoclonal, may be produced in response to the fully translated mutant polypeptide or to synthetic peptides as described above.","Such antibodies are conveniently made using the methods and compositions disclosed in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, other references cited herein, as well as immunological and hybridoma technologies known to those in the art.","Importantly, the antibodies raised against translation products from nucleic acids sequences carrying at least one mutatation associated with type 2 diabetes should distinguish between the mutant amino acid sequence and the wild-type amino acid sequence.","In particular, antibodies should have very little or no cross-reactivity for the wild-type sequence.","Preferably the anti-mutant protein antibodies should bind with higher affinity to the mutant polypeptide than to wild-type polypeptide, with binding to the mutant polypeptide at levels 500:1, more preferably 1,000:1, greater than binding the wild-type polypeptide.","Isolated polypeptides corresponding to the entire length of the mutant polypeptide or a peptide of at least 12 amino acids in length containing a mutation of interest may be used in accordance with conventional methods to immunize a mammal, (e. g., mouse or higher mammal, primate, or chimeric or transgenic animals which produce human immunoglobulins) in accordance with conventional procedures.","See for example, U.S. Pat.","Nos.","4,172,124; 4,350,683; 4,361,549; and 4,464,465.Hybridomas may be prepared by fusing available myeloma lines, e. g., NS/I, Ag8.6.5.3, etc., with peripheral blood lymphocytes, splenocytes or other lymphocytes of the immunized host and the resulting immortalized B-lymphocytes (e.g., hybridomas, heteromyelomas, EBV transformed cells, etc.)","selected, cloned and screened for binding to a mutant polypeptide of a wild-type protein involved in insulin secretion or glucose metabolism.","Monoclonal antibodies raised against a mutant polypeptide sequence of interest may be of any immunoglobulin class such as IgA, IgD, IgE, IgG and IgM, preferably IgG or IgM, and may be of any one of the subclasses of the classes.","Whole antibodies, or fragments thereof which retain binding activity, may be employed, such as Fab, F(ab′)2, or the like.","Once the antibodies with binding specificity for the mutant polypeptide are available, these antibodies may be used for screening.","Antibodies that distinguish between normal and mutant forms of HNF-1α, glucokinase, amylin or other mutant/wild-type pairs of proteins involved in insulin secretory function may be used in diagnostic tests employing ELISA, EMIT, CEDIA, SLIFA, and the like.","For an assessment of total risk of developing disease or in designing individualized treatments of diagnosed patients, identified mutations and polymorphisms that are indicative of a Chinese individual to develop type 2 diabetes are correlated with phenotypic parameters of screened patients and interpreted with consideration of a positive or negative family history of the disease.","Genetic studies will be correlated with data from individuals indicating hormone levels (growth hormone, adrenaline, cortisol, noradrenaline, insulin), anthropometry (body-mass index; waist-to-hip ratio), hemodynamics (blood pressure), cardiovascular risk factors (HDL, LDL, cholesterol, triglycerides) and autoimmunity (anti-glutamic acid decarboxylase antibodies).","For instance, a patient with a single mutation in the glucokinase gene may never develop symptoms, whereas the likelihood of a patient with a mutation in both the glucokinase gene and the HNF-1α gene or a mutation in the glucokinase gene and the phenotypic attribute of obesity to develop overt type 2 diabetes is relatively higher.","A positive family history of the disease would increase the predicted predisposition even more.","Obtaining a genotypic assessment while a patient shows no signs of developing disease, or while showing preliminary signs of disease such as impaired glucose tolerance (IGT), can enable a physician to initiate therapy or suggest lifestyle changes that prevent the onset or progression of overt symptoms.","For example, a patient identified as having a mutation in the HNF-1α gene and IGT, can be treated with diet and/or oral drugs and/or insulin early enough that hyperglycemic toxicity of pancreatic β-cells and further insulin secretory dysfunction due to their death is prevented or ameliorated.","In this way, severe complications associated with progressive type 2 diabetes, such as nephropathy, retinopathy and sensorineural loss, can be more commonly averted.","In addition to allowing a clinician to better tailor traditional therapies for treating type 2 diabetes, such as diet, oral drugs and insulin, identification of associative mutations can enable a clinician to design tailored therapies, such as introducing a wild-type gene into a patient to replace a mutant gene that encodes a malfunctioning protein.","For gene therapy methods, transfection in vivo is obtained by introducing a therapeutic transcription or expression vector into the mammalian host, either as naked DNA, complexed to lipid carriers, particularly cationic lipid carriers, or inserted into a viral vector, for example a recombinant adenovirus.","The introduction into the mammalian host can be by any of several routes, including intravenous or intraperitoneal injection, intratracheally, intrathecally, parenterally, intraarticularly, intranasally, intramuscularly, topically, transdermally, application to any mucous membrane surface, corneal installation, etc.","Of particular interest is the introduction of a therapeutic expression vector into a circulating bodily fluid or into a body orifice or cavity, such as the heart.","Thus, intravenous administration and intrathecal administration are of particular interest since the vector may be widely disseminated following such routes of administration, and aerosol administration finds use with introduction into a body orifice or cavity.","Particular cells and tissues can be targeted, depending upon the route of administration and the site of administration.","For example, a tissue which is closest to the site of injection in the direction of blood flow can be transfected in the absence of any specific targeting.","An arterial catheter can be used to introduce the expression vector into an organ such as the heart or kidney.","The eye can be accessed directly either by the use of ocular drops or by injecting into the eye.","For accessing nerves, this can be by injection into the nerve or injection into the region of the cell body.","If lipid carriers are used, they can be modified to direct the complexes to particular types of cells using site-directing molecules.","Thus, antibodies or ligands for particular receptors or other cell surface proteins may be employed, with a target cell associated with a particular surface protein.","An amino terminal mitochondrial targeting sequence joined to a nucleic acid can be used to target the nucleic acid to the mitochondria.","See Taylor et al, Nature Genetics 15: 212-215,1997.Any physiologically acceptable medium may be employed for administering the DNA, recombinant viral vectors or lipid carriers, such as deionized water, saline, phosphate-buffered saline, 5% dextrose in water, and the like as described above for the pharmaceutical composition, depending upon the route of administration.","Other components may be included in the formulation such as buffers, stabilizers, biocides, etc.","These components have found extensive exemplification in the literature and need not be described in particular here.","Any diluent or components of diluents that would cause aggregation of the complexes should be avoided, including high salt, chelating agents, and the like.","The amount of therapeutic vector used will be an amount sufficient to provide for a therapeutic level of expression in a target tissue susceptible to diabetic complications or for adequate dissemination to a variety of tissues after entry into the bloodstream and to provide for a therapeutic level of expression in susceptible target tissues.","A therapeutic level of expression is a sufficient amount of expression to prevent, treat, or palliate one or more diabetic complication or the symptoms of diabetic complications.","In addition, the dose of the nucleic acid vector used must be sufficient to produce a desired level of transgene expression in the affected tissue or tissues in vivo.","Other DNA sequences, such as adenovirus VA genes can be included in the administration medium and be co-transfected with the gene of interest.","The presence of genes coding for the adenovirus VA gene product may significantly enhance the translation of mRNA transcribed from the expression cassette if this is desired.","A number of factors can affect the amount of expression in transfected tissue and thus can be used to modify the level of expression to fit a particular purpose.","Where a high level of expression is desired, all factors can be optimized, where less expression is desired, one or more parameters can be altered so that the desired level of expression is attained.","For example, if high expression would exceed the therapeutic window, then less than optimum conditions can be used.","The level and tissues of expression of the recombinant gene may be determined at the mRNA level as described above, and/or at the level of polypeptide or protein.","Gene product may be quantitated by measuring its biological activity in tissues.","For example, protein activity can be measured by immunoassay as described above, by biological assay such as inhibition of ROS, or by identifying the gene product in transfected cells by immunostaining techniques such as probing with an antibody which specifically recognizes the gene product or a reporter gene product present in the expression cassette.","Typically, the therapeutic cassette is not integrated into the patient's genome.","If necessary, the treatment can be repeated on an ad hoc basis depending upon the results achieved.","If the treatment is repeated, the patient can be monitored to ensure that there is no adverse immune or other response to the treatment.","The following examples are offered by way of illustration of the present invention, not limitation.","EXPERIMENTAL EXAMPLE 1 Identification of Mutations in Glucokinase and Hepatocyte Nuclear Factor 1α Genes in Chinese Patients with Early-Onset Type 2 Diabetes Mellitus/MODY This example illustrates mutations identified in the glucokinase, HNF-1α and HNF-4α genes in a cohort of Chinese patients.","Mutations in the glucokinase and HNF-1α genes are relatively common in early-onset diabetes and they account for about 3% and 5%, respectively, of the present Chinese early-onset diabetic patients.","Experimental Design and Methods Subjects The study group consisted of 92 unrelated patients (age 34±5 years (mean±SD) range 18-40 years; 30 males and 62 females) who were diagnosed with Type 2 diabetes before 40 years of age and who had a positive family history (at least one first degree relative with Type 2 diabetes).","The mean age at diagnosis was 30±5 years (range 16-40 years).","Thirteen (14%) of these patients met the minimal criteria of MODY (age at diagnosis before 25 years old and presence of diabetes in two consecutive generations).","These patients were selected from a database containing 1800 cases recruited in the Diabetes and Endocrine Centre of the Prince of Wales Hospital.","Family members of probands with MODY gene mutations, if available, were recruited and underwent a 75-gram oral glucose tolerance test (OGTT).","One hundred healthy Chinese (age 33±10 years, 40 males and 60 females) without a history of diabetes were recruited as controls amongst hospital staff and students.","Informed consent was obtained from each subject for a blood sample to be taken for DNA isolation and measurement of clinical parameters.","This study was approved by the Clinical Research Ethics Committee of The Chinese University of Hong Kong.","Screening of Glucoknase, HNF-1α and HNF-4α Genes for Mutations The minimal promoter region and exons of the glucokinase (β cell form), HNF-1α and HNF-4α (HNF-4α 2 form) genes were screened for mutations by direct sequencing of polymerase chain reaction (PCR) products as described (Froguel, et al (1993) N Engl J Med 328: 697; Yamagata, et al (1996) Nature 384: 455; Yamagata, et al (1996) Nature 384: 458).","The occurrence of putative mutations in other family members and controls was determined by PCR-restriction fragment length polymorphism (RFLP).","An artificial restriction site was introduced into either the wild-type or mutant sequence if the nucleotide substitution did not lead to gain or loss of a restriction site.","Briefly, direct sequencing identified 5 mutations in the HNF-1α gene (G20R, R203H; S432C, I618M and IVS2nt-1G→A) and 3 mutations in the glucokinase gene (I110T, A119D and G385V) that are unique to Chinese subjects.","They were screened as follows.","HNF-1α G20R was screened by using the forward primer 5′-GGCAGGC-AAACGCAACCCACG-3′ (SEQ ID NO:14) and modified reverse primer 5′-CAGTGCCTCTTTGCTCAGGC-3′ (SEQ ID NO:15) for PCR amplification followed by digestion with StuI.","The wild-type allele showed 19 and 140 bp products whereas the mutant allele showed a 159 bp product.","HNF-1α R203H was screened by using the forward primer 5′-TGCCTGCAGAGTTCA-CCCATG-3′ (SEQ ID NO:16) and modified reverse primer5′-ATCTGCTGGGATGCTGGG-CCCCACTTGCAA-3′ (SEQ ID NO:17) for PCR amplification followed by digestion with BsrDI.","The wild-type allele showed a 121 bp product whereas the mutant allele showed 26 and 95 bp products.","HNF-1α S432C was screened by using the forward primer 5′-TGGAGCAGTCCCTAG-GGAGGC-3′ (SEQ ID NO:18) and reverse primer 5′-GTTGCCCCATGAGCCTCCCAC-3′ (SEQ ID NO:19) for PCR amplification followed by digestion with Cac81.The wild-type allele showed 104 and 218 bp products whereas the mutant allele showed 37,67 and 218 bp products.","HNF-1α 1618M was screened by using the forward primer 5′-GTACCCCTAGGGACAGG-CAGG-3′ (SEQ ID NO:20) and reverse primer 5′-ACCCCCCAAGCAGGCAGTACA-3′ (SEQ ID NO:21) for PCR amplification followed by digestion with TaqI.","The wild-type allele showed 88 and 160 bp products whereas the mutant allele showed a 248 bp product.","HNF-1α IVS2nt-1G→A was screened by using the forward primer 5′-GGGC-AAGGTCAGGGGAATGGA-3′ (SEQ ID NO:22) and reverse primer 5′-CAGCCCAGACCAAACCAGCAC-3′ (SEQ ID NO:23) for PCR amplification followed by digestion with PstI.","The wild-type allele showed 73 and 231 bp products whereas the mutant allele showed a 304 bp product.","Glucokinase I110T was screened by using the forward primer 5′-GTCCCTGAGGCTG-ACACACTT-3′ (SEQ ID NO:24) and reverse primer 5′-AGCTGGGCCCTGAGATCCTGCA-3′ (SEQ ID NO: 25) for PCR amplification followed by digestion with FokI.","The wild-type allele showed 108 and 142 bp products whereas the mutant allele showed a 250 bp product.","Glucokinase A 119D was screened by using the forward primer 5′-ACCTGGGTGGCA-CTAACTTCA-3′ (SEQ ID NO:26) and modified reverse primer 5′-CGGCCCCTGCGCTG-CTCACCATCTGA-3′ (SEQ ID NO:27) for PCR amplification followed by digestion with BclI.","The wild-type allele showed a 150 bp product whereas the mutant allele showed 28 and 122 bp products.","Glucokinase G385V was screened by using the forward primer 5′-GGACTGTCG-GAGCGACACTCA-3′ (SEQ ID NO:28) and modified reverse primer 5′-GCGGTTGATGAC-GCCTGCCAG-3′ (SEQ ID NO:29) for PCR amplification followed by digestion with FauI.","The wild-type allele showed 5,22,44 and 137 bp products whereas the mutant allele showed 5,44 and 159 bp products.","Mutations in the amylin gene (S20G) and mitochondrial DNA (A3243G) were screened as follows.","Amylin S20G was screened by using the forward primer 5′-TCACAT-TTGTTCCATGTTAC-3′ (SEQ ID NO:30) and reverse primer 5′-CAATAACTATAGAG-TTACATTG-3′ (SEQ ID NO:31) for PCR amplification followed by digestion with MspI.","The wild-type allele showed a 239 bp product whereas the mutant allele showed 99 and 140 bp products.","Mitochondrial DNA A3243G was screened by using the forward primer 5′-AAGGTTCGTT-TGTTCAACGA-3′ (SEQ ID NO:32) and reverse primer 5′-AGCGAAGGGTTGTAGTAGCC-3′ (SEQ ID NO:33) for PCR amplification and labeling of PCR product with α32PdATP at the last cycle.","The PCR products were then digested with ApaI and analysed on 8% denaturing polyacrylamide gels.","The wild-type allele showed a 427 bp product whereas the mutant allele showed 213 and 214 bp products.","Clinical Studies All patients underwent a structured assessment including documentation of family history, age at diagnosis and body mass index (BMI) (Piwernetz, et al (1993) Diabetic Med 10: 371; Chan, et al (1997) Hong Kong Auth Qual Bull 2: 3).","Family history was documented in two generations only since the diabetic status of grandparents was usually unknown.","A fasting blood sample was taken for the measurement of glucose, C-peptide and glycosylated haemoglobin (HbA1c).","Obesity was defined as a BMI≧27 kg/m2in men and ≧25 kg/m2in women (National Diabetes Data Group (1979) Diabetes 28: 1039).","Assays Plasma glucose concentrations were measured by a glucose oxidase method (Diagnostic Chemicals, Charlottetown, Prince Edward Island, Canada).","C-peptide was measured by radioimmunoassay (Novo-Nordisk, Copenhagen, Denmark).","HbA1c was measured by gel electrophoresis (Ciba Coming Diagnostics Corp, Palo Alto, Calif.).","Data Analysis Data are expressed as mean ±SD if normally distributed.","Otherwise, data are expressed as median and range.","Results Mututions and Polymorphisms in the Glucokinase, HNF-1α and HNF-4α Genes Screening of the promoter region and exons 1a, 2-10 of-the glucokinase gene (Stoffel, et al (1992) Proc Nratl Acad Sci 89: 7698; Tanizawa, et al (1992) 6:1070) revealed three novel missense mutations: I110T, A119D and G385V.","In addition to these mutations, three uncommon variants (two of which had not been previously described) and two polymorphisms were found in the 5′-untranslared region of the mRNA and intron regions (Table 1).","The brother and mother of subject HK84 (Table 1) also inherited the I110T mutation (FIG.","7).","The mother was diagnosed with diabetes at the age of 64 years upon screening.","The brother aged 25 years, when tested with a 75 g OGTT had a plasma glucose at 0 and 120 min of 6.3 mmol/1 and 6.9 mmol/1, respectively.","These results were inconclusive, suggesting impaired fasting glucose (IFG) by the 1997 ADA criteria but not reaching that of impaired glucose tolerance (IGT) by the 1998 WHO criteria.","Screening of the HNF-1α gene revealed four missense mutations (G20R, R203H, S432C and 1618M) and one splice acceptor site mutation (IVS2nt-1G→A) (Table 2).","All of these represent mutations in the HNF-1α gene unique to Chinese patients.","Subject HK10 (Table 2) had three siblings (ages 26-36 years) with diabetes.","The affected siblings all had inherited the IVS2nt-IG→A mutation while another sibling and the father with IGT had not.","Moreover, the maternal grandparents, uncle and mother of HK10 were diabetic but they were not available for screening (FIG.","7) (Chan, et al (1990) Diabetic Med 7: 211).","Subject HK54 (Table 2) had four siblings (age 33-43 years) with normal glucose tolerance and one sibling (age 39 years) with IGT.","The father and mother were diagnosed as having diabetes at the ages of 50 and 60 years, respectively.","Neither the mother and nor any of the siblings had inherited the R203H mutation (FIG.","7).","In addition to the putative diabetes-associated mutations in HNF-1α two substitutions resulting in common amino acid polymorphisms, four silent mutations and nine variants/polymorphisms in introns were identified (Table 2).","Family members of the other five probands (Tables 1 and 2) with glucokinase or HNF-1α missense mutations were not available for screening.","None of the mutations in the glucokinase and HNF-1α genes were found in 100 healthy controls.","Analysis of the promoter region and exons 1a, 2-10 of the HNF-4α gene (Furuta, et al (1997) Diabetes 46: 1652) revealed no obvious diabetes-associated mutations.","Three patients were heterozygous for a previously described amino acid polymorphism, T/1130 (Yamagata et al, (1997) Nature 384: 458).","Two patients were heterozygous for a silent mutation in the codon for L211, and one patient was heterozygous for a silent mutation in the codon for P441 (Table 3).","There were two polymorphisms in the intron-upstream of exon 2 (intron 1B) and a G→A substitution in the promoter was found in the heterozygous state in one patient.","The G→A substitution in the promoter at nucleotide-462 was not located in a known cis-acting regulatory region of the gene (Furuta, et al (1997) supra) and its effect on the regulation of expression of HNF-4α remains to be determined.","Clinical Features of Patients with MODY or Unknown Etiology The clinical features of the patients with mutations in the glucokinase and HNF-1α genes or with unknown etiology are shown in Table 4.Of the 92 patients, 54 (59%) were non-obese at the time of study.","The mean age at diagnosis of the patients with glucokinase mutation-associated diabetes (‘glucokinase diabetes’) was 28 years.","All three subjects had mild hyperglycemia and satisfactory glycemic control (fasting glucose ≦7.4 mmol/1; HbA1c, 6.7%; non-diabetic range: 5.1-6.4%).","These patients had varving degrees of basal pancreatic β cell secretory function as indicated by their fasting C-peptide levels (0.28-1.60 nmol/1) (Chan, et al (1990) supra).","All were treated with diet or oral drugs.","No diabetic complications were observed in the three patients with glucokinase mutation (Froguel, et al (1993) supra ; Page, et al (1995) 12: 209; Velho, et al (1997) 40: 217).","The mean age at diagnosis of the patients with HNF-1α mutation-associated diabetes was 31 years.","Among the four patients (HK30, 54,90 and 92) with missense mutations, all had mild hyperglycemia and satisfactory glycemic control (fasting glucose ≦7.4 mmol/1; HbA1c≦7.1%) but exhibited varying degrees of basal pancreatic β cell secretory function (fasting C-peptide, 0.10-0.49 nmol/1).","They did not have diabetic complications and were treated with diet or oral drugs.","The subject (HK10) with the splice-site IVS2nt-1G→A mutation was not overweight when diagnosed at the age of 19 years (Fajans (1990) Diabetes Care 13: 49-64) and presented with proliferative retinopathy and clinical proteinuria.","She was treated with insulin continuously for three months after the diagnosis.","She eventually developed neuropathy and renal failure.","TABLE 1 Mutations and polymorphisms in the glucokinase gene in Chinese subjects with early-onset Type 2 diabetes mellitus Subject Location Codon/nt Nucleotide change Designation Frequency Mutations HK84 Exon 3 110 ATC (Ile)→ACC (Thr) I110T HK38 Exon 3 119 GCT(Ala)→GAT(Asp) A1I9D HK15 Exon 9 385 GGG(Gly)→GTG(Val) G385V Polymorphisms 5′-UT* −213 A→G 5′-UT β − 213 A/G A 0.96, G 0.04 5′-UT −84 C→G 5′-UT β − 84 C/G C 0.94, G 0.06 Intron 1c nt − 13 C→G IVS1nt − 13 C/G C 0.99, C 0.01 Intron 9 nt + 8 C→T IVS9nt + 8C/T C 0.50, T 0.50 Intron 9* nt + 49 G→A IVS9nt + 49G/A G 0.99, A 0.01 nt indicates the nucleotide location relative to the first nucleotide of codon 1 (ATG) for polymorphisms in the 5′-untranslated region (5′-UT) of the β cell specific exon 1 α/1 β, and splice donor (+) or acceptor site (−).","Intron 1c is the intron between exon 1c, which encodes the amino terminal 14 amino acids of the minor liver isoform of glucokinase, and exon 2 (Velho, et al (1996) 19: 915).","The asterisks indicate polymorphisms that were reported by Ng, et al (Diabetic Med (1999) 16: 956) and that have not been reported in studies of other populations (Veiga-deCunha, et al (1996) J Biol Chem 271: 6292; Zhang, et al (1995) 38: 1055).","TABLE 2 Mutations and polymorphisms in the HNF-1 α gene in Chinese subjects with early-onset Type 2 diabetes mellitus Subject Location Codon/n Nucleotide change Designation Frequency Mutations HK90 Exon 1 20 GGG (Gly)→AGG (Arg) G20R HK10 Intron 2/ nt − 1 AG→AA at splice IVS2nt − 1G→A Exon 3 acceptor site HK54 Exon 3 203 CGT (Arg)→CAT (His) R203H HK30 Exon 6 432 TCC (Ser)→TGC (Cys) S432C HK92 Exon 10 618 ATC (Ile)→ATG (Met) I618M Silent mutations/polymorphisms Exon 1 17 CTC (Leu)→CTG (Leu) L17C/G C 0.63, C 0.37 Exon 1 27 ATC (Ile)→CTC (Leu) I/L27 A 0.57, C 0.43 Intron 1 nt − 42 G→A IVS1nt − 42G/A C 0.58, A 0.42 Intron 2* nt + 53 C→G IVS2nt + 53C/G C 0.99, C 0.01 Intron 2 nt − 51 T→A IVS2nt − 51T/A T 0.77, A 0.23 Intron 2 nt − 23 C→T IVS2nt − 23C/T C 0.48, T 0.52 Intron 5 nt + 9 C→G IVS5nt + 9C/G C 0.98, G 0.02 Intron 5 nt − 42 G→T IVS5nt − 42G/T G 0.87, T 0.13 Intron 6* nt + 26 C→T IVS6nt + 26C/T C 0.99, T 0.01 Exon 7 459 CTG (Leu)→TTG(Leu) L459C/T C 0.48, T 0.52 Exon 7 459 CTG (Leu)→CTA (Leu) L459G/A C 0.99, A 0.01 Exon 7 487 AGC (Ser)→AAC (Asn) S/N487 G 0.48, A 0.52 Intron 7 nt + 7 G→A IVS7nt + 7G/A G 0.48, A 0.52 Exon 8* 531 AGC (Ser)→AGT (Ser) S531 C/T C 0.99, T 0.01 Intron 9 nt − 24 T→C IVS9nt − 24T/C T 0.48, C 0.52 nt indicates the nucleotide location relative to the splice donor (+) or acceptor site (−).","The asterisks indicate polymorphisms reported by Ng, et al (Diabetic Med (1999) 16: 956) and that have not been reported in studies of other populations.","TABLE 3 Mutations and polymorphisms in the HNF-4 α gene in Chinese subjects with early-onset Type 2 diabetes mellitus Location Codon/nt Nucleotide change Designation Frequency Silent mutations/ polymorphisms Promoter* nt-462 G→A Ptr-462G/A G 099, A 0.01 Intron lB nt-38 C→T IVS1nt-38C/T C 0.80, T 0.20 Intron lB nt-5 C→T IVS1nt-5C/T C 0.79, T 0.21 Exon 4 130 ACT (Thr)→ATT (Ile) T/I130 C 0.98, T 0.02 Exon 6* 211 CTC (Leu)→CTT (Leu) L211C/T C 0.99, T 0.01 Exon.","10* 441 CCG (Pro)→CCA (Pro) P441G/A G 0.99, A 0.01 G 0.99, A 0.01 nt indicates the nucleotide location relative to the first nucleoride of codon 1 (ATG) for polymorphisms in the promoter region and splice donor (+) or acceptor site (−).","The sequence context of the nt-464 polymorphism is GATA(G/A)TATC.","The asterisks indicate polymorphisms that have not been reported in studies of other populations.","TABLE 4 Clinical features of Chinese patients with early-onset Type 2 diabetes of unknown etiology as compared with those with diabetes as a result of mutations in glucokinase and HNF-1 α genes Unknown Glucokinase Etiology diabetes (n = 3) HNF-1 α diabetes (n = 5) (n = 84) HK1 HK38 HK84 HK10 HK30 HK54 HK90 HK92 Age at diagnosis (year) 30 ± 5 18 29 38 19 30 33 36 38 Interval since diagnosis (year) 3 (0-16) 15 2 1 9 3 6 0 0 Sex(M/F)(%) 32/68 F F F F M F M M Family history(fa/mot/sib)(%) 45/63/25 fa mot, sib mot gparent, fa fa, mot mot, sib mot uncle, mot, sib BMI (kg/m2) 26 ± 5 nd* 15 28 26 23 20 20 29 HbA1c(%) 7.5 ± 1.9 6.7 6.0 6.6 8.7 7.1 6.0 6.9 6.1 Fasting glucose (mmol/l) 8.5 ± 3.3 7.2 7.4 6.6 13.9 7.4 4.9 4.9 6.6 Fasting C-peptide (nmol/l) 0.43 (0.03-4.96) nd 1.60 0.28 0.47 0.49 0.11 0.16 0.10 Treatment (D/O/I)(%) 49/43/8 O O D I O O D O gparent, grandparent affected; uncle, uncle affected; fa, father affected; mot, mother affected; sib, siblings affected; D, diet; O, oral drugs; I, insulin; nd, not done.","Data are expressed as mean ± SD median (range) or n. *BMI was not measured because this patient had spinomuscular atrophy.","The mean age at diagnosis of the 84 patients with unknown etiology was 30 years, similar to those with glucokinase or HNF-α gene mutations.","Large variations in the degree of hyperglycemia (fasting glucose 8.5±3.3 mmol/1) and basal pancreatic β cell secretory function (fasting C-peptide 0.03-4.96 nmol/1) were observed.","Most of these pateints were treated with oral drugs or diet (92%).","EXAMPLE 2 Mitochondrial DNA A3243G Mutation in Patients with Early-or Late-Onset Type 2 Diabetes Mellitus in Hong Kong Chinese This example illustrates the prevalence of the mitochondrial DNA A3243G mutation in the Hong Kong Chinese population as represented by a large cohort of type 2 diabetic patients with differing ages of diagnosis and clinical phenotypes.","Experimental Design and Methods Subjects The study group consisted of 906 unrelated type 2 diabetic patients diagnosed according to the 1985 WHO criteria (World Health Organization, 1985).","This cohort included four groups of patients selected according to the age of diagnosis and the presence or absence of family history of diabetes.","Groups 1 and 2 consisted of 219 and 128 patients, respectively, with an early age of diagnosis (≦40 years) and with (Group 1) or without (Group 2) a family history of diabetes.","Groups 3 and 4 consisted of 211 and 348 patients, respectively, with an older age of diagnosis (>40 years) and with (Group 3) or without (Group 4) a family history of diabetes.","Patients in each of these groups were randomly selected from a cohort recruited in the Diabetes and Endocrine Centre of the Prince of Wales Hospital, which has a catchment of 1.2 million population in Hong Kong.","All the patients underwent a structured assessment based on the Europe DiabCare Protocol (Piwernetz, et al (1993) Diabetic Med 10: 371; Chan, et al (1997) Hosp Auth Qual Bull 2: 3).","Family members of mt3243 mutation carriers, if available, were recruited and underwent a 75 grain oral glucose tolerance test (OTT) Two hundred and thirteen healthy Chinese without a history of diabetes were recruited as control subjects amongst hospital staff and students.","The present study group included 75 early onset type 2 diabetic (two of whom had an mt3243 mutation) and 95 control subjects who were included in a previous report (see Smith, et al (1997) Diabetic Med 14: 1026).","Informed consent was obtained from each subject for a blood sample to be taken for DNA isolation and measurement of clinical partners.","This study was approved by the Clinical Research Ethics Committee of The Chinese University of Hong Kong.","Mt3243 Mutation Analysis Leukocyte DNA was extracted by standard methods involving proteinase K and phenol/chloroform (Sambrook, et al (1989) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York, incorporated herein by reference).","The mt3243 genotype was determined by polymerase chain reaction (PCR) amplification and ApaI digestion as described (Smith, et al, (1997) supra).","In brief, the DNA region spanning nucleotide 3029 and 3456 was amplified by PCR and labelled with α32PDATP at the last cycle.","This method prevents the underestimation of the proportion of mutant mtDNA as a consequence of heteroduplex formation during the PCR (Schoffner, et al (1990) Cell 61: 931).","The PCR products were then digested with ApaI (Gibco BRL, Gaithersburg, Md., USA) for 2 h at 30 C. Digested PCR products were electrophoresed on 8% denaturing polyacrylamide gels and visualized by autoradiography.","The presence of mt3243 led to the cleavage of the 427 bp product into 213 and 214 bp fragments.","Standards containing 0-100% mutant mt3243 (made by mixing a cloned DNA carrying no mt3243 mutation and another cloned DNA carrying >99% mutant mt3243 in different proportions, kindly given by Dr J. van den Ouweland, Leiden University) were also included in the assay.","The 100% mutant DNA was used as a positive control to evaluate completeness of PCR product digestion.","The intensity of bands was quantified by a Bio-Rad Model GS-670 imaging densitometer and a Molecular Analyst software (version 1.3) (Bio-Rad, Hercules, Calif., USA).","The proportion of mt3243 in a sample was calculated by dividing the intensity at mutant bands (213 and 214 bp) by the total intensity of both wild-type and mutant bands.","Clinical Studies All patients underwent a structured assessment including documentation of family history, age of diagnosis, body mass index (BMI) and waist-to-hip ratio.","Audiometry was performed by a technician at the otolaryngology department to assess the sensorineural status in subjects carrying the mt3243 mutation.","A fasting blood sample was taken for the measurement of glucose, C-peptide, insulin and glycosylated haemoglobin (HbA1c).","Insulin resistance (IR) was estimated using the homeostasis model assessment (HOMA) where IR=fasting insulin×fasting glucose/22.5 (Matthews et al, (1985) Diabetologia 28: 412).","Obesity was defined as a BMI≧27 kg/m2 in men and ≧25 kg/m2 in women (National Diabetes Data Group, 1979).","The basal pancreatic β-cell reserve was also assessed by plasma fasting C-peptide level.","Patients with a C-peptide level≦0.2 nmol/l were considered to be insulin deficient (Service et al,.","(1997) Diabetes Care 20: 198).","Biochemical Assays Plasma glucose was measured by a glucose oxidase method (Diagnostic Chemicals, Charlottetown, PEI, Canada).","HbA1c was measured by gel electrophoresis (Ciba Coming Diagnostics Corp, Palo Alto, Calif. USA) C-peptide was measured by radioimmunoassay (Novo-Nordisk, Copenhagen, Denmark) with an intra-assay coefficient of variance (CV) of 3.4% and interassay CV of 9.6%.","Insulin was measured by radioimmunoassay (Pharmacia, Uppsala, Sweden) with in intra-assay CV of 6% and interassay CV of 13.8%.","Statistical Analysis Data are expressed as mean ±SD or median (range) as appropriate.","The X2 test was used for analysing categorical data.","Spearman correlation was used for measurement of association between variables.","All statistics was performed with the Statistical Package for Social Sciences (SPSS) for Windows, version 6.1.A P-value<0.05 was considered as significant.","Results The clinical details of the 906 type 2 diabetic patients are shown in Table 5.A significantly higher prevalence of maternal over paternal history of diabetes was found in both early- (Group 1) and late-onset (Group 3) diabetic patients with a positive family history (Table 5).","Amongst the 906 type 2 diabetic patients, in addition to the two patients reported previously (Smith, et al (1997) supra), three more patients carrying the mt3243 mutation were identified.","In Group 1, this mutation was found in 1.8% of (four of 219) early onset patients with a positive family history.","This prevalence increased to 3% (four of 133) if only those with a positive maternal family history were considered.","In addition, one of the 348 late-onset patients without a family history (Group 4) was found to have this mutation (0.3%).","None of the 128 early onset patients who had no family history (Group 2) or 211 late-onset patients with a positive family history (Group 3) or 213 control subjects had the mutation.","Amongst the five probands with the mt3243 mutation, three families were recruited (FIG.","8).","In family A, family members with diabetes or IGT were identified but none of them carried the mutation.","In Family E, two more subjects were found to have the mutation of whom one had diabetes.","The clinical and biochemical characteristics of subjects carrying the mt3243 mutation are summarized in Table 6.The percentage of mt3243 varied from 1% to 14% (Table 6).","There was no correlation of heteroplasmy level of mutation with levels of HbA1c fasting plasma glucose, C-peptide, insulin, insulin resistance or the presence of sensorineural impairment (P>0.05) (Table 6).","Families A and B These two families have been reported in a previous study (Smith, et al, (1997) supra).","The 37-year-old proband (II 4) in family A had been treated with oral drugs since diagnosed at the age of 32 years.","The mother (I-2) and two siblings (II-1 and II-3) were diabetic while the father (I-1) and one sister (II-2) had IGT.","However, none of the family members had the mt3243 mutation although the mother had a history of hearing loss.","The proband of family B was diagnosed as having diabetes at the age of 22 years and had been treated with oral drugs.","The mother was diabetic and deaf but was not available for screening.","Family C The 38-year-old proband (III-3) was diagnosed with diabetes at the age of 31 years.","She was treated with diet and oral drugs for 6 years before being changed to insulin therapy.","Audiometry revealed bilateral high tone sensorineural impairment.","The father (II-1) had normal glucose tolerance and did not have the mt3243 mutation.","The older sister (III-2) and the mother (II-2) both developed diabetes at about 40 years of age and the grandmother had diabetes at the age of 50 years.","The mother became deaf at the age of 59 years.","None of these affected members were available for screening.","TABLE 5 The clinical and biochemical features of 902 Chinese patients with type 2 diabetes Group 1 Group 2 Group 3 Group 4 (n = 219) (n = 128) (n = 211) (n = 348) Age of diagnosis (year) 32 ± 6 32 ± 7 52 ± 8 57 ± 9 Duration of disease (years) 4 (0-31) 2 (0-41) 5 (0-24) 4 (0-26) Sex (M/F) (%) 36/64 36/64 44/56 41/59 Family history of diabetes Father (%) 45* 0 21** 0 Mother(%) 61 0 38 0 Siblings(%) 35 0 57 0 At least 1 parent and 1 sibling(%) 26 0 19 0 Body mass index (kg/m2) 25.7 ± 4.8 24.9 ± 4.4 24.4 ± 3.8 24.3 ± 3.8 HbAlc (%) 7.3 (4.1-15.3) 7.1 (3.8-16.8) 7.7 (4.0-16.0) 7.6 (4.2-19.7) Fasting plasma glucose (nmol/l) 7.4 (4.4-23.0) 7.7 (2.8-21.4) 7.8 (3.9-34.0) 8.1 (3.0-24.5) Fasting plasma C-peptide (nmol/l) 0.47 (0.03-4.96) 0.56 (0.09-1.62) 0.57 (0.01-9.40) 0.51 (0.01-8.22) Insulin deficiency (%)† 16 11 12 16 Insulin treatment(%) 11 13 9 11 Mean ± SD or median (range).","Group 1: early onset (40 years) patients with a family history of diabetes; Group 2: early onset patients without a family history of diabetes; Group 3: late-onset patient (>40 years) with a family history of diabetes; Group 4: late-onset patients without a family history of diabetes.","†Insulin deficiency defined as fasting plasma C-peptide 0.2 nmol/l (Service, et al (1997) supra); *P < 0.005 and **P < 0.0001 for comparison between prevalence rates of paternal vs. maternal family histories.","TABLE 6 The clinical and biochemical characteristics of Chinese subjects carrying the mt3243 mutation in the mitochondrial tRNALeu gene Family A Family B Family C Family D Family E proband proband proband proband proband II-2 II-4 Age of diagnosis (yr) 32 22 31 70 33 38 — Duration of disease (yr) 2 1 7 9 2 1 — Sex F M F M M F F Body mass index (kg/m2) 18.2 22.4 18.6 25.3 27.6 21.6 19.6 Waist-to-hip ratio 0.79 0.81 0.75 0.90 0.86 0.79 0.75 HbA1c (%) 5 7.7 6.8 5.9 11.3 7.5 4.3 Fasting glucose(mmol/l) 5.3 11.6 9.8 5.8 15.7 7.1 4.4 Fasting C-peptide (nmol/l) 0.43 0.23 0.30 0.7 0.72 0.83 0.4 Fasting insulin (mIU/I) 13.4 26.0 ND ND 20.6 16.8 13.2 Insulin resistance* 3.2 13.4 5.5 ND 14.4 5.3 2.6 Treatment Oral drugs Oral drugs insulin Oral drugs Oral drugs Oral drugs — Audiogram Normal Normal High tone High tone Normal ND ND impairment Impairment Mt3243 level (%) 13 1 14 1 5 9 4 *HOMA method, ND, not determined Family D The 79-year-old proband was diagnosed with diabetes at the age of 70 years.","An audiometry test revealed high tone sensorineural impairment.","Neither of the parents and nor any siblings were available for screening or known to have diabetes.","Family E The 35-year-old proband (II-3) was diagnosed with diabetes at the age of 33 years and was treated with diet.","One of the sisters (II-4) had normal glucose tolerance while two sisters (II-1 and II-2) were diagnosed to have diabetes at the age of 30 and 38 years, respectively.","The father (I-1) and mother (I-2) also had diabetes at the age of 50 and 35 years, respectively.","All the diabetic and nondiabetic sisters who came for screening had the mt3243 mutation.","One of the diabetic sisters (II-1) had high tone sensorineural impairment whereas the audiogram of the proband was normal.","EXAMPLE 3 The Role of the Amylin Gene S20G Mutation in Early Onset Type 2 Diabetes and in the Regulation of Cholesterol Metabolism in Chinese This example illustrates the distribution of the amylin gene S20G mutation in Hong Kong Chinese with or without Type 2 diabetes, and its influences on β-cell function and metabolic profiles.","The data are consistent with the conclusion that the S20G mutation in the amylin gene may contribute to early occurrence of Type 2 diabetes, and that it may also influence lipid metabolism in the Chinese population.","Experimental Design and Methods Subjects The study protocol was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong.","Informed consent was obtained from each of the participants.","For the study, 227 early-and 235 late-onset Type 2 diabetic patients (defined as age at diagnosis ≦40 and >40 years respectively), as well as 126 non-diabetic subjects (defined as fasting plasma glucose <6 mmol/1), were consecutively recruited at the Diabetes Centre of the Prince of Wales Hospital.","Type 2 diabetes was asscertained according to the World Health Organisation criteria (Anonymous (1997) Diabetes Care 20: 1183).","None of the patients had typical presentations of Type 1 diabetes, such as acute symptoms and heavy ketonuria (>3+), history of diabetic ketoacidosis or continuous need for insulin treatment within I year of diagnosis.","Patients who had anti-glutamic acid decarboxylase autoantibody (Ko, et al (1998) Ann Clin Biochem 35: 761) diabetes-causing mutations in the glucokinase and hepatonuclear factor-1α and 4-α genes (Ng et al (1999) Diabetic Medicine 16: 956) were excluded from the study.","Clinical and Biochemical Measurements Patients fasted at least 8 hours prior to their clinical examinations.","Blood pressures were taken, after they remained sitting for at least for 5 min using a standard mercury sphygmomanometer.","Body height and weight, and waist and hip circumferences were taken while the patients were standing in light clothing but wearing no shoes.","Measurements of fasting plasma glucose and lipids were performed by routine assays in the Department of Chemical Pathology at the Prince of Wales Hospital.","Levels of total cholesterol and triglyceride were assayed enzymatically with commercial reagents(Centrichem, chemistry system, Baker Instrument Co., Allentown, Pa.).","HDL-cholesterol was determined after fractional precipitation with dextran sulfate-MgCl2 and LDL-cholesterol, calculated by the Friedewald's equation (Friedewald et al (1972) Clin Chem 18:499).","HbA1c was measured using an automated ion-exchange chromatographic method (BioRad, Hercules, Calif., USA; normal range: 5.1-6.4%).","Plasma levels of C-peptide were measured by radioimmunoassays using commercial kits (#7350104 and #141 respectively, Novo Nordisk, Denmark).","The detection range was from 0.01 to 1.0 pmol/1.Insulin deficiency was defined as fasting plasma C-peptide level <0.2 pmol/1 (Service et al (1997) Diabetes Care 21: 987).","Mutation Detection The S20G mutation creates a MSP I restriction fragment length polymorphism (RFLP), which can be detected using PCR-RFLP analysis (Sakagashira et al (1996) Diabetes 45: 1279).","Briefly, DNA fragments spanning the mutation site were amplified by PCR using the primers 5′-TCACATTTGTTCCATGTTAC-3′ (SEQ ID NO: 30) and 5′-CAATAACTATAGAGTTACATTG-3′ (SEQ ID NO: 31), at the annealing temperature of 56° C. Each of the PCR products was then digested overnight with 5 units of the restriction enzyme MSP I (#R6401, Promega, Wis., USA) at 37° C. Alleles were separated on 2.5% agarose gel.","The wild-type allele showed a 239 bp product whereas the mutant allele showed 99 and 140 bp products.","Statistical Analysis Continuous variants were expressed as mean ±SD.","Chi-square test was used for the analysis of proportions.","Differences between continuous variables were analysed by the student's t-test using the statistical package for social sciences (SPSS Inc., Chicago, USA).","ap value <0.05 was considered to be statistically significant.","Results Table 7 summaries the demographic data of the subjects involved in the study.6 early-and 1 late-onset patients heterozygous for the amylin S20G mutation (2.6% vs 0.4%, p=0.055) were identified.","None of the non-diabetic subjects had the S20G mutation (Table 7).","In the early-onset group, 5 out of the 6 mutation-carrying patients had satisfactory glycemic control with diet and/or oral drug medications, and had fasting plasma C-peptide concentrations of greater than 0.2 pmol/1 (Table 8).","Moreover, the mutation carriers had lower total cholesterol (4.3±0.9 vs 5.3 1.1, p=0.02) and LDL-cholesterol (2.3±0.7 vs 3.2±0.9, p=0.01) (Table 9) than those without the mutation.","The patients with or without the S20G mutation were of a comparable age (34±6 vs 35±8, p>0.05).","TABLE 7 Clinical characteristics of the early and late-onset patients as well as non-diabetic subjects, and the distribution of the amylin gene S20G mutation.","Type 2 diabetes Control subjects Early-onset Late-onset Clinical characteristics n 126 227 235 Age (years) 34.9 ± 10.4 36.8 ± 6.7 59.4 ± 10.1 Sex ratio (M/F) 1:1.75 1:1.97 1:1.33 Age of diagnosis (years) NA 31.7 ± 4.6 54.3 ± 9.8 Body mass index (kg/m2) 22.3 ± 3.4 25.1 ± 4.5 24.2 ± 3.6 Waist to hip ratio 0.77 ± 0.05 0.85 ± 0.07 0.89 ± 0.06 Systolic blood 114 ± 10 119 ± 17 137 ± 21 pressure (mmHg) Diastolic blood 64 ± 9 76 ± 10 82.0 ± 11 pressure (mmHg) HbAlc (%) — 7.6 ± 2.0 8.1 ± 2.2 Total cholesterol (mmol/l) 4.7 ± 0.9 5.3 ± 1.2 5.6 ± 1.2 HDL-cholesterol (mmol/l) 1.4 ± 0.3 1.3 ± 0.4 1.3 ± 0.4 LDL-cholesterol (mmol/l) 2.9 ± 0.8 3.2 ± 0.9 3.6 ± 1.1 Triglyceride (mmol/l) 0.9 ± 0.5 1.7 ± 1.8 1.8 ± 1.5 Genotypes Wild-type allele 126 221 234 homozygotes Heterozygotes 0 6 1 Mutant allele 0 0 0 homozygote S20G allele 0 2.6‡ 0.4 frequency (%) Mean ± SD; ‡p = 0.055 TABLE 8 Fasting plasma levels of glucose, HbA1c and C-peptide in early-onset Type 2 diabetic patients with an amylin gene S20G mutation C- Onset Duration peptide HbA1c Glucose Patient age (years) Treatment (pmol/l) (%) (mmol/l) Index a 29 1 Diet >1.0 6.2 5.3 Index b 25 18 Oral drugs >1.0 7.2 7.2 Index c 35 3 Diet >1.0 8.2 9.3 Index d 28 2 Diet + oral 0.02 5.4 4.9 Index e 36 1 Diet + oral 0.51 5.8 14.0 Index f 13 13 Insulin — 11.5 7.2 TABLE 9 Comparisons of clinical characteristics and biochemical measurements between early-onset S20G mutation-carrying patients and early-onset patients without the S20G mutation.","Patients Age Sex BMI WHR SBP DBP HbA1c Triglyceride Total-C HDL-C LDL-C Index a 30 F 23.2 0.76 114 79 6.2 0.76 3.8 1.04 2.5 Index b 43 F 17.9 0.78 138 74 7.2 0.46 5.8 2.21 3.3 Index c 38 M 28.2 0.88 128 90 8.2 2.90 4.7 1.06 2.3 Index d 30 F 23.2 0.75 105 66 5.4 0.99 4.0 1.37 2.2 Index e 37 F 24.0 0.89 126 80 5.8 2.76 3.3 0.87 1.2 Index f 26 M 29.1 0.89 120 81 11.5 0.59 4.2 1.71 2.2 Mutation + (n = 6) 34 ± 6 — 24 ± 4 0.8 ± 0.1 122 ± 16 78 ± 8 7.4 ± 2.0 1.4 ± 1.1 4.3 ± 0.9 1.3 ± 0.5 2.3 ± 0.7 Mutation − (n = 221 35 ± 8 — 25 ± 4 0.9 ± 0.1 119 ± 17 76 ± 10 7.6 ± 2.3 1.7 ± 2.5 5.3 ± 1.1* 1.3 ± 0.4 3.2 ± 0.9** Mean ± SD; *p = 0.02; **p = 0.01 BMI, body mass index (kg/m2); WHR, waist to hip ratio; SBP, systolic blood pressure (mmHg); DBP, diastolic blood pressure (mmHg); Total-C, total cholesterol (mmol/l); HDL-C, HDL-cholesterol (mmol/l); LDL-cholesterol (mmol/l).","The genetic association between the S20G mutation and early-onset Type 2 diabetes (Table 7 ; Sakagashira et al (1996) supra) is consistent with the physiological data that amylin may play a role in the pathogenesis of the disease (Cooper (1994) Proc Natl Acad Sci 84: 8628).","Early onset of Type 2 diabetes is in fact not uncommon (Rosenbloom et al (1999) Diabetes Care 22: 345), although type 2 diabetes is classically a late-onset disease.","However, early-onset patients appear to be heterogenous in etiology.","In Hong Kong Chinese, in particular, maturity-onset Type 2 diabetes of the young as well as atypical autoimmune diabetes are present, but accounting for only a small proportion of the overall early-onset population (Ng et al (1999) supra; Ko et al (1998) supra).","The S20G mutation may also explain some of the early-onset cases.","The S20G mutation carriers usually did not require insulin for glycemic control, and did not appear to be insulin deficient (Table 8).","These findings are different from previous observations that the S20G mutation may be associated with poor glycemic control as well as β cell dysfunction (Sakagashira et al (1996) supra; Chuang et al (1998) supra).","The reported Japanese S20G carriers (Sakagashira et al (1996) supra) had an average diabetes duration of approximately 20 years at the time they were tested.","That they commonly required insulin treatment may be due to the deterioration in glycemic control during their long diabetes course, not necessary the presence of the mutation.","Moreover, the mutation appears to be associated with lower plasma levels of total cholesterol and LDL-cholesterol (Table 9).","This is in keeping with the recent finding that pramlintide (a synthetic human amylin analog) was able to lowers plasma levels of total cholesterol and LDL-cholesterol in Type 2 diabetic patients (Thompson et al (1998) Diabetes Care 21: 987).","Few studies to date have been focused on the relationships between amylin action and lipid profiles These data and those from Thompson and co-workers are consistent with the conclusion that amylin may also play a role in the regulation of cholesterol metabolism.","EXAMPLE 4 The Significant Roles of Genetics and Obesity in Familial Early-Onset Type 2 Diabetes in Chinese Patients This example illustrates the prevalence of known molecular defects in separate cohorts of Chinese patients with early-and late-onset familial Type 2 diabetes.","The genes studied are those that have been found to be associated with diabetes and which may contribute to early onset of the disease under gene-gene and gene-environmental influences, including glucokinase (MODY2), HNF-1α (MODY3), and the A3243G mutation in the mitochondrial DNA coding for tRNALeu(UUR) (mt3243) that has been associated with a form of diabetes characterized by maternal inheritance and deafness (van den Ouweland, et al (1992) Nature Genet.","1: 368).","Experimental Design and Methods Subjects The Prince of Wales Hospital (PWH) is a regional teaching hospital in Hong Kong.","Its catchment area has a population of 1.2 million, accounting for 20% of the total population in Hong Kong.","There is a lack of long term health care programs in Hong Kong, and medical insurance is not widely available.","Many patients with chronic diseases such as diabetes are managed in public hospitals or clinics where they pay only a nominal fee.","Hence, except for high social classes, the patients are largely representative of the diabetic population in Hong Kong.","Since 1995, all patients attending the diabetes clinic of the PWH have been entered into the PWH Diabetes Registry after undergoing a structured assessment (Piwemetz, et al (1993) Diabetic Med 10:371; Chan, et al (1997) Hosp Auth Qual Bull 2:3).","During the study period, a separate cohort of 150 young patients with early-onset diabetes (age ≦40 years and age at diagnosis ≦35 years) who underwent the structured assessment were recruited consecutively from the diabetes clinics at the PWH to form the Young Chinese Diabetes Database (Ko, et al (1998) Ann Clin Biochem 35: 761).","The 150 cases in the Young Chinese Diabetes Database, 92 and 53 patients, respectively, were selected for the present study as they satisfied the following criteria: All these 145 young patients had early-onset (current age and age at diagnosis ≦40 years) Type 2 diabetes (1985 WHO criteria, Geneva) and a positive family history for diabetes (at least 1 first degree relative with diabetes).","Patients with classical Type 1 diabetes (acute ketotic presentation or continuous requirement of insulin within 1 year of diagnosis) were excluded from the study.","The prevalence of anti-GAD (Ko, et al (1998) supra), mt3243 (Smith, et al (1997) Diabetic Med 14: 1026; Ng, et al (2000) 52: 557) and amylin gene mutations (Lee, et al (2001) J. Endocrinol) amongst patients from the Young Chinese Diabetes Database has been reported.","Additionally, the prevalence of mt3243 (Ng, et al (2000) supra), amylin (Lee, et al (2001) supra), glucokinase, HNF-1α and HNF-4α gene mutations (Ng, et al(1999) Diabetic Med 16:956) in a separate cohort from the PWH Diabetes Registry has been reported.","In this study, screening for glucokinase and HNF-1α gene mutations was extended to the 53 patients from the Young Chinese Diabetes Database.","The HNF-4α gene was not screened in this cohort due to the expected low frequency of mutations.","(None were found in the 92 patients from the PWH Diabetes Registry (Ng, et al (1999) supra)).","Screening for anti-GAD was extended to the 92 patients from the PWH Diabetes Registry.","Nineteen (13%) of these 145 young patients with familial diabetes met the minimal criteria for MODY (age at diagnosis ≦25 years and presence of diabetes in two consecutive generations).","Altogether 10 out of 20 families with probands carrying putative diabetogenic gene mutations were recruited for a 75-gram OGTT and clinical assessment.","The 1999 WHO classification was used to define the glycemic status of the family members (WHO, Geneva, 1999).","For comparison of clinical characteristics of the early-onset patients, 290 sex-matched patients with late-onset diabetes (age at diagnosis >40 years) and a positive family history of diabetes were randomly selected from the current 1800 cases in the PWH Diabetes Registry.","One hundred healthy Chinese (age 33±10 years, 40 males and 60 females) were selected as control subjects from hospital staff and students for screening for the gene variants identified in the study patients.","Informed consent was obtained from each subject for a blood sample to be taken for DNA extraction and measurement of biochemical indices.","This study was approved by the Clinical Research Ethics Committee of The Chinese University of Hong Kong.","Clinical Studies All patients underwent a structured assessment based on the Europe DiabCare Protocol.","They had documentation of their family history of diabetes, age at diagnosis and anthropometric indices (Piwemetz, et al (1993) supra; Chan, et al (1997) supra).","Body mass index (BMI) was used as an index of general obesity.","Waist circumference, which is highly correlated in Chinese with visceral fat accumulation measured by magnetic resonance imaging (Anderson, et al (1997) Diabetes Care 20: 1854), was used as an index of central obesity.","After an overnight fast, venous blood was sampled for measurement of plasma glucose, insulin, HbA1c, total cholesterol (TC), HDL-C, LDL-C (calculated), triglyceride (TG) and anti-GAD.","A morning spot urine sample was collected for assessment of albuminuria.","Retinopathy and sensory neuropathy were assessed as previously described (Ko, et al (1999) J Diabetes Complications 13: 300).","General obesity was defined as a BMI≧25 kg/m2 using the recent Asian criteria (WHO, Western Pacific Region, 2000).","Albuminuria was defined as an albumin: creatinine ratio (ACR)≧3.5 mg/mmol in a spot urine sample (Schwab, et al (1992) Diabetes Care 15: 1581).","The HOMA IR index (fasting plasma insulin×glucose/22.5) derived from the HOMA equation was used to assess insulin resistance (Matthews, et al (1985) Diabetologia 28: 412).","Biochemical Assays Plasma glucose, HbA1c, lipids, urinary albumin and creatinine were measured by routine assays in the Department of Chemical Pathology at the PWH (see Chan, et al (1996) Diabetic Med 13:150).","Plasma insulin was measured in non-insulin treated patients by a radioimmunoassay (Pharmacia, Sweden) with intra-and inter-assay CVs of 6% and 13.8%, respectively.","Anti-GAD was measured by a radioimmunoprecipitation assay (Chen, et al (1993) Pediatr Res 34: 785).","The upper normal limit of 18 units, is applicable to Asian and European subjects (Tuomi, et al (1995) Clin Immunol Immunopath 74: 202, Chen, et al (1993) supra).","Genetic Analysis The minimal promoter regions and exons of the glucokinase (β-cell form), HNF-1α and HNF-4α (HNF-4α 2 form) genes were screened for mutations by direct sequencing of PCR products (see Froguel, et al (1993) N Engl J Med 328: 697; Yamagata, et al (1996) Nature 384: 455; Yamagata, et al (1996) Nature 384: 458).","One previously unreported mutation in HNF-1α (A116V) and two previously unreported mutations in glucokinase (V101M and Q239R) were identified in this study.","HNF-1α A116V was screened by using the forward primer 5′-CATGCACAGTCCCCACCCTCA-3′ (SEQ ID NO:34) and reverse primer 5′-TCCCACTG ACTTCCTTTCC-3′ (SEQ ID NO:35) for PCR amplification followed by digestion with HphI.","The wild-type allele showed 44 and 397 bp products whereas the mutant allele showed 44, 136 and 261 bp products.","Glucokinase V101M was screened by using the forward primer 5′-GTCCCTGA-GGCTGACACACTT-3′ (SEQ ID NO: 24) and reverse primer 5′-AGCTGGGCCCTGAGATCC-TGCA-3′ (SEQ ID NO: 25) for PCR amplification followed by digestion with Hsp9211.The wild-type allele showed 20, 56 and 174 bp products whereas the mutant allele showed 20,42,56, and 132 bp products.","Glucokinase Q239R was screened by using the forward primer 5′-AGGAACC-AGGCCCTACTCCG-3′ (SEQ ID NO: 36) and reverse primer 5′-TACTCCAGCAGGAACTC-GTCC-3′ (SEQ ID NO: 37) for PCR amplification followed by digestion with Acil.","The wild-type allele showed 70 and 134 bp products whereas the mutant allele showed 33,70 and 101 bp products.","The occurrence of putative mutations in family members of the probands (Table 10) and control subjects was determined by PCR-RFLP.","Mt3243A→G and amylin gene S20G mutations were determined by PCR-RFLP as described (Sakagashira, et al (1996) Diabetes 45: 1279; Smith, et al (1997) supra).","Statistical Analysis Normally distributed data are expressed as mean ± SD.","Data with skewed distributions were normalised by logarithmic transformation.","The resultant means were antilogarithmically transformed and expressed as geometric mean together with 25 and 75 percentiles.","Chi square test and Student's unpaired t tests were used for between-group comparisons.","A p value<0.05 (2-tailed) was considered to be significant.","All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS for Windows, version 9.0).","Results Prevalence of Putative Gene Mutations and Anti-Gad in Patients with Familial Early-Onset Diabetes Amongst the 145 patients with familial early-onset diabetes, there were 20 (14%) with putative mutations, involving the HNF-1α gene, 7 (5%), glucokinase gene, 6 (4%), mt3243,4 (3%), and amylin S20G, 3 (2%).","Anti-GAD was positive in 6 (4%).","No mutation in the HNF-4α gene was found in the 92 patients from the PWH Diabetes Registry.","All mutations identified in the HNF-1α and glucokinase genes were previously unreported (Table 10).","The HNF-1α G20R and glucokinase Q239R mutations were found in 4 unrelated patients.","None of these mutations were found in 100 healthy control subjects.","Family Cosegregation Study of Gene Mutations Amongst the 20 patients carrying putative gene mutations, 10 families were recruited for cosegregation study (FIG.","9).","Cosegregation of a mutation with clinical diabetes or glucose intolerance were observed in 4 families: HK10 with HNF-1α IVS2nt-1G→A, YDM142 with glucokinase V101M, HK84 with glucokinase I110T and HK50 with mt3243.Segregation was inconclusive in the other 6 families.","For the families HK54 with HNF-1α R203H, YDM83 with mt3243 and CX216 with amylin S20G mutations, only the probands, and none of the diabetic or non-diabetic family members who presented for screening, carried the gene mutations.","For YDM67 with glucokinase Q239R, HK61 with mt3243 and YDM99 with amylin S20G mutations, the mutations were found in both diabetic and non-diabetic family members.","Amongst the 3 families with glucokinase mutations, all mutation carriers from the families YDM142 and HK84 had higher fasting plasma glucose concentrations (5.8-8.9 mmol/1) than those with no mutation (4.2-5.3 mmol/1).","On the other hand, the 4 mutation-carrying siblings of proband YDM67 had a normal fasting plasma glucose concentration (4.0-5.6 mmol/1) irrespective of their glycemic status.","TABLE 10 Mutations in the HNF-1 α and glucokinase genes in Chinese subjects with early-onset diabetes mellitus Subject Location Codon/nt Nucleotide change Designation HNF-1 αmutation HK90*, YDM42† Exon 1 20 GGG (GLy)→AGG (Arg) G20R YDM20† Exon 2 116 GCG (Ala)→GTG(Val) A116V HK10* Intron2/Exon3 nt-1 AG→AA at splice acceptor site IVS2nt-1G→A HK54* Exon 3 203 CGT (Arg)→CAT (His) R203H HK30* Exon 6 432 TCC (Ser) →TGC (Cys) S432C HK92* Exon 10 618 ATC (Ile)→ATG (Met) 1618M Glucokinase mutation YDM142† Exon 3 101 GTG (Val)→ATG (Met) V101M HK84* Exon 3 110 ATC (Ile)→ACC (Thr) I110T HK38* Exon 3 119 GCT (Ala)→GAT (Asp) A119D YDM67†, YDM144† Exon 7 239 CAG (Gln)→CGG (Arg) Q239R HK15* Exon 9 385 GGG (Gly)→GTG(Val) G385V *reported in previous studies (Ng, et al (1999) Diabetic Med 16: 956; Ng, et al (2000) Diabetologia 43: 816) †newly found in the present study Clinical Characteristics of Patients with Familial Early-Onset Diabetes of Unknown Cause Compared with Familial Late-Onset Diabetes Although 26 of the patients with early-onset diabetes carried putative gene mutations associated with diabetes or the autoimmune indicator, anti-GAD antibodies, the causes of diabetes in the other 119 patientsremain to be determined.","These young patients with diabetes of unknown cause (age at diagnosis 30±6 years) differed clinically from the 290 late-onset patients (age at diagnosis 52±8 years) (Table 11).","Thus, despite a positive family history of diabetes in all patients in both groups, those with early-onset diabetes more frequently had a father with diabetes (39% vs. 22%) and a mother with diabetes (63% vs. 41%), but less frequently a sibling with diabetes (30% vs. 53%) (p<0.001).","The early-onset patients had a higher BMI but lower BP and increased prevalence of retinopathy and neuropathy as compared to the late-onset patients.","The early-onset patients had better glycemic control (glucose and HbA1c) as well as higher fasting insulin concentrations than the late-onset patients.","Notwithstanding similar mean disease duration of only 4 years, both the early-and late-onset patients had a disproportionately high prevalence of albuminuria, 40% and 38%, respectively, as compared with the prevalence rates of other microangiopathic complications.","Insulin resistance, as assessed by the HOMA IR index, was similar between the two groups of non-insulin treated patients.","The proportion of patients treated with insulin was similar in both groups (8% vs. 7%) but fewer patients with early-onset diabetes were treated with oral drugs (33% vs. 61%, p<0.001) as compared to the late-onset group.","Clinical Characteristics of the Patients with Familial Early-Onset Diabetes of Unknown cause and Familial Late-Onset Diabetes Classified According to Obesity Index Due to the high prevalence of general obesity in both early-onset patients of unknown cause and late-onset patients (55% and 46%, respectively), the association of obesity with cardiovascular risk factors and complications in these patients was further analyzed (Table 11).","Amongst the early-onset patients, the obese patients had worse glycemic control (HbA1c) as well as a higher systolic BP, a more adverse lipid profile (higher TG, lower HDL-C and higher TC/HDL-C), and higher fasting insulin than the non-obese patients.","They were also more insulin resistant (HOMA IR index) and had a higher prevalence of retinopathy and albuminuria than the non-obese patients.","Amongst the late-onset patients, the obese patients had better glycemic control (glucose and HbA1c) than the non-obese patients.","However, they had a higher systolic and diastolic BP, and a higher fasting insulin than the non-obese patients.","The degree of insulin resistance and prevalence of complications were similar in the two groups.","TABLE 11 Comparison of clinical features of Chinese patients with familial Type 2 diabetes according to age of diagnosis of diabetes and obesity status Early-onset Late-onset Early-onset patients Late-onset non-obese Early-onset non-obese Late-onset with unknown etiology patients patients obese patients patients obese patients N 119 290 54 65 156 134 Sex Male (%) 37 (31) 98 (34) 12 (22) 25 (38) 56 (36) 42 (31) Female (%) 82 (69) 192 (66) 42 (78) 40 (62) 100 (64) 92 (69) Current age (yr) 34 ± 5 56 ± 9‡ 34 ± 5 33 ± 5 56 ± 10 55 ± 9 Age at diagnosis (yr) 30 ± 6 52 ± 8‡ 31 ± 5 29 ± 6 52 ± 8 52 ± 8 Duration of disease (yr) 4.0 ± 3.9 4.0 ± 4.2 3.9 ± 3.8 4.2 ± 4.0 4.5 ± 4.4 3.5 ± 3.9† Family history Father 46 (39) 64 (22)‡ 21 (39) 27 (42) 28 (18) 36 (27) Mother 75 (63) 119 (41)‡ 38 (70) 39 (60) 65 (42) 54 (40) Sibling 36 (30) 154 (53)‡ 15 (28) 20 (31) 85 (54) 69 (51) BMI (kg/m2) 26.2 ± 4.7 25.0 ± 3.7† 22.3 ˜ 1.8 29.5 ± 3.8‡ 22.4 ± 1.8 28.0 ± 3.1‡ Waist circumference (cm) Male 90 ± 11 87 ± 9 78 ± 6 95 ± 9‡ 81 ± 6 94 ± 7‡ Female 81 ± 11 83 ± 9 74 ± 5 89 ± 10‡ 78 ± 6 89 ± 8‡ Systolic BP (mmHg) 117 ± 14 136 ± 22‡ 114 ± 13 120 ± 14† 134 ± 23 139 ˜ 21† Diastolic BP (mmHg) 75 ± 9 83 ± 11‡ 74 ± 9 77 ± 10 80 ± 11 86 ± 12‡ Triglyceride (mmol/l) 1.4 (0.9-2.0) 1.4 (1.0-2.0) 1.0 (0.7-1.5) 1.7 (1.0-2.4)‡ 1.4 (0.9-1.9) 1.6 (1.1-2.1) Total cholesterol (mmol/l) 5.3 ± 1.2 5.6 ± 1.3 5.1 ± 1.0 5.4 ± 1.4 5.6 ± 1.3 5.5 ± 1.2 HDL-C (mmol/l) 1.2 ± 0.3 1.2 ± 0.3 1.3 ± 0.3 1.1 ± 0.3‡ 1.3 ± 0.4 1.2 ± 0.3 TC/HDL-C 4.7 ± 1.8 4.7 ± 1.5 4.0 ± 1.0 5.3 ± 2.2‡ 4.7 ± 1.7 4.7 ± 1.3 LDL-C (mmol/l) 3.3 ± 0.9 3.5 ± 1.0 3.2 ± 0.8 3.4 ± 1.0 3.5 ± 1.0 3.5 ± 1.0 Fasting glucose (mmol/l) 8.2 ± 3.1 9.1 ± 3.6† 7.6 ± 2.8 8.7 ± 3.3 9.6 ± 3.8 8.6 ± 3.3† HbA1c (%) 7.5 ± 1.8 8.0 ± 1.9† 7.1 ± 1.8 7.9 ± 1.8† 8.2 ± 2.1 7.7 ± 1.5† Fasting insulin (pmol/l)* 105 (72-164) 87 (51-146)† 89 (60-149) 120 (78-179)† 76 (43-129) 99 (57-157)† HOMA IR index* 34.7 (22.9-55.8) 33.1 (19.2-62.8) 27.5 (16.0-46.7) 42.7 (27.6-58.2)† 29.9 (14.7-61.9) 36.4 (19.7-64.2) Urinary albumin creatinine 2.6 (0.7-6.1) 2.8 (0.8-7.1) 1.2 (0.6-1.9) 5.1 (0.9-24.1)‡ 2.6 (0.9-5.8) 3.2 (0.8-8.4) ratio (mg/mmol) Treatment (%) Diet 71 (60) 93 (32) 35 (65) 36 (55) 55 (35) 38 (28) Oral drugs 39 (33) 176 (61)‡ 17 (31) 22 (34) 89 (57) 87 (65) Insulin 9 (8) 21 (7) 2 (4) 7 (11) 12 (8) 9 (7) Retinopathy (%) 10 (8) 62 (21)† 1 (2) 9 (14)† 38 (24) 24 (18) Albuminuria (%) 48 (40) 110 (38) 8 (15) 40 (62)‡ 53 (34) 57 (43) Neuropathy (%) 4 (3) 29 (10)† 2 (4) 2 (3) 13 (8) 16 (12) Data are compared between early- and late-onset patients, between early-onset non-obese and obese patients, and between late-onset non-obese and obese patients Data are expressed as n (%), mean ± SD or geometric mean (25 and 75 percentiles) *only measured in patients not treated with insulin †p < 0.05 ‡p < 0.001 EXAMPLE 5 An Illustration of a Chinese Family with Hepatocyte Nuclear Factor-1α Diabetes (MODY3) that Emphasizes the Need for Early Diagnosis and Appropriate Treatment This example reports the clinical course of HNF-1α diabetes/MODY 3 in a Chinese family with early-onset diabetes and severe complications (FIG.","10) (Chan, et al (1990) Diabetic Medicine 7: 211).","This family highlights the importance of early diagnosis and prompt treatment in the improvement of clinical outcome even in genetically susceptible subjects.","Three family members in the proband's family had severe diabetic complications when they were referred for treatment.","The proband (III-5), 19 years of age, had severe proliferative retinopathy, heavy proteinuria (1.4 g protein a day) and necrobiosis lipoidica.","She had been diagnosed with Type 2 (non-insulin-dependent) diabetes mellitus 3 months earlier and was treated with glibenclamide.","Retinal photocoagulation treatment was initiated and she was started on insulin and an ACE inhibitor.","She subsequently developed hypertension and progressed to end-stage renal disease requiring dialysis by the age of 30 years.","Her mean HbA1c was 8.0% over the years.","She is currently receiving 42 units of insulin.","Her older sister (III-2) had a vitreous haemorrhage and had been treated with insulin since diagnosis at the age of 24 years.","She became blind and had nephropathy (0.8 g protein a day) 2 years later.","She is currently treated with insulin (16 units) and an ACE inhibitor, and has a mean HbA , of 6.4%.","The subject's mother (II-3) had a glycosuria complicated pregnancy when she was 33 years old.","She was diagnosed to have Type 2 diabetes at the age of 38 years and was then treated with glibenclamide for 10 years.","At the time of the study she had proliferative retinopathy, nephropathy, peripheral neuropathy, necrobiosis lipoidica, hypertension and cataracts.","Insulin treatment (20 units) was commenced and her HbA1c was reduced from 17.2% to 9.2% within 8 months.","Two months later, she had a myocardial infarction followed by progressive deterioration of cardiac and renal functions.","She died of pulmonary edema and septicaemia with a gangrenous foot at the age of 52 years.","The fourth daughter (III-6) had been treated with insulin since her incidental diagnosis of diabetes at the age of 12 years after a nasal polypectomy.","She is currently receiving 68 units of insulin, and has mean HbA1c of 8.8%.","Two other family members underwent screening by OGTT.","The second daughter (III-3) has fluctuated between having normal glucose tolerance and IGT over the last 11 years.","A brother (III-7) had overt diabetes on screening with an initial HbA1c of 10.5%.","Insulin was started after 3 months of dietary treatment.","He is currently receiving 26 units of insulin, with a mean HbA1c of 5.3%.","One maternal uncle (II-4) was diagnosed with diabetes and hyperlipidnemia with thirst and polyuria at the age of 39 years.","He has been treated with oral drugs since diagnosis, and has mean HbA1c of 8.4%.","His children were not available for detailed genetic testing and clinical assessment.","The affected members II-4, III-6 and III-7 (FIG.","10) have remained free of complications despite all having had diabetes for more than 10 years.","The father was also diagnosed with IGT.","He was non-obese and had hyperlipidaemia.","Sequencing of the HNF-1α gene in this family showed a novel splice acceptor site mutation (AG→AA) in intron 2 (IVS2nt-1G→A) which cosegregated with diabetes (FIG.","10) (Ng, et al (1999) Diabetic Medicine 16: 956).","This mutation is expected to produce a nonfunctional mRNA.","All the diabetic members, including the maternal uncle, (II4, III-2, III-5, III-6 and III-7) were heterozygous for this mutation but the father (II-2) and the daughter (III-3) with IGT did not have the mutation.","Thus it is very likely that the mother (II-3) for whom no DNA sample was available also carried this mutation.","As with other patients with HNF-1α diabetes (Byrne, et al (1996) Diabetes 45: 1503), most affected family members exhibited defective pancreatic beta-cell function as assessed by the glucagon stimulation test.","The mother and all the affected siblings, except subject III-2, were insulin deficient based on a definition of post-glucagon (1 mg intravenously) stimulated plasma C peptide at 6 min of less than 0.6 nmol/1 (0.24-0.55 nmol/I respectively) (Service, et al (1997) Diabetes Care 20: 198).","The brother, III-7, who was diagnosed with diabetes by OGTT was also insulin deficient.","All the HNF-1α mutant carriers, except II-4, required insulin treatment for glycemic control.","Although all affected family members carried the same HNF-1α gene mutation, their clinical courses have varied tremendously.","Severe complications were present in those family members whose diagnosis was delayed and who presumably had poor glycemic control before diagnosis (II-3, III-2 and III-5).","Complications were, however, absent in the uncle (II-4) and the younger siblings (III-6 and III-7) despite now having had diabetes for more than 10 years (FIG.","10), who were promptly diagnosed and received treatment.","This is in accordance with a recent report suggesting that poor glycemic control is associated with a twofold to threefold increased risk among MODY3 patients of developing microalbuminuria and retinopathy, respectively (Isomaa, et al (1998) Diabetologia 41: 467).","It is noteworthy that both maternal grandparents (I-3 and I-4) were diagnosed with diabetes diagnosed in their late 50 s. The effect of this bilineality on the natural course of HNF-1α diabetes in this family is uncertain.","It is, however, possible the non-MODY maternal grandparent transmitted a modifier gene affecting the age at onset or severity of the diabetes in carriers with the HNF-1α amutation.","The age at diagnosis of diabetes in this family was increasingly younger with successive generations despite all carriers being relatively non-obese.","This earlier diagnosis could be due to ascertainment bias or, more likely, an epiphenomenon due to increasing westernisation of the Hong Kong lifestyle with increased intake of high fat food and decreased physical activity (Chan and Cockram (1997) Diabetes Care 20:1785).","This highlights the important influence of environment interacting with genetics in the natural course of HNF-1α diabetes.","In conclusion, this report emphasizes the need for early diagnosis by glucose tolerance testing or genetic screening, and appropriate treatment in patients who have a strong family history of diabetes, especially those with early onset disease and insulin deficiency.","The data presented in Examples 1-5 demonstrate a combination of genetic mutations that are uniquely associated with the increased risk of a Chinese individual to develop type 2 diabetes.","The mutations are exemplified by, but are not limited to G20R, A116V, IVS2nt→GA, R203H, S432C and I618M of HNF-1α; V101M, I110T, A119D, Q239R and G385V of glucokinase; S20G of amylin; and A3243G of mitochondrial tRNALeu(UUR).","Mutations correlative with a genetic predisposition of a Chinese individual to develop type 2 diabetes are efficiently identified in Chinese families with a positive family history of the disease, but find use in screening any Chinese individual that is asymptomatic but at risk of developing diabetes.","Methods for identification of a combination of at least two genetic mutations correlative with type 2 diabetes in a Chinese individual offers an important tool for clinicians, not only to initiate prophylactic therapies before the onset of overt diabetic symptoms, but also to design therapies that are directed to the specific etiology of the disease in each individual.","All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains.","All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporate by reference."]],"string":"[\n [\n \"Method and compositions for evaluating risk of developing type 2 diabetes in people of chinese descent\",\n \"Methods and compositions for identifying mutations and polymorphisms in mutant genes encoding gene product involved in insulin secretion, for example, hepatocyte nuclear factor-1∝, glucokinase, amylin and mitochondrial DNA are disclosed.\",\n \"Specifically, a microchip comprising a combination of at least two different mutant genes wherein each gene comprises at least one mutation indicative of a predisposition for type-2 diabetes in a member of a Chinese population is disclosed.\",\n \"A kit comprising the microchip, an isolated nucleic acid, primers and probes which are specifically used to screen or identify the mutations in genes of hepatocyte nuclear factor-1∝, glucokinase, amylin and mitochondrial DNA are also disclosed.\"\n ],\n [\n \"1.A microchip comprising: a combination of at least two different mutant nucleic acid sequences of a wild-type nucleic acid sequence, wherein each wild-type nucleic acid sequence encodes a protein involved in insulin secretion, wherein said gene comprises at least one mutation indicative of a predisposition for type 2 diabetes in a member of a Chinese population.\",\n \"2.The microchip according to claim 1, wherein said nucleic acid sequences comprise nucleic acid selected from the group consisting of genomic DNA, complementary DNA and messenger RNA.\",\n \"3.The microchip according to claim 1, wherein said type 2 diabetes is maturity onset diabetes of the young.\",\n \"4.The microchip according to claim 1, wherein said microchip further comprises a genetic marker that uniquely identifies a member of a Chinese population.\",\n \"5.A microchip comprising: a combination of at least two different nucleic acid sequences, wherein each nucleic acid sequence encodes a gene product involved in insulin secretion wherein said gene comprises at least one mutation indicative of a predisposition for type 2 diabetes in a human subject of a Chinese population, wherein said gene product is selected from the group consisting of a glucokinase, a hepatocyte nuclear factor 1α, an amylin and a mitochondrial tRNA (Leu) (UUR).\",\n \"6.A microchip comprising: at least one each of a combination of different nucleic acid sequences, wherein each nucleic acid sequence encodes a protein selected from the group consisting of glucokinase, hepatocyte nuclear factor la, amylin and mitochondrial tRNA(Leu)(UTR), wherein said glucokinase gene comprises at least one mutation selected from the group consisting of V101M, I110T, A119D, Q239R, and G385V, and said hepatocyte nuclear factor la gene comprises at least one mutation selected from the group consisting of G20R, A116V, IVS2nt-G→A, R203H, S432C, and I618M, and said amylin gene comprises the mutation S20G, and said mitochondrial tRNA(Leu)(UUR) gene comprises the mutation A3243G.\",\n \"7.A microchip comprising at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:10.8.A microassay system comprising a microchip according to claim 1.9.A kit comprising a microchip according to claim 1.10.A nucleic acid primer comprised of SEQ ID NO: 34.11.A nucleic acid primer comprised of SEQ ID NO: 35.12.A nucleic acid primer comprised of SEQ ID NO: 36.13.A nucleic acid primer comprised of SEQ ID NO: 37.14.A nucleic acid probe that specifically anneals to a nucleic acid encoding a mutant gene of a wild-type gene involved in insulin secretion, wherein said mutant gene comprises at least one mutation indicative of increased risk for type 2 diabetes in a human subject of a Chinese population, and wherein said nucleic acid probe does not bind to said wild-type gene.\",\n \"15.An isolated nucleic acid encoding a mutant gene of a wild-type gene that encodes a protein involved in the secretion of insulin, wherein said mutant gene comprises at least one mutation associated with increased risk for type 2 diabetes in a subject of a Chinese population.\",\n \"16.The isolated nucleic acid according to claim 15, wherein said mutation is a single nucleotide polymorphism.\",\n \"17.The isolated nucleic acid according to claim 15, wherein said mutation is selected from the group consisting of a missense, a nonsense, an insertion and a deletion mutation.\",\n \"18.The isolated nucleic acid according to claim 15, wherein said wild-type gene encodes hepatocyte nuclear factor la, and said mutation is A116V.\",\n \"19.The isolated nucleic acid according to claim 15, wherein said wild-type gene encodes glucokinase, and said mutation is selected from the group consisting of V101M and Q239R.\",\n \"20.An isolated nucleic acid encoding a mutant gene of a wild-type gene that encodes a protein involved in the secretion of insulin, wherein said mutant gene is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 10.21.An isolated amino acid sequence encoded by a mutant gene of a wild-type gene encoding a protein involved in the secretion of insulin, wherein said mutant gene comprises at least on mutation associated with increased risk for type 2 diabetes in a member of a Chinese population.\",\n \"22.An antibody that specifically binds a protein encoded by a mutant gene of a wild type gene encoding a protein involved in the secretion of insulin, wherein said mutant gene comprises at least on mutation associated with increased risk for type 2 diabetes in a member of a Chinese population, and wherein said antibody does not bind to a protein encoded by said wild-type gene.\",\n \"23.A method of determining a genetic predisposition of a member of a Chinese population to develop type 2 diabetes, said method comprising the step of: contacting a sample comprising nucleic acid from said member with a combination of at least two nucleic acid sequences, wherein each nucleic acid sequence encodes a mutant gene of a wild-type gene encoding a protein involved in insulin secretion, wherein each mutant gene comprises at least one mutation indicative of a predisposition of a member of a Chinese population to develop type 2 diabetes, whereby identification of at least one of said mutations in said sample is indicative of a genetic predisposition for type 2 diabetes in said member of a Chinese population.\",\n \"24.A method for detecting an increased risk of an individual of a Chinese population with decreased insulin secretory function to develop type 2 diabetes, said method comprising the step of: contacting a sample comprising nucleic acid from said individual with a combination of at least two different nucleic acid sequences, wherein each nucleic acid sequence encodes a mutant gene of a wild-type gene encoding a protein involved in insulin secretion, wherein each mutant gene comprises at least one mutation indicative of a predisposition of a member of a Chinese population to develop type 2 diabetes, wherein identification of at least one of said mutations in said sample is indicative of an increased risk for type 2 diabetes in said individual of a Chinese population.\",\n \"25.The method according to claim 23, wherein said combination of at least two different nucleic acid sequences are attached to a microchip.\",\n \"26.The method according to claim 23, wherein said nucleic acid sample is obtained from bodily fluid or tissue.\",\n \"27.The method according to claim 23, wherein said wild-type gene encodes a gene product selected from the group consisting of hepatocyte nuclear factor 1α, glucokinase, amylin and mitochondrial tRNA(Leu)(UUR).\",\n \"28.A method of determining a genetic predisposition of a member of a Chinese population to develop type 2 diabetes, said method comprising the step of: contacting a sample comprising nucleic acid from said member with a combination of at least two different nucleic acid sequences selected from the group consisting of G20R, A116V, IVS2nt-G→A, R203H, S432C, and I618M of hepatocyte nuclear factor 1α; V101M, I110T, A119D, Q239R, and G385V of glucokinase; S20G of amylin, and A3243G of mitochondrial tRNA(Leu)(UUR), wherein each nucleic acid sequence encodes a mutant gene of a wild-type gene encoding a protein involved in insulin secretion, wherein each mutant gene comprises at least one mutation indicative of a predisposition of a member of a Chinese population to develop type 2 diabetes, and wherein said identification of one of said mutations in said sample is indicative of a genetic predisposition for type 2 diabetes in said member of a Chinese population.\",\n \"29.A method for detecting an increased risk of an individual of a Chinese population with decreased insulin secretory function to develop type 2 diabetes, said method comprising the step of: contacting a sample from said individual with a combination of at least two different nucleic acid sequences selected from the group consisting of G20R, A116V, IVS2nt-G→A, R203H, S432C, and I618M of hepatocyte nuclear factor 1α;V101M, I110T, A119D, Q239R, and G385V of glucokinase; S20G of amylin, and A3243G of mitochondrial tRNA(Leu)(UUR), wherein each nucleic acid sequence encodes a mutant gene of a wild-type gene encoding a protein involved in insulin secretion, wherein each mutant gene comprises at least one mutation indicative of a predisposition of an individual of a Chinese population to develop type 2 diabetes, and wherein the identification of at least one of said mutations in said sample is indicative of an increased risk for type 2 diabetes in said individual of a Chinese population.\",\n \"30.A method for screening for genetic mutations in an individual of a Chinese population diagnosed with type 2 diabetes, said method comprising the steps of: contacting a sample from said individual with a combination of at least two different nucleic acid sequences, wherein each nucleic acid sequence encodes a mutant gene of a wild-type gene encoding a protein involved in insulin secretion, wherein each mutant gene comprises at least one mutation indicative of a predisposition of a member of a Chinese population to develop type 2 diabetes, and wherein identification of at least one of said mutations in said sample is indicative of an etiology of said type 2 diabetes in said individual of a Chinese population.\",\n \"31.The method according to claim 30, wherein said individual has been diagnosed with maturity onset diabetes of the young.\",\n \"32.The method according to claim 30, wherein said individual has at least one primary family member that has been diagnosed with maturity onset diabetes of the young.\",\n \"33.The method according to claim 30, wherein said mutation is selected from the group consisting of a missense, a nonsense, an insertion and a deletion mutation.\",\n \"34.A method for screening for genetic mutations indicative of increased risk of an individual of a Chinese population to develop type 2 diabetes, said method comprising the steps of: contacting a sample from said individual with a combination of at least two different nucleic acid sequences selected from the group consisting of G20R, A116V, IVS2nt-G→A, R203H, S432C, and I618M of hepatocyte nuclear factor 1α;V101M, I110T, A119D, Q239R, and G385V of glucokinase; S20G of amylin, and A3243G of mitochondrial tRNA(Leu)(UTR), wherein each nucleic acid sequence encodes a mutant gene of a wild-type gene encoding a protein involved in insulin secretion, wherein each mutant gene comprises at least one mutation indicative of a predisposition of an individual of a Chinese population to develop type 2 diabetes.\",\n \"35.A method for screening for a genetic predisposition to develop type 2 diabetes in an individual of a Chinese population having at least one primary family member that has been diagnosed with type 2 diabetes, said method comprising the steps of: contacting a sample comprising nucleic acid from said individual with a combination of at least two different nucleic acid sequences, wherein each nucleic acid sequence encodes a mutant gene of a wild-type gene encoding a protein involved in insulin secretion, wherein each mutant gene comprises at least one mutation indicative of a predisposition of a member of a Chinese population to develop type 2 diabetes, and wherein identification of at least one of said mutations in said sample is indicative of a genetic predisposition to develop type 2 diabetes in said individual of a Chinese population.\"\n ],\n [\n \"<SOH> BACKGROUND <EOH>Although people of Chinese ancestry account for >20% of the world's population (Chan, et al(1997) 20: 1785), very little is known about the genetic factors that contribute to the development of diabetes in this population.\",\n \"The prevalence of diabetes amongst Chinese people varies from <1% in some rural areas in mainland China to 6-12% in Hong Kong, Singapore, and Taiwan (Chan, et al (1997), supra).\",\n \"Hong Kong can be regarded as a paradigm of future China.\",\n \"The prevalence of diabetes mellitus is reaching epidemic proportions amongst Hong Kong Chinese, with type 2 diabetes being the predominant form in pateints with early-or late-onset of disease (Chan and Cockram (1997) Diabetes Care 20: 1785).\",\n \"Type 2 diabetes mellitus is a heterogeneous disease that is caused by both genetic and environmental factors.\",\n \"The age-adjusted prevalence of diabetes in the Chinese population has increased from 7.7% in 1990 (Cockram, et al (1993) Diabetes Res and Clin Practice 21: 67) to 8.9% in 1995 (Cockram and Chan (1999) In: Diabetes in the New Millennium, Pot Still Press, Sydney, pp.\",\n \"11-22).\",\n \"In a population-based study conducted in 1995, the crude prevalence of diabetes mellitus was 9.6%, rising from 1.7% in those aged under 40 years to 25% in those older than 60 years (Janus (1997) Clin Exp Pharmacol Physiol 24: 987).\",\n \"There is a high prevalence of obesity (43%) and positive family history of diabetes (50%) in Chinese patients presenting with acute or early onset diabetes (Chan, et al (1993) Postgrad Med J 69: 204; Ko, et al (1998) 35: 761).\",\n \"These findings indicate that genetic factors, in addition to environmental factors, can be an important cause of early onset diabetes in this population.\",\n \"Because type 2 diabetes is an insidious disease, it is estimated that as many as half of the individuals in Hong Kong that would be considered diabetic remain undiagnosed.\",\n \"Most patients are finally diagnosed only when presenting with overt symptoms that often are the consequence of advanced disease.\",\n \"Clinic as well as population-based studies reveal that about 17% of diabetic patients in Hong Kong are diagnosed before age 35 years (Chan, et al (1993) Postgrad Med 69: 204; Janus (1996) The Hong Kong cardovascular risk factor prevalence study 1995-1996 Dept of Clin Biochem, Queen Mary Hospital of Hong Kong, Hong Kong, 1997).\",\n \"Due to their anticipated long duration of disease, it is important to classify and characterize the nature of diabetes in these young patients to facilitate early diagnosis and appropriate treatments.\",\n \"Current methods of diagnosing type 2 diabetes generally involve assessing phenotypic parameters, such as measuring fasting serum glucose levels by administering an oral glucose tolerance test (OGTT) to determine impaired glucose tolerance (IGT) or impaired fasting glucose (IFG).\",\n \"Phenotypic assessments of persons suspected of having type 2 diabetes are important, but they are limited in that patients generally receive a diagnosis only after presentation with overt symptoms.\",\n \"Furthermore, because the common symptoms of type 2 diabetes are a consequence of a combination heterogenous genetic and environmental causes, the therapies provided are general with regard to the disease rather than targetted to the specific etiology of the individual patient.\",\n \"Numerous studies have attempted to correlate the increased risk for development of type 2 diabetes with a mutation of a specific gene, but the results of these studies repeatedly demonstrate that no one mutated gene can be attributed as the major cause of type 2 diabetes, emphasizing the heterogeneous nature of this disease.\",\n \"Furthermore, a mutation in a particular gene that correlates with increased risk for developing type 2 diabetes in individuals of one ethnic population is not relevant to individuals of a second ethnic population, wherein the risk for type 2 diabetes in individuals of the second ethnic population will correlate with a different mutation or a mutation in a completely different gene.\",\n \"It is therefore of interest to identify additional genetic mutations and polymorphisms that are indicative of an increased risk for developing type 2 diabetes in people of Chinese ancestry, and to develop methods that can be effectively employed to prophylactically identify asymptomatic Chinese individuals with a genetic predisposition for type 2 diabetes.\",\n \"Relevant Literature Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes characterized by autosomal dominant inheritance, early onset (usually before 25 years of age) and a primary defect in pancreatic β-cell function (Fajans (1990) Diabetes Care 13: 49; Chan, et al (1990) Diabetic Med 7: 211; Byrne, et al (1996) Diabetes 45: 1503).\",\n \"This form of diabetes can result from mutations in at least five different genes including those encoding the glycolytic enzyme glucokinase (Froguel, et al (1993) New Engl J Med 328: 697), the liver-enriched transcription factors expressed in the pancreatic β-cell, which are hepatocyte nuclear factors HNF-1α (Yamagata, et al (1996) Nature 384: 455), HNF-1β (Horikawa, et al (1997) Nature Genet 17: 384), and HNF-4α (Yamagata, et al (1996) Nature 384: 458), and insulin promoter factor-1 (IPF-1) (Stoffers, et al (1997) Nature Genet 17: 138).\",\n \"Some mutations and polymorphisms in the glucokinase and HNF-1α genes that are associated with the genetic predisposition of a Chinese individual to develop type 2 diabetes mellitus have been initially identified in Ng, et al ( Diabetic Medicine 1999, 16: 956, herein incorporated by reference), but this manuscript does not disclose how these mutations and polymorphisms might be used to identify Chinese individuals with increased risk of developing type 2 diabetes.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,541,060 discloses the results of screening a cohort of sixteen French families having MODY and the identification of several missense mutations in the glucokinase gene, however none of the mutations identified are relevant to individuals of Chinese descent.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,800,998 discloses a point mutation at nucleotide 414 of human HNF 1α, but this single point mutation is not associated with a genetic predisposition of a Chinese individual to develop type 2 diabetes.\",\n \"Major susceptibility loci for non-insulin dependent diabetes have been identified through genome scans of individuals in Mexican-American (Hanis, et al (1996) Nature Genet 13: 161) and Finnish (Mahtani, et al (1996) Nature Genet 14: 90) populations, but not in individuals of a Chinese population.\",\n \"Specific microsatellite regions of genomic DNA can be correlated with major susceptibility loci that closely associate with the increased risk of a Chinese subject to develop type 2 diabetes.\",\n \"For instance, Le Stunff, et al ( Nature Genet.\",\n \"(2000) 26: 444) have reported that particular alleles of the insulin gene variable number of tandem repeat (VNTR) locus are associated with obesity and type 2 diabetes.\",\n \"Also, microsatellite polymorphisms flanking the glucokinase have been associated with type 2 diabetes in a Taiwanese population (Wu, et al (1995) Diabetes Res Clin Pract 30: 21).\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>Compositions and methods are provided, wherein a unique combination of genetic markers indicative of a genetic predisposition for developing type 2 diabetes in members of a Chinese population is described.\",\n \"The invention is exemplified by a combination of mutated gene sequences from wild-type genes that are involved in insulin secretory function, including hepatocyte nuclear factor 1α (HNF-1α), glucokinase, amylin and mitochondrial DNA.\",\n \"The combination of representative mutations include G20R, A116V, IVS2nt→GA, R203H, S432C and 1618M of HNF-1α; V101M, 1110T, A119D, Q239R and G385V of glucokinase; S20G of amylin; and A3243G of mitochondrial tRNA Leu(UUR) .\",\n \"The combination of the mutated genes of interest will be most efficiently used for screening individuals at increased risk by attaching them to a microchip.\",\n \"Embodiments of methods for determining or detecting the genetic predisposition of a Chinese individual to develop type 2 diabetes include obtaining a sample containing genomic nucleic acid from a Chinese patient, such as a tissue biopsy or a blood sample, and contacting that sample with a representative combination of at least two mutated genes of interest, then subjecting the sample DNA together with the patient's DNA to hybridization conditions stringent enough to detect nucleotide differences of at least one base pair.\",\n \"Alternatively, particular genes of interest from the genomic DNA of a Chinese individual at risk are screened using PCR primer pairs and PCR-RFLP techniques to identify the presence or absence of a mutation known to be associated with type 2 diabetes.\",\n \"The methods further encompass screening the genomic DNA of Chinese individuals who have been diagnosed with type 2 diabetes or who have a primary family member with type 2 diabetes for additional associative mutations in identified genes or for mutations correlative with the predisposition of a member of a Chinese population to develop type 2 diabetes in additional candidate genes, such as those associated with diabetic kidney disease and obesity.\",\n \"The invention further provides for nucleic acid primers and probes that are specifically used to identify mutations, for instance by PCR or hybridization, of wild-type genes involved in insulin secretion that are associated with an increased risk of a Chinese subject to develop type 2 diabetes.\",\n \"Additionally, proteins translated from genes carrying at least one mutation associated with increased risk of a Chinese individual to develop type 2 diabetes find use in functional diagnostic assays and in the production of diagnostic antibodies that bind to the mutant but not the wild-type protein.\",\n \"The prophylactic detection of mutations and polymorphisms that are indicative of a genetic predisposition of a Chinese individual to develop type 2 diabetes finds application in providing clinicians with information that allows for early detection and therapy initiation before the onset of overt symptoms or complications, and that enables clinicians to administer specifically targetted therapies that address the etiology of an individual's disease.\"\n ],\n [\n \"FIELD OF THE INVENTION This subject invention relates to the identification and use of mutations and polymorphisms in mutant genes of wild-type genes involved in insulin secretory function that are associated with the increased risk of a Chinese individual to develop type 2 diabetes.\",\n \"The invention is exemplified by a combination of mutations, uniquely identified in Chinese individuals with a positive family history of type 2 diabetes, in the genes encoding hepatocyte nuclear factor-1∝, glucokinase, amylin and mitochondrial DNA.\",\n \"The combination of mutated genes finds use in screening Chinese individuals at risk of developing type 2 diabetes and in providing physicians with information to enable them to apply patient tailored therapies.\",\n \"BACKGROUND Although people of Chinese ancestry account for >20% of the world's population (Chan, et al(1997) 20: 1785), very little is known about the genetic factors that contribute to the development of diabetes in this population.\",\n \"The prevalence of diabetes amongst Chinese people varies from <1% in some rural areas in mainland China to 6-12% in Hong Kong, Singapore, and Taiwan (Chan, et al (1997), supra).\",\n \"Hong Kong can be regarded as a paradigm of future China.\",\n \"The prevalence of diabetes mellitus is reaching epidemic proportions amongst Hong Kong Chinese, with type 2 diabetes being the predominant form in pateints with early-or late-onset of disease (Chan and Cockram (1997) Diabetes Care 20: 1785).\",\n \"Type 2 diabetes mellitus is a heterogeneous disease that is caused by both genetic and environmental factors.\",\n \"The age-adjusted prevalence of diabetes in the Chinese population has increased from 7.7% in 1990 (Cockram, et al (1993) Diabetes Res and Clin Practice 21: 67) to 8.9% in 1995 (Cockram and Chan (1999) In: Diabetes in the New Millennium, Pot Still Press, Sydney, pp.\",\n \"11-22).\",\n \"In a population-based study conducted in 1995, the crude prevalence of diabetes mellitus was 9.6%, rising from 1.7% in those aged under 40 years to 25% in those older than 60 years (Janus (1997) Clin Exp Pharmacol Physiol 24: 987).\",\n \"There is a high prevalence of obesity (43%) and positive family history of diabetes (50%) in Chinese patients presenting with acute or early onset diabetes (Chan, et al (1993) Postgrad Med J 69: 204; Ko, et al (1998) 35: 761).\",\n \"These findings indicate that genetic factors, in addition to environmental factors, can be an important cause of early onset diabetes in this population.\",\n \"Because type 2 diabetes is an insidious disease, it is estimated that as many as half of the individuals in Hong Kong that would be considered diabetic remain undiagnosed.\",\n \"Most patients are finally diagnosed only when presenting with overt symptoms that often are the consequence of advanced disease.\",\n \"Clinic as well as population-based studies reveal that about 17% of diabetic patients in Hong Kong are diagnosed before age 35 years (Chan, et al (1993) Postgrad Med 69: 204; Janus (1996) The Hong Kong cardovascular risk factor prevalence study 1995-1996 Dept of Clin Biochem, Queen Mary Hospital of Hong Kong, Hong Kong, 1997).\",\n \"Due to their anticipated long duration of disease, it is important to classify and characterize the nature of diabetes in these young patients to facilitate early diagnosis and appropriate treatments.\",\n \"Current methods of diagnosing type 2 diabetes generally involve assessing phenotypic parameters, such as measuring fasting serum glucose levels by administering an oral glucose tolerance test (OGTT) to determine impaired glucose tolerance (IGT) or impaired fasting glucose (IFG).\",\n \"Phenotypic assessments of persons suspected of having type 2 diabetes are important, but they are limited in that patients generally receive a diagnosis only after presentation with overt symptoms.\",\n \"Furthermore, because the common symptoms of type 2 diabetes are a consequence of a combination heterogenous genetic and environmental causes, the therapies provided are general with regard to the disease rather than targetted to the specific etiology of the individual patient.\",\n \"Numerous studies have attempted to correlate the increased risk for development of type 2 diabetes with a mutation of a specific gene, but the results of these studies repeatedly demonstrate that no one mutated gene can be attributed as the major cause of type 2 diabetes, emphasizing the heterogeneous nature of this disease.\",\n \"Furthermore, a mutation in a particular gene that correlates with increased risk for developing type 2 diabetes in individuals of one ethnic population is not relevant to individuals of a second ethnic population, wherein the risk for type 2 diabetes in individuals of the second ethnic population will correlate with a different mutation or a mutation in a completely different gene.\",\n \"It is therefore of interest to identify additional genetic mutations and polymorphisms that are indicative of an increased risk for developing type 2 diabetes in people of Chinese ancestry, and to develop methods that can be effectively employed to prophylactically identify asymptomatic Chinese individuals with a genetic predisposition for type 2 diabetes.\",\n \"Relevant Literature Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes characterized by autosomal dominant inheritance, early onset (usually before 25 years of age) and a primary defect in pancreatic β-cell function (Fajans (1990) Diabetes Care 13: 49; Chan, et al (1990) Diabetic Med 7: 211; Byrne, et al (1996) Diabetes 45: 1503).\",\n \"This form of diabetes can result from mutations in at least five different genes including those encoding the glycolytic enzyme glucokinase (Froguel, et al (1993) New Engl J Med 328: 697), the liver-enriched transcription factors expressed in the pancreatic β-cell, which are hepatocyte nuclear factors HNF-1α (Yamagata, et al (1996) Nature 384: 455), HNF-1β (Horikawa, et al (1997) Nature Genet 17: 384), and HNF-4α (Yamagata, et al (1996) Nature 384: 458), and insulin promoter factor-1 (IPF-1) (Stoffers, et al (1997) Nature Genet 17: 138).\",\n \"Some mutations and polymorphisms in the glucokinase and HNF-1α genes that are associated with the genetic predisposition of a Chinese individual to develop type 2 diabetes mellitus have been initially identified in Ng, et al (Diabetic Medicine 1999, 16: 956, herein incorporated by reference), but this manuscript does not disclose how these mutations and polymorphisms might be used to identify Chinese individuals with increased risk of developing type 2 diabetes.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,541,060 discloses the results of screening a cohort of sixteen French families having MODY and the identification of several missense mutations in the glucokinase gene, however none of the mutations identified are relevant to individuals of Chinese descent.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,800,998 discloses a point mutation at nucleotide 414 of human HNF 1α, but this single point mutation is not associated with a genetic predisposition of a Chinese individual to develop type 2 diabetes.\",\n \"Major susceptibility loci for non-insulin dependent diabetes have been identified through genome scans of individuals in Mexican-American (Hanis, et al (1996) Nature Genet 13: 161) and Finnish (Mahtani, et al (1996) Nature Genet 14: 90) populations, but not in individuals of a Chinese population.\",\n \"Specific microsatellite regions of genomic DNA can be correlated with major susceptibility loci that closely associate with the increased risk of a Chinese subject to develop type 2 diabetes.\",\n \"For instance, Le Stunff, et al (Nature Genet.\",\n \"(2000) 26: 444) have reported that particular alleles of the insulin gene variable number of tandem repeat (VNTR) locus are associated with obesity and type 2 diabetes.\",\n \"Also, microsatellite polymorphisms flanking the glucokinase have been associated with type 2 diabetes in a Taiwanese population (Wu, et al (1995) Diabetes Res Clin Pract 30: 21).\",\n \"SUMMARY OF THE INVENTION Compositions and methods are provided, wherein a unique combination of genetic markers indicative of a genetic predisposition for developing type 2 diabetes in members of a Chinese population is described.\",\n \"The invention is exemplified by a combination of mutated gene sequences from wild-type genes that are involved in insulin secretory function, including hepatocyte nuclear factor 1α (HNF-1α), glucokinase, amylin and mitochondrial DNA.\",\n \"The combination of representative mutations include G20R, A116V, IVS2nt→GA, R203H, S432C and 1618M of HNF-1α; V101M, 1110T, A119D, Q239R and G385V of glucokinase; S20G of amylin; and A3243G of mitochondrial tRNALeu(UUR).\",\n \"The combination of the mutated genes of interest will be most efficiently used for screening individuals at increased risk by attaching them to a microchip.\",\n \"Embodiments of methods for determining or detecting the genetic predisposition of a Chinese individual to develop type 2 diabetes include obtaining a sample containing genomic nucleic acid from a Chinese patient, such as a tissue biopsy or a blood sample, and contacting that sample with a representative combination of at least two mutated genes of interest, then subjecting the sample DNA together with the patient's DNA to hybridization conditions stringent enough to detect nucleotide differences of at least one base pair.\",\n \"Alternatively, particular genes of interest from the genomic DNA of a Chinese individual at risk are screened using PCR primer pairs and PCR-RFLP techniques to identify the presence or absence of a mutation known to be associated with type 2 diabetes.\",\n \"The methods further encompass screening the genomic DNA of Chinese individuals who have been diagnosed with type 2 diabetes or who have a primary family member with type 2 diabetes for additional associative mutations in identified genes or for mutations correlative with the predisposition of a member of a Chinese population to develop type 2 diabetes in additional candidate genes, such as those associated with diabetic kidney disease and obesity.\",\n \"The invention further provides for nucleic acid primers and probes that are specifically used to identify mutations, for instance by PCR or hybridization, of wild-type genes involved in insulin secretion that are associated with an increased risk of a Chinese subject to develop type 2 diabetes.\",\n \"Additionally, proteins translated from genes carrying at least one mutation associated with increased risk of a Chinese individual to develop type 2 diabetes find use in functional diagnostic assays and in the production of diagnostic antibodies that bind to the mutant but not the wild-type protein.\",\n \"The prophylactic detection of mutations and polymorphisms that are indicative of a genetic predisposition of a Chinese individual to develop type 2 diabetes finds application in providing clinicians with information that allows for early detection and therapy initiation before the onset of overt symptoms or complications, and that enables clinicians to administer specifically targetted therapies that address the etiology of an individual's disease.\",\n \"BRIEF DESCRIPTION OF DRAWINGS FIG.\",\n \"1A shows the nucleic acid sequence of human nuclear factor 1a (HNF-1α) exon 1 with the G20R mutation (SEQ ID NO: 1).\",\n \"The wild-type sequence is GenBank number U72612.FIG.\",\n \"1B shows the nucleic acid sequence HNF-1α exon 2 with the A116V mutation (SEQ ID NO: 2).\",\n \"The wild-type sequence is GenBank number U72613.FIG.\",\n \"2 shows the nucleic acid sequence of HNF-1α exons 3 and 4 depicting the splice acceptor site mutation IVS2nt-1G→A (SEQ ID NO: 3) and the missense mutation R203H (SEQ ID NO: 4).\",\n \"The wild-type sequence is GenBank number U72614.FIG.\",\n \"3A shows the nucleic acid sequence of HNF-1α exons 5 and 6 with the S432C mutation (SEQ ID NO : 5).\",\n \"The wild-type sequence is GenBank number U72615.FIG.\",\n \"3B shows the nucleic acid sequence of HNF-1α exon 10 with the I618M mutation (SEQ ID NO: 6).\",\n \"The wild-type sequence is GenBank number U72618.FIG.\",\n \"4A shows the nucleic acid sequence of human glucokinase exon 3, depicting the mutations V101M (SEQ ID NO: 7), I110T (SEQ ID NO: 8) and A119D (SEQ ID NO: 9).\",\n \"The wild-type sequence is GenBank number AF041016.FIG.\",\n \"4B shows the nucleic acid sequence of human glucokinase exon 7 with the Q239R mutation (SEQ ID NO: 10).\",\n \"The wild-type sequence is GenBank AF041019.FIG.\",\n \"4C shows the nucleic acid sequence of human glucokinase exon 9 with the G385V mutation (SEQ ID NO: 11).\",\n \"The wild-type sequence is GenBank number AF041021.FIG.\",\n \"5 shows the nucleic acid sequence of the human amylin gene exon 3 with the S20G mutation (SEQ ID NO: 12).\",\n \"The wild-type sequence is GenBank number X52819.FIG.\",\n \"6 shows the nucleic acid sequence base pairs 3001-3480 of the human mitochondrion complete genome, depicting the A3243G mutation (SEQ ID NO: 13).\",\n \"The wild-type sequence is GenBank number J01415.FIG.\",\n \"7 shows the pedigrees of families with mutations/polymorphisms in the glucokinase (HK84) or HNF-1α gene (HK10 and HK54).\",\n \"Individuals with diabetes are noted by filled symbols; individuals with impaired fasting glucose (IFG) or impaired glucose tolerance (IGT) by grey symbols; non-diabetic individuals by open symbols and untested by hatched symbols.\",\n \"The arrow indicates the proband.\",\n \"Present age, age at diagnosis and genotype of glucokinase or HNF-1α of tested individuals are noted: N, normal; M, mutation/polymorphism.\",\n \"FIG.\",\n \"8 shows the pedigrees of families with an mt3243 mutation.\",\n \"Individuals with diabetes are noted by filled symbols, IGT by grey symbols, non-diabetic individuals by open symbols, and untested individuals by hatched symbols.\",\n \"The arrow indicates the proband.\",\n \"Present age, age of diagnosis, audiogram and genotype are also shown.\",\n \"N, normal; M, mutant allele.\",\n \"FIG.\",\n \"9A-9J show the pedigrees of 10 families carrying the HNF-1α (9A-9B), glucokinase (9C-9E), mt3243 (9F-9H) or amylin S20G (9I-9J) gene mutations/polymorphisms.\",\n \"Subjects with diabetes are noted by black symbols, subjects with IFG or IGT by grey symbols, non-diabetic and untested subjects by open symbols.\",\n \"The genotype of the family members is indicated by: N, wild-type allele; and M, mutant/variant allele.\",\n \"Present age, age at diagnosis, therapy and complications are stated in this order.\",\n \"The proband is indicated by an arrow.\",\n \"Abbreviations: Oral, oral drugs; Ins, insulin; R, retinopathy; K, albuminuria; U, neuropathy; H, hearing impairment.\",\n \"FIG.\",\n \"10 shows the pedigree of a Chinese family with HNF-1α IVS2nt-1G→A mutation.\",\n \"Subjects with diabetes are represented by black symbols, subjects with IGT by grey symbols and untested ones by open symbols.\",\n \"The genotype of family members is indicated: N, normal allele; and M, mutant allele.\",\n \"The proband is indicated by an arrow.\",\n \"CP, C-peptide; GST, glucagon stimulation test; Complications: R, retinopathy; K, nephropathy; U, neuropathy.\",\n \"DESCRIPTION OF THE SPECIFIC EMBODIMENTS Compositions and methods are provided, wherein a unique combination of genetic markers indicative of a genetic predisposition for developing type 2 diabetes in members of a Chinese population is described.\",\n \"The invention comprises as compositions: (1) a combination of nucleic acid sequences from wild-type genes that encode proteins important for insulin secretory function, each nucleic acid sequence having a mutation uniquely associated with the genetic predisposition of a Chinese individual to develop type 2 diabetes, (2) nucleic acid sequences encoding glucokinase and HNF-1α and carrying previously unreported mutations indicative of the increased risk of a Chinese person to develop type 2 diabetes, (3) a microchip having attached to it at least two of the mutated genes of interest, and (4) nucleic acid primers used to detect the unique mutations in the genes of interest.\",\n \"The methods involve: (1) obtaining genomic DNA from a Chinese subject, (2) combining the genomic DNA with either a combination of the mutated nucleic acids of interest or a combination of primers used to identify the presence or absence of a mutation in a gene of interest, and (3) detecting for the presence or absence of mutations, either by identifying mismatches between the patient's DNA and a wild-type or mutant nucleic acid sequence by hybridization techniques, or by amplifying regions of the patients DNA that contain putative mutations by PCR, and subjecting the amplicons to restriction endonucleases and/or DNA sequencing.\",\n \"Advantages of the present invention include that the method of screening uses genetic markers shown to cosegregate with type 2 diabetes in persons of Chinese ancestry to assess whether a given patient is at increased risk for developing type 2 diabetes.\",\n \"The mutations and polymorphisms used for screening are specifically applicable to individuals of Chinese descent.\",\n \"As a further advantage, the screening can be based upon the presence or absence of a combination of at least two different mutations or polymorphisms to provide for even more accurate and reliable evaluations because the contributing factors to development of type 2 diabetes are heterogeneous.\",\n \"Identification of particular mutations or polymorphisms in an individual offers the advantage that with this information physicians are able to provide more specific and appropriate therapies for individual patients, and to guide a patient in making lifestyle adjustments to ameliorate or delay symptoms of diabetes and associated complications.\",\n \"Because type 2 diabetes is often an insidious disease, representative combinations of genetic markers indicative of a predisposition in a Chinese individual to develop the disease can be used to screen populations of individuals who may be at increased risk for developing type 2 diabetes so that they can be given appropriate therapy before overt diabetic symptoms or complications are realized.\",\n \"Likewise, family members of an individual diagnosed with type 2 diabetes can be screened for the particular mutants/polymorphisms of the affected individual to quickly identify family members also at increased risk of developing type 2 diabetes.\",\n \"By a member of a Chinese population is intended to include any individual of Chinese ancestry.\",\n \"In certain cases, for instance when a mutation in a gene involved in the secretion of insulin is dominant for increasing the risk of a Chinese individual to develop type 2 diabetes, a member of a Chinese population will encompass those individuals with at least one parent of Chinese descent.\",\n \"A member of a Chinese population may be more specifically identified by HLA haplotyping.\",\n \"For example, HLA class I and class II frequencies among a Hong Kong Chinese population have been studied by Chang and Hawkins (Hum Immunol (1997) 56: 125).\",\n \"Numerous studies have been carried out to determine HLA class I and class II alleles that are more frequently or even uniquely found in members of a Chinese population, and alleles with strong associations.\",\n \"Shaw et al and Shen et al have studied HLA polymorphism and allele frequency and association of Chinese populations in Taiwan (Tissue Antigens (1997) 50: 610; Tissue Antigens (1999) 53: 51; J Formos Med Assoc (1999) 98: 11).\",\n \"Allele frequency and associations found in Chinese individuals of mainland China have been reported by Trejaut et al (Eur J Immunogenet (1996) 23: 437), Shieh, et al (Transfusion (1996) 36: 818), Zhao et al (Eur J Immunogenet (1993) 20: 293), Wang, et al (Tissue Antigens (1993) 41: 223; Hum Immunol (1992) 33: 129), Lee, et al (Eur J. Immunogenet (1999) 26: 275), and Gao et al (Hum Immunol (1991) 32: 269; Tissue Antigens (1991) 38: 24; Immunogenetics (1991) 34: 401).\",\n \"Additionally, a Chinese individual may be objectively defined by “DNA fingerprinting” techniques well known to those in the art, where microsatellite, short tandem repeat (STR) and variable number tandem repeat (VNTR) loci specific to individuals of Chinese descent are identified.\",\n \"Numerous examples of such ethnic genotyping studies have been reported (Meng, et al (1999) J Forensic Sci 44: 1273; Yoshimoto, et al (1999) Int J Legal Med 113: 15; Wu, et al (1999) J Forensic Sci 44: 1039; Evett, et al (1996) Am J Hum Genet 58: 398; Gill and Evett (1995) Genetica 96: 69; Balazs (1993) EXS 67: 193; Lan, et al (1992) Arch Kriminol 189: 169; and Hwu, et al (1992) J Formos Med Assoc 91: 839).\",\n \"All of these above references are incorporated herein by reference.\",\n \"Whereas insulin resistance is a strong predictor of type 2 diabetes, it is not sufficient for manifestation of the disease (So, et al (2000) Hong Kong J. Med 6: 69-76).\",\n \"A relative insulin deficiency is essential to the development of hyperglycemia, setting up a vicious cycle wherein elevated glucose levels are toxic to pancreatic β-cells, thereby inducing insulin resistance and decreased β-cell secretory function.\",\n \"Based in the intrinsic interconnection between insulin secretion and action, the invention is exemplified by a combination of mutated gene sequences from wild-type genes that are involved in insulin secretory function, including hepatocyte nuclear factor 1α (HNF-1α), glucokinase, amylin and mitochondrial DNA.\",\n \"By “genes involved in insulin secretory function” and “genes involved in insulin secretion” is intended genes in which a heterozygous mutation has a dominant-negative effect on normal pancreatic β-cell secretory function.\",\n \"The invention is primarily concerned with a representative array of gene markers, the combination of which is uniquely indicative of the genetic predisposition of a member of a Chinese population to develop type 2 diabetes (referred to hereinafter as “genes of interest”).\",\n \"The combination of representative mutations is exemplified by G20R, A116V, IVS2nt→GA, R203H, S432C and 1618M of HNF-1α; V101M, I110T, A119D, Q239R and G385V of glucokinase; S20G of amylin; and A3243G of mitochondrial tRNALeu(UUR)”.\",\n \"The mutation IVS2nt→GA represents a splice acceptor site mutation that likely results in a truncated translation product.\",\n \"A representative combination of the mutated genes of interest finds particular use in the prophylactic screening of (i) Chinese individuals who have been diagnosed with maturity onset diabetes of the young (MODY) to determine the etiology of their disease, (ii) Chinese individuals that have a positive family history of type 2 diabetes to determine their likelihood of developing diabetic symptoms, and (iii) and Chinese individuals deemed to be at greater risk of developing diabetic symptoms because of correlative phenotypic characteristics (i. e. obese individuals).\",\n \"The combination of the mutated genes of interest will be most efficiently used for screening-individuals at increased risk by attaching them to a microchip or other solid support.\",\n \"A specific kind of microchip is not critical, except that it must be able to present a representative array of at least two different nucleic acid sequences, each with a mutation or polymorphism indicative of the increased risk of a Chinese individual to develop type 2 diabetes.\",\n \"Additionally, it will be useful to attach representative wild-type nucleic acid sequences to the chip as comparative controls.\",\n \"The microarray will normally involve a plurality of different nucleic acid sequences, usually be at least 10, more usually at least 20, frequently at least 50, but may have as many as 100 or more.\",\n \"Chips that will find use with the present invention are known in the art (for example, see U.S. Pat.\",\n \"Nos.\",\n \"5,741,644, 5,837,832 and 6,183,970).\",\n \"Additionally, other solid substrates may be used for the covalent attachment of representative combinations of mutated nucleic acid sequences of interest, including beads and slides.\",\n \"Solid supports can be made out of glass or silicon oxide or other materials that can be adapted to be covalently attached to oligonucleotide sequences by the introduction of functionalities which react with oligonucleotides.\",\n \"One may use a variety of approaches to bind the nucleic acid to the solid substrate.\",\n \"By using chemically reactive solid substrates, one may provide for a chemically reactive group to be present on the nucleic acid, which will react with the chemically active solid substrate surface.\",\n \"For example, by using silicate esters, halides or other reactive silicon species on the surface, the nucleic acid may be modified to react with the silicon moiety.\",\n \"One may form silicon esters for covalent bonding of the nucleic acid to the surface.\",\n \"Instead of silicon functionalities, one may use organic addition polymers.\",\n \"e. g. styrene, acrylates and methacrylates, vinyl ethers and esters, and the like, where functionalities are present which can react with a functionality present on the nucleic acid.\",\n \"For example, amino groups, activated halides, carboxyl groups, mercaptan groups, epoxides, and the like, may be provided in accordance with conventional ways.\",\n \"The linkages may be amides, amidines, amines, esters, ethers, thioethers, dithioethers, and the like.\",\n \"Methods for forming these covalent linkages may be found in U.S. Pat.\",\n \"No.\",\n \"5,565,324 and U.S. Pat.\",\n \"No.\",\n \"6,156,501.The invention also contemplates a microassay system and a kit that comprises a solid support having attached to it a representative array of nucleic acid sequences, each with a mutation or polymorphism associated with the genetic disposition of a Chinese individual to develop type 2 diabetes.\",\n \"The microassay system or kit would contain, for example, a microchip or beads to which are attached wild-type and mutant nucleic acid sequences from genes that encode proteins involved in insulin secretory function, preferentially wild-type and mutant sequences from HNF-1α, glucokinase, amylin and mitochondrial DNA.\",\n \"Additionally, mutant and wild-type sequences known to hybridize and known not to hybridize under stringent conditions to those sequences immobilized on the support would be included as positive and negative controls, respectively.\",\n \"A microassay system or kit with nucleic sequences immobilized on a solid support would involve screening by hybridization detection (fluorescent or radioactive signal upon duplex formation).\",\n \"Alternatively, another microassay system or kit would include primer pairs that anneal to nucleic acid sequences encoding proteins involved in insulin secretion.\",\n \"The primer pairs specifically anneal to flanking regions of the genes that putatively contain mutations associated with type 2 diabetes, such that PCR amplification with such primers would reveal the presence or absence of an associative mutation of interes.\",\n \"Such a kit or microassay system would also contain representative mutant and wild-type sequences as controls, and screening would be carried out using PCR and sequencing or through PCR restriction fragment length polymorphism analysis (RFLP) and electrophoresis.\",\n \"Type 2 diabetes is a heterogeneous disease, and no single mutation or single mutated gene can be fully attributed to the manifestation of its symptoms.\",\n \"Therefore, a combination of at least two different nucleic acid sequences encoding mutations or polymorphisms of closely associated with increased risk of a person of Chinese descent to develop type 2 diabetes is attached to a microchip or is individually screened.\",\n \"By “at least two different nucleic acid sequences” is intended two different nucleic acid sequences from the same wild-type gene having different mutations, or two different mutant nucleic acid sequences from two different wild-type genes.\",\n \"Preferably, at least one of the mutant; sequences A116V of HNF-1α (SEQ ID NO: 2), V101M (SEQ ID NO: 7) or Q239R (SEQ D NO: 10) are attached to the microchip or solid support.\",\n \"By wild-type gene is intended one that is not associated with type 2 diabetes, and this would include any allelic variant of the wild-type gene, at any frequency, and that encodes a protein that functions in its expected manner without inducing pathological symptoms.\",\n \"By mutant gene is intended one whose sequence has been modified by insertions, deletions, or substitutions of at least one nucleic acid base pair, wherein the modification may result in detectable changes in the expression or function of the mutant gene product as compared to the wild-type gene product.\",\n \"In the genes of interest for the invention, a mutant gene is associated with type 2 diabetes.\",\n \"The nucleic acid sequences may be from genomic DNA, complementary DNA (cDNA) or from messenger RNA (mRNA).\",\n \"They may be synthetic or isolated from human bodily tissue or fluid.\",\n \"The mutations preferably occur, but do not need to occur, in a translated region of a nucleic acid sequence that encodes a protein that in wild-type form is involved in glucose metabolism or insulin secretion.\",\n \"Within a translated nucleic acid sequence, a mutation can be a missense mutation, replacing one amino acid with another amino acid, or a nonsense mutation, replacing an amino acid with a stop codon.\",\n \"Mutations can also be insertions or deletions of at least one nucleic acid in either a coding or in non-coding region, such as a region that controls the transcription of a gene, including promoters, enhancers, response elements, signal sequences and polyadenylation signals, and the like.\",\n \"Single nucleotide polymorphisms (SNPs), preferably but not necessarily occurring within the translated regions of nucleic acid sequences that encode proteins involved in glucose metabolism or insulin secretion and that correlate with increased risk of type 2 diabetes are also contemplated by the present invention.\",\n \"Such SNPs can be identified by correlating mutations in known genes that cosegregate with development of type 2 diabetes in members of families with a positive history of the disease.\",\n \"Additionally, SNPs that occur in non-translated and translated regions can be identified through genome-wide scans and correlate linkage analyses of family pedigrees.\",\n \"The use of microarray technologies also can be conveniently applied to identifying SNPs of interest.\",\n \"Embodiments of methods for determining or detecting the genetic predisposition of a Chinese individual to develop type 2 diabetes include obtaining a sample containing genomic nucleic acid from a Chinese patient, such as tissue from autopsy or biopsy, or a blood sample, and contacting that sample with a representative combination of at least two mutated genes of interest, then subjecting the sample DNA together with the patient's DNA to hybridization conditions stringent enough to detect nucleotide differences of at least one base pair.\",\n \"Alternatively, particular genes of interest from the genomic DNA of a Chinese individual at risk are subjected to restriction fragmentation and then screened using PCR primer pairs and PCR-RFLP techniques to identify the presence or absence of a mutation known to be associated with type 2 diabetes.\",\n \"The methods further encompass screening the genomic DNA of Chinese individuals that have been diagnosed with type 2 diabetes or that have a primary family member with type 2 diabetes for additional associative mutations in identified genes or for mutations correlative with the predisposition of a member of a Chinese population to develop type 2 diabetes in additional candidate genes, such as those associated with diabetic kidney disease and obesity.\",\n \"Mutations are most efficiently identified in Chinese families with a positive history of developing type 2 diabetes (i. e. families with members that develop MODY).\",\n \"However, identified associative mutations are useful for identifying the increased risk in any member of a Chinese population.\",\n \"In practicing a method of identifying the mutations associated with the genotype of a Chinese individual who is at increased risk for developing type 2 diabetes, Chinese subjects with (i) a confirmed diagnosis of type 2 diabetes, (ii) a positive familial history of type 2 diabetes or (iii) phenotypically determined elevated risk factors (e. g. obesity) are identified by clinical testing, pedigree analysis, and linkage analysis, using standard diagnostic criteria and interview procedures, and DNA or RNA samples are obtained from the subjects.\",\n \"A sample of genomic DNA is obtained from any nucleated cell source or body fluid.\",\n \"Examples of cell sources available in clinical practice include blood cells, buccal cells, cervicovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy.\",\n \"Body fluids include blood, urine, cerebrospinal fluid, amniotic fluid, and tissue exudates at the site of infection or inflammation.\",\n \"DNA is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art.\",\n \"It will be understood that the particular method used to extract DNA will depend on the nature of the source.\",\n \"A variety of techniques are then employed to identify the presence or absence of new or known mutant sequences.\",\n \"First, the sequences of genes known to be involved in insulin secretory function may be subjected to direct DNA sequencing, using methods that are standard in the art.\",\n \"Mutations may be detected using a PCR-RFLP, in which pairs of olignucleotides are used to prime amplification reactions and the sizes of the amplification products, cleaved or uncleaved by restriction endonucleases, are compared with those of control products.\",\n \"Other useful techniques include Single-Strand Conformation Polymorphism analysis (SSCP), denaturing gradient gel electrophoresis, and two dimensional gel electrophoresis, EMC, and the like.\",\n \"Detection of known mutations, such as those exemplified by the invention, may alternatively be detected using nucleic acid probes that contain mutations of interest in sufficiently stringent hybridization conditions.\",\n \"Appropriate stringency conditions for identifying mutations of at least one base pair in a mutant sequence of a gene involved in insulin secretory function, for example, in 6× sodium chloride/sodium citrate (SSC) at at least 42° C., preferably at about 43,44 or 45° C., followed by a wash of 2×SSC at 50° C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y.\",\n \"(1989).\",\n \"To optimize conditions, both salt and temperature may be varied, or either the temperature or salt concentration may be held constant while the other variable is changed.\",\n \"For example, the salt concentration in the wash step can be selected from a low stringency of about 2×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. The temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C. to high stringency conditions at about 65° C. Optimal conditions will additionally depend on the length of the nucleic acid probe used, and the scale at which the hybridization takes place.\",\n \"High stringency hybridization conditions using nucleotides attached to a microchip may require lower temperatures.\",\n \"One can perform a series of routine thermal equilibrium experiments to determine optimal hybridization discrimination between wild-type and mutant gene sequences of interest, by starting a low stringency temperature of about 20° C. and increasing the temperature in successive 5° C. temperature intervals.\",\n \"The nucleic acid probes of the invention are nucleic acid sequences from the mutated genes of interest.\",\n \"They are at least 8,12,15 or 20 base pairs in length, but can be 50,80 or 100 base pairs in length, and may even be 250 or 500 base pairs in length, and include at least one associative mutation but may include multiple mutations, and can be as long as the length of the transcribed gene.\",\n \"The length of the probe chosen will be optimized based on the better base pair mismatch discrimination of shorter probes and the better duplex stability of longer probes (see U.S. Pat.\",\n \"No.\",\n \"6,156,601 and U.S. Pat.\",\n \"No.\",\n \"6,197,506, herein incorporated by reference).\",\n \"The length of the probe used should enable discrimination between a mutant and wild-type gene with at least one base-pair mutation.\",\n \"For detection of hybridized probes, light detectable means are preferred, although other methods of detection may be employed, such as radioactivity, atomic spectrum, and the like.\",\n \"For light detectable means, one may use fluorescence, phosphoresence, absorption, chemiluminescence, or the like.\",\n \"The most convenient will be fluorescence, which may take many forms.\",\n \"One may use individual fluorescers or pairs of fluorescers, particularly where one wishes to have a plurality of emission wavelengths with large Stokes shifts.\",\n \"Illustrative fluorescers which have found use include fluorescein, rhodamine, Texas red, cyanine dyes, phycoerythrins, thiazole orange and blue, etc.\",\n \"When using pairs of dyes, one may have one dye on one molecule and the other dye on another molecule which binds to the first molecule.\",\n \"For example, one may have one dye on the first or bound component and the other dye on the second or complexing component.\",\n \"The important factor is that the two dyes when the two components are bound are close enough for efficient energy transfer (see U.S. Pat.\",\n \"No.\",\n \"5,992,617).\",\n \"The identification of the presence or absence of known mutations can also conveniently be detected by PCR followed by restriction analysis and/or sequencing using techniques well known to those in the art.\",\n \"PCR analysis furthermore offers an efficient technique for identifying new mutations in genes already known to contain mutations that correlate with the predisposition of a Chinese individual to develop type 2 diabetes, or in identifying associative mutations in additional candidate genes.\",\n \"PCR primers should be at least 12 base pairs in length, preferably 15-18 base pairs in length, and may be as long as 25-30 base pairs in length.\",\n \"They can be designed to anneal to the wild-type gene sequence in regions that flank a mutation in a gene of interest, such that extension from the primer amplifies a region that allows the detection of the presence or absence of a mutation of interest.\",\n \"Primers can also be designed such that their extension results in an amplified sequence only in the presence of either a wild-type or mutant gene, as desired.\",\n \"This can be accomplished by designing a primer with at least one nucleotide at the 3′end that is mismatched with the wild-type sequence, but matched to a mutant sequence.\",\n \"The invention is exemplified by primer pairs used to screen HNF-1α, glucokinase, amylin and human mitochondrial DNA for mutations.\",\n \"Of particular interest are nucleic acid primers that can be used to screen mutations in HNF-1α and glucokinase that have not yet been previously reported (see for example, SEQ ID Nos: 34-36).\",\n \"Simultaneous sequencing of several nucleic acid samples can also be carried out on a microchip (see U.S. Pat.\",\n \"No.\",\n \"6,197,506) For SSCP, primers are designed that amplify DNA products of about 250-300 bp in length across non-duplicated segments of the gene of interest.\",\n \"For each amplification product, one gel system and two running conditions are used.\",\n \"Each amplification product is applied to a 10% polyacrylamide gel containing 10% glycerol.\",\n \"Separate aliquots of each amplimer are subjected to electrophoresis at 8 W at room temperature for 16 hours and at 30 W at 4° C. for 5.4 hours.\",\n \"These conditions were previously shown to identify 98% of the known mutations in the CFTR gene (Ravnik-Glavac et al, (1994) Hum Mol Genet 3: 801).\",\n \"As with identification of associative mutations of interest, identification of associative SNPs that correlate with the increased risk of a Chinese individual to develop type 2 diabetes can be accomplished by nucleic acid sequencing of desired regions of genomic or complementary DNA.\",\n \"Screening for SNPs is pursued most efficiently using microarray technologies where attached nucleic acid sequences attached to a solid support such as a microchip are exposed to hybridization conditions that allow the discrimination between two nucleic acid sequences that differ at one nucleotide (see for example, Wang, et al (1998) Science 280: 1077; and Hacia, et al (1998) Nature Genet.\",\n \"18: 155).\",\n \"Alternatively, mass spectrophotometers can be used to identify small mass differences in PCR products that have single nucleotide polymorphisms (see Kirpekar, et al (1998) Nucleic Acids Res 26: 2554).\",\n \"A further means of analyzing genetic information is” dynamic allele specific hybridization” (DASH).\",\n \"This technique uses labeled oligonucleotides in a multiwell format that will fluoresce when the oligonucleotide exists in a double-stranded form, but not when it is in single-stranded form.\",\n \"Adding a single strand of the DNA to be tested allows the strands to hybridize.\",\n \"The temperature at which the strands denature will allow identification of the base at the SNP.\",\n \"The DASH technique has the advantages of being technically simple, and not requiring expensive equipment.\",\n \"Additional techniques that can be used in the screening for SNPs associated with the genetic predisposition of a Chinese person to develop type 2 diabetes include exonuclease resistance, microsequencing, solution-phase or solid phase extension of ddNTPs, and oligonucleotide ligation assay (as described in U.S. Pat.\",\n \"No.\",\n \"5,952,174, herein incorporated by reference).\",\n \"After the presence of an associative mutation or SNP is detected by any of the above techniques, the specific nucleic acid alteration comprising the mutation is identified by direct DNA sequence analysis or restriction analysis or a combination of both.\",\n \"In this manner, previously unidentified mutations in genes that encode proteins involved in insulin secretion, or in genes associated with obesity or diabetic kidney disease may be defined.\",\n \"For instance, new mutations could be identified with other genes known to closely correlate with familial type 2 diabetes in Chinese subjects (e.g., other MODY genes).\",\n \"Examples of additional MODY genes include hepatocyte nuclear factor 4α (HNF-4α), hepatocyte nuclear factor 1β (HNF-1β), and insulin promoter factor 1 (IPF-1).\",\n \"Additional candidate genes of particular interest for screening because mutations or polymorphisms of the wild type genes are positively associated with type 2 diabetes and nephropathy in Chinese individuals include those that encode angiotensin converting enzyme (ACE)/angiotensinogen (AGT) (Tomino, et al (1999) Nephron 82: 139; Hsieh, et al (2000) Nephrol Dial Transplant 15: 1008; Thomas, et al (2001) Diabetes Care 24; 356), aldose reductase (Ko, et al (1995) Diabetes 44: 727; Moczulski, et al (1999) Diabetologia 42: 94) and plasminogen activator inhibitor-1 (PAI-1) (Wong, et al (2000) Kidney Int 57: 632).\",\n \"Nucleic acid sequences that encode genes involved with glucose metabolism, insulin resistance, obesity and diabetic kidney can also be screened to identify mutations in, for example, proteins that influence insulin binding to its receptor, that are involved in the insulin signaling pathway, that influence glucose uptake and cell metabolism.\",\n \"Specific examples include associative mutations in the α or β chain of the insulin receptor, the insulin receptor substrate proteins (IRS-1 and IRS-2), glucose transporter proteins GLUT2 and GLUT4, and transcription factors HNF-3β and NeuroDI/Beta2 and to correlated any identified mutation an/or polymorphism with indidence of type 2 diabetes.\",\n \"Examples of candidate genes where mutations or polymorphisms have been shown to be associated with type 2 diabetes and obesity in other populations include genes that encode the transporter GLUT4 (Abel, et al (2001) Nature 409: 729), the beta-3-adrenergic receptor (Oeveren van-Dybicz, et al (2001) Diabetes Obes Metab 3: 47), the hormone resistin (Steppan, et al2001) Nature 409: 307), the peroxisome proliferator-activated receptor gamma2 PPARgamma) (Hasstedt, et al (2001) J Clin Endocrinol Metab 86: 536), uncoupling protein-1 (UCP-1) (Heilbronn, et al (2000) Diabetologia 43: 242), leptin (Ohshiro, et al (2000) J Mol Med 78: 516), G protein beta 3 subunit and insulin receptor substrate-1 (Rosskopf, et al (2000) 5: 484), and the dopamine D2 receptor (Jenkinson, et al (2000) Int J Obes Relat Metab Disord 24: 1233).\",\n \"Additionally, mutations or polymorphisms shown to be closely associated with type 2 diabetes and nephropathy in other populations include genes that encode the G protein beta 3 subunit (Beige, et al (2000) Nephrol Dial Transplant 15: 1384; Zychma, et al (2000) Am J Nephrol 20: 305), methylenetetrahydrofolate reductase (MTHFR) (Shpichinetsky, et al (2000) J Nutr 130: 2493, the glucose transporter GLUT1 (Grzeszczak, et al (2001) Kidney Int 59: 631), and paraoxonase (PON1) (Inoue, et al (2000) Metabolism 49: 1400).\",\n \"Additionally, proteins translated from genes carrying at least one mutation associated with increased risk of a Chinese individual to develop type 2 diabetes are contemplated by the invention and find use in functional diagnostic assays and in the production of diagnostic antibodies that bind to the mutant but not the wild-type protein.\",\n \"The polypeptides may be the translational products of the entire mutant gene, as well as peptides of twelve or more amino acids derived therefrom that contain at least one mutation of interest.\",\n \"The polypeptide (s) may be isolated from human tissues obtained by biopsy or autopsy, or may be produced in a heterologous cell by recombinant DNA methods, well known to those in the art (as disclosed in Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor, 1989), or Current Protocols in Molecular Biology (Eds.\",\n \"Aufubel, Brent, Kingston, More, Feidman, Smith and Stuhl,Greene Publ.\",\n \"Assoc., Wiley-Interscience, NY, N.Y., 1992), both references herein incorporated by reference).\",\n \"Peptides comprising HNF-1α-, glucokinase- or amylin specific sequences may be derived from isolated larger polypeptides described above, using proteolytic cleavages by e. g. proteases such as trypsin and chemical treatments such as cyanogen bromide that are well-known in the art.\",\n \"Alternatively, peptides up to 60 residues in length can be routinely synthesized in milligram quantities using commercially available peptide synthesizers.\",\n \"Recombinant translational products are expressed from vectors comprising mutant nucleic acid sequences of wild-type nucleic acid sequences that encode proteins involved in insulin secretion.\",\n \"Exemplified mutant nucleic acid sequences of interest include those that encode HNF-1α, glucokinase or amylin with single amino acid residue changes, as depicted in SEQ ID Nos: 1-13, and particularly SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 10.A large number of vectors, including plasmid and fungal vectors, have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.\",\n \"Vectors used for expression will also include a promoter operably linked to the mutant polypeptide encoding portion, that is preferably the cDNA sequence of the mutated gene of interest or a part thereof that encodes a polypeptide of at least 12 amino acids.\",\n \"The encoded polypeptide may be expressed by using any suitable commercially available vectors, and any suitable host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.\",\n \"The particular choice of vector/host is not critical to the operation of the invention.\",\n \"Appropriate host cells include bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells.\",\n \"Of particular interest are E.coli, B.Subtilis, Saccharomyces cerevisiae, SF9 cells, C129 cells, 293 cells, Neurospora, and CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoid cell lines.\",\n \"Preferred replication systems include M13, ColEI, SV40, baculovirus, lambda, adenovirus, and the like.\",\n \"A large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts.\",\n \"Examples of these regions, methods of isolation, manner of manipulation, etc.\",\n \"are known in the art.\",\n \"Under appropriate expression conditions, host cells can be used as a source of recombinantly produced mutant polypeptides of interest.\",\n \"The translational products of mutant HNF-1α, glucokinase or amylin, and/or fragments or portions thereof may be used to produce specific antibodies.\",\n \"The antibodies may be polyclonal or monoclonal, may be produced in response to the fully translated mutant polypeptide or to synthetic peptides as described above.\",\n \"Such antibodies are conveniently made using the methods and compositions disclosed in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, other references cited herein, as well as immunological and hybridoma technologies known to those in the art.\",\n \"Importantly, the antibodies raised against translation products from nucleic acids sequences carrying at least one mutatation associated with type 2 diabetes should distinguish between the mutant amino acid sequence and the wild-type amino acid sequence.\",\n \"In particular, antibodies should have very little or no cross-reactivity for the wild-type sequence.\",\n \"Preferably the anti-mutant protein antibodies should bind with higher affinity to the mutant polypeptide than to wild-type polypeptide, with binding to the mutant polypeptide at levels 500:1, more preferably 1,000:1, greater than binding the wild-type polypeptide.\",\n \"Isolated polypeptides corresponding to the entire length of the mutant polypeptide or a peptide of at least 12 amino acids in length containing a mutation of interest may be used in accordance with conventional methods to immunize a mammal, (e. g., mouse or higher mammal, primate, or chimeric or transgenic animals which produce human immunoglobulins) in accordance with conventional procedures.\",\n \"See for example, U.S. Pat.\",\n \"Nos.\",\n \"4,172,124; 4,350,683; 4,361,549; and 4,464,465.Hybridomas may be prepared by fusing available myeloma lines, e. g., NS/I, Ag8.6.5.3, etc., with peripheral blood lymphocytes, splenocytes or other lymphocytes of the immunized host and the resulting immortalized B-lymphocytes (e.g., hybridomas, heteromyelomas, EBV transformed cells, etc.)\",\n \"selected, cloned and screened for binding to a mutant polypeptide of a wild-type protein involved in insulin secretion or glucose metabolism.\",\n \"Monoclonal antibodies raised against a mutant polypeptide sequence of interest may be of any immunoglobulin class such as IgA, IgD, IgE, IgG and IgM, preferably IgG or IgM, and may be of any one of the subclasses of the classes.\",\n \"Whole antibodies, or fragments thereof which retain binding activity, may be employed, such as Fab, F(ab′)2, or the like.\",\n \"Once the antibodies with binding specificity for the mutant polypeptide are available, these antibodies may be used for screening.\",\n \"Antibodies that distinguish between normal and mutant forms of HNF-1α, glucokinase, amylin or other mutant/wild-type pairs of proteins involved in insulin secretory function may be used in diagnostic tests employing ELISA, EMIT, CEDIA, SLIFA, and the like.\",\n \"For an assessment of total risk of developing disease or in designing individualized treatments of diagnosed patients, identified mutations and polymorphisms that are indicative of a Chinese individual to develop type 2 diabetes are correlated with phenotypic parameters of screened patients and interpreted with consideration of a positive or negative family history of the disease.\",\n \"Genetic studies will be correlated with data from individuals indicating hormone levels (growth hormone, adrenaline, cortisol, noradrenaline, insulin), anthropometry (body-mass index; waist-to-hip ratio), hemodynamics (blood pressure), cardiovascular risk factors (HDL, LDL, cholesterol, triglycerides) and autoimmunity (anti-glutamic acid decarboxylase antibodies).\",\n \"For instance, a patient with a single mutation in the glucokinase gene may never develop symptoms, whereas the likelihood of a patient with a mutation in both the glucokinase gene and the HNF-1α gene or a mutation in the glucokinase gene and the phenotypic attribute of obesity to develop overt type 2 diabetes is relatively higher.\",\n \"A positive family history of the disease would increase the predicted predisposition even more.\",\n \"Obtaining a genotypic assessment while a patient shows no signs of developing disease, or while showing preliminary signs of disease such as impaired glucose tolerance (IGT), can enable a physician to initiate therapy or suggest lifestyle changes that prevent the onset or progression of overt symptoms.\",\n \"For example, a patient identified as having a mutation in the HNF-1α gene and IGT, can be treated with diet and/or oral drugs and/or insulin early enough that hyperglycemic toxicity of pancreatic β-cells and further insulin secretory dysfunction due to their death is prevented or ameliorated.\",\n \"In this way, severe complications associated with progressive type 2 diabetes, such as nephropathy, retinopathy and sensorineural loss, can be more commonly averted.\",\n \"In addition to allowing a clinician to better tailor traditional therapies for treating type 2 diabetes, such as diet, oral drugs and insulin, identification of associative mutations can enable a clinician to design tailored therapies, such as introducing a wild-type gene into a patient to replace a mutant gene that encodes a malfunctioning protein.\",\n \"For gene therapy methods, transfection in vivo is obtained by introducing a therapeutic transcription or expression vector into the mammalian host, either as naked DNA, complexed to lipid carriers, particularly cationic lipid carriers, or inserted into a viral vector, for example a recombinant adenovirus.\",\n \"The introduction into the mammalian host can be by any of several routes, including intravenous or intraperitoneal injection, intratracheally, intrathecally, parenterally, intraarticularly, intranasally, intramuscularly, topically, transdermally, application to any mucous membrane surface, corneal installation, etc.\",\n \"Of particular interest is the introduction of a therapeutic expression vector into a circulating bodily fluid or into a body orifice or cavity, such as the heart.\",\n \"Thus, intravenous administration and intrathecal administration are of particular interest since the vector may be widely disseminated following such routes of administration, and aerosol administration finds use with introduction into a body orifice or cavity.\",\n \"Particular cells and tissues can be targeted, depending upon the route of administration and the site of administration.\",\n \"For example, a tissue which is closest to the site of injection in the direction of blood flow can be transfected in the absence of any specific targeting.\",\n \"An arterial catheter can be used to introduce the expression vector into an organ such as the heart or kidney.\",\n \"The eye can be accessed directly either by the use of ocular drops or by injecting into the eye.\",\n \"For accessing nerves, this can be by injection into the nerve or injection into the region of the cell body.\",\n \"If lipid carriers are used, they can be modified to direct the complexes to particular types of cells using site-directing molecules.\",\n \"Thus, antibodies or ligands for particular receptors or other cell surface proteins may be employed, with a target cell associated with a particular surface protein.\",\n \"An amino terminal mitochondrial targeting sequence joined to a nucleic acid can be used to target the nucleic acid to the mitochondria.\",\n \"See Taylor et al, Nature Genetics 15: 212-215,1997.Any physiologically acceptable medium may be employed for administering the DNA, recombinant viral vectors or lipid carriers, such as deionized water, saline, phosphate-buffered saline, 5% dextrose in water, and the like as described above for the pharmaceutical composition, depending upon the route of administration.\",\n \"Other components may be included in the formulation such as buffers, stabilizers, biocides, etc.\",\n \"These components have found extensive exemplification in the literature and need not be described in particular here.\",\n \"Any diluent or components of diluents that would cause aggregation of the complexes should be avoided, including high salt, chelating agents, and the like.\",\n \"The amount of therapeutic vector used will be an amount sufficient to provide for a therapeutic level of expression in a target tissue susceptible to diabetic complications or for adequate dissemination to a variety of tissues after entry into the bloodstream and to provide for a therapeutic level of expression in susceptible target tissues.\",\n \"A therapeutic level of expression is a sufficient amount of expression to prevent, treat, or palliate one or more diabetic complication or the symptoms of diabetic complications.\",\n \"In addition, the dose of the nucleic acid vector used must be sufficient to produce a desired level of transgene expression in the affected tissue or tissues in vivo.\",\n \"Other DNA sequences, such as adenovirus VA genes can be included in the administration medium and be co-transfected with the gene of interest.\",\n \"The presence of genes coding for the adenovirus VA gene product may significantly enhance the translation of mRNA transcribed from the expression cassette if this is desired.\",\n \"A number of factors can affect the amount of expression in transfected tissue and thus can be used to modify the level of expression to fit a particular purpose.\",\n \"Where a high level of expression is desired, all factors can be optimized, where less expression is desired, one or more parameters can be altered so that the desired level of expression is attained.\",\n \"For example, if high expression would exceed the therapeutic window, then less than optimum conditions can be used.\",\n \"The level and tissues of expression of the recombinant gene may be determined at the mRNA level as described above, and/or at the level of polypeptide or protein.\",\n \"Gene product may be quantitated by measuring its biological activity in tissues.\",\n \"For example, protein activity can be measured by immunoassay as described above, by biological assay such as inhibition of ROS, or by identifying the gene product in transfected cells by immunostaining techniques such as probing with an antibody which specifically recognizes the gene product or a reporter gene product present in the expression cassette.\",\n \"Typically, the therapeutic cassette is not integrated into the patient's genome.\",\n \"If necessary, the treatment can be repeated on an ad hoc basis depending upon the results achieved.\",\n \"If the treatment is repeated, the patient can be monitored to ensure that there is no adverse immune or other response to the treatment.\",\n \"The following examples are offered by way of illustration of the present invention, not limitation.\",\n \"EXPERIMENTAL EXAMPLE 1 Identification of Mutations in Glucokinase and Hepatocyte Nuclear Factor 1α Genes in Chinese Patients with Early-Onset Type 2 Diabetes Mellitus/MODY This example illustrates mutations identified in the glucokinase, HNF-1α and HNF-4α genes in a cohort of Chinese patients.\",\n \"Mutations in the glucokinase and HNF-1α genes are relatively common in early-onset diabetes and they account for about 3% and 5%, respectively, of the present Chinese early-onset diabetic patients.\",\n \"Experimental Design and Methods Subjects The study group consisted of 92 unrelated patients (age 34±5 years (mean±SD) range 18-40 years; 30 males and 62 females) who were diagnosed with Type 2 diabetes before 40 years of age and who had a positive family history (at least one first degree relative with Type 2 diabetes).\",\n \"The mean age at diagnosis was 30±5 years (range 16-40 years).\",\n \"Thirteen (14%) of these patients met the minimal criteria of MODY (age at diagnosis before 25 years old and presence of diabetes in two consecutive generations).\",\n \"These patients were selected from a database containing 1800 cases recruited in the Diabetes and Endocrine Centre of the Prince of Wales Hospital.\",\n \"Family members of probands with MODY gene mutations, if available, were recruited and underwent a 75-gram oral glucose tolerance test (OGTT).\",\n \"One hundred healthy Chinese (age 33±10 years, 40 males and 60 females) without a history of diabetes were recruited as controls amongst hospital staff and students.\",\n \"Informed consent was obtained from each subject for a blood sample to be taken for DNA isolation and measurement of clinical parameters.\",\n \"This study was approved by the Clinical Research Ethics Committee of The Chinese University of Hong Kong.\",\n \"Screening of Glucoknase, HNF-1α and HNF-4α Genes for Mutations The minimal promoter region and exons of the glucokinase (β cell form), HNF-1α and HNF-4α (HNF-4α 2 form) genes were screened for mutations by direct sequencing of polymerase chain reaction (PCR) products as described (Froguel, et al (1993) N Engl J Med 328: 697; Yamagata, et al (1996) Nature 384: 455; Yamagata, et al (1996) Nature 384: 458).\",\n \"The occurrence of putative mutations in other family members and controls was determined by PCR-restriction fragment length polymorphism (RFLP).\",\n \"An artificial restriction site was introduced into either the wild-type or mutant sequence if the nucleotide substitution did not lead to gain or loss of a restriction site.\",\n \"Briefly, direct sequencing identified 5 mutations in the HNF-1α gene (G20R, R203H; S432C, I618M and IVS2nt-1G→A) and 3 mutations in the glucokinase gene (I110T, A119D and G385V) that are unique to Chinese subjects.\",\n \"They were screened as follows.\",\n \"HNF-1α G20R was screened by using the forward primer 5′-GGCAGGC-AAACGCAACCCACG-3′ (SEQ ID NO:14) and modified reverse primer 5′-CAGTGCCTCTTTGCTCAGGC-3′ (SEQ ID NO:15) for PCR amplification followed by digestion with StuI.\",\n \"The wild-type allele showed 19 and 140 bp products whereas the mutant allele showed a 159 bp product.\",\n \"HNF-1α R203H was screened by using the forward primer 5′-TGCCTGCAGAGTTCA-CCCATG-3′ (SEQ ID NO:16) and modified reverse primer5′-ATCTGCTGGGATGCTGGG-CCCCACTTGCAA-3′ (SEQ ID NO:17) for PCR amplification followed by digestion with BsrDI.\",\n \"The wild-type allele showed a 121 bp product whereas the mutant allele showed 26 and 95 bp products.\",\n \"HNF-1α S432C was screened by using the forward primer 5′-TGGAGCAGTCCCTAG-GGAGGC-3′ (SEQ ID NO:18) and reverse primer 5′-GTTGCCCCATGAGCCTCCCAC-3′ (SEQ ID NO:19) for PCR amplification followed by digestion with Cac81.The wild-type allele showed 104 and 218 bp products whereas the mutant allele showed 37,67 and 218 bp products.\",\n \"HNF-1α 1618M was screened by using the forward primer 5′-GTACCCCTAGGGACAGG-CAGG-3′ (SEQ ID NO:20) and reverse primer 5′-ACCCCCCAAGCAGGCAGTACA-3′ (SEQ ID NO:21) for PCR amplification followed by digestion with TaqI.\",\n \"The wild-type allele showed 88 and 160 bp products whereas the mutant allele showed a 248 bp product.\",\n \"HNF-1α IVS2nt-1G→A was screened by using the forward primer 5′-GGGC-AAGGTCAGGGGAATGGA-3′ (SEQ ID NO:22) and reverse primer 5′-CAGCCCAGACCAAACCAGCAC-3′ (SEQ ID NO:23) for PCR amplification followed by digestion with PstI.\",\n \"The wild-type allele showed 73 and 231 bp products whereas the mutant allele showed a 304 bp product.\",\n \"Glucokinase I110T was screened by using the forward primer 5′-GTCCCTGAGGCTG-ACACACTT-3′ (SEQ ID NO:24) and reverse primer 5′-AGCTGGGCCCTGAGATCCTGCA-3′ (SEQ ID NO: 25) for PCR amplification followed by digestion with FokI.\",\n \"The wild-type allele showed 108 and 142 bp products whereas the mutant allele showed a 250 bp product.\",\n \"Glucokinase A 119D was screened by using the forward primer 5′-ACCTGGGTGGCA-CTAACTTCA-3′ (SEQ ID NO:26) and modified reverse primer 5′-CGGCCCCTGCGCTG-CTCACCATCTGA-3′ (SEQ ID NO:27) for PCR amplification followed by digestion with BclI.\",\n \"The wild-type allele showed a 150 bp product whereas the mutant allele showed 28 and 122 bp products.\",\n \"Glucokinase G385V was screened by using the forward primer 5′-GGACTGTCG-GAGCGACACTCA-3′ (SEQ ID NO:28) and modified reverse primer 5′-GCGGTTGATGAC-GCCTGCCAG-3′ (SEQ ID NO:29) for PCR amplification followed by digestion with FauI.\",\n \"The wild-type allele showed 5,22,44 and 137 bp products whereas the mutant allele showed 5,44 and 159 bp products.\",\n \"Mutations in the amylin gene (S20G) and mitochondrial DNA (A3243G) were screened as follows.\",\n \"Amylin S20G was screened by using the forward primer 5′-TCACAT-TTGTTCCATGTTAC-3′ (SEQ ID NO:30) and reverse primer 5′-CAATAACTATAGAG-TTACATTG-3′ (SEQ ID NO:31) for PCR amplification followed by digestion with MspI.\",\n \"The wild-type allele showed a 239 bp product whereas the mutant allele showed 99 and 140 bp products.\",\n \"Mitochondrial DNA A3243G was screened by using the forward primer 5′-AAGGTTCGTT-TGTTCAACGA-3′ (SEQ ID NO:32) and reverse primer 5′-AGCGAAGGGTTGTAGTAGCC-3′ (SEQ ID NO:33) for PCR amplification and labeling of PCR product with α32PdATP at the last cycle.\",\n \"The PCR products were then digested with ApaI and analysed on 8% denaturing polyacrylamide gels.\",\n \"The wild-type allele showed a 427 bp product whereas the mutant allele showed 213 and 214 bp products.\",\n \"Clinical Studies All patients underwent a structured assessment including documentation of family history, age at diagnosis and body mass index (BMI) (Piwernetz, et al (1993) Diabetic Med 10: 371; Chan, et al (1997) Hong Kong Auth Qual Bull 2: 3).\",\n \"Family history was documented in two generations only since the diabetic status of grandparents was usually unknown.\",\n \"A fasting blood sample was taken for the measurement of glucose, C-peptide and glycosylated haemoglobin (HbA1c).\",\n \"Obesity was defined as a BMI≧27 kg/m2in men and ≧25 kg/m2in women (National Diabetes Data Group (1979) Diabetes 28: 1039).\",\n \"Assays Plasma glucose concentrations were measured by a glucose oxidase method (Diagnostic Chemicals, Charlottetown, Prince Edward Island, Canada).\",\n \"C-peptide was measured by radioimmunoassay (Novo-Nordisk, Copenhagen, Denmark).\",\n \"HbA1c was measured by gel electrophoresis (Ciba Coming Diagnostics Corp, Palo Alto, Calif.).\",\n \"Data Analysis Data are expressed as mean ±SD if normally distributed.\",\n \"Otherwise, data are expressed as median and range.\",\n \"Results Mututions and Polymorphisms in the Glucokinase, HNF-1α and HNF-4α Genes Screening of the promoter region and exons 1a, 2-10 of-the glucokinase gene (Stoffel, et al (1992) Proc Nratl Acad Sci 89: 7698; Tanizawa, et al (1992) 6:1070) revealed three novel missense mutations: I110T, A119D and G385V.\",\n \"In addition to these mutations, three uncommon variants (two of which had not been previously described) and two polymorphisms were found in the 5′-untranslared region of the mRNA and intron regions (Table 1).\",\n \"The brother and mother of subject HK84 (Table 1) also inherited the I110T mutation (FIG.\",\n \"7).\",\n \"The mother was diagnosed with diabetes at the age of 64 years upon screening.\",\n \"The brother aged 25 years, when tested with a 75 g OGTT had a plasma glucose at 0 and 120 min of 6.3 mmol/1 and 6.9 mmol/1, respectively.\",\n \"These results were inconclusive, suggesting impaired fasting glucose (IFG) by the 1997 ADA criteria but not reaching that of impaired glucose tolerance (IGT) by the 1998 WHO criteria.\",\n \"Screening of the HNF-1α gene revealed four missense mutations (G20R, R203H, S432C and 1618M) and one splice acceptor site mutation (IVS2nt-1G→A) (Table 2).\",\n \"All of these represent mutations in the HNF-1α gene unique to Chinese patients.\",\n \"Subject HK10 (Table 2) had three siblings (ages 26-36 years) with diabetes.\",\n \"The affected siblings all had inherited the IVS2nt-IG→A mutation while another sibling and the father with IGT had not.\",\n \"Moreover, the maternal grandparents, uncle and mother of HK10 were diabetic but they were not available for screening (FIG.\",\n \"7) (Chan, et al (1990) Diabetic Med 7: 211).\",\n \"Subject HK54 (Table 2) had four siblings (age 33-43 years) with normal glucose tolerance and one sibling (age 39 years) with IGT.\",\n \"The father and mother were diagnosed as having diabetes at the ages of 50 and 60 years, respectively.\",\n \"Neither the mother and nor any of the siblings had inherited the R203H mutation (FIG.\",\n \"7).\",\n \"In addition to the putative diabetes-associated mutations in HNF-1α two substitutions resulting in common amino acid polymorphisms, four silent mutations and nine variants/polymorphisms in introns were identified (Table 2).\",\n \"Family members of the other five probands (Tables 1 and 2) with glucokinase or HNF-1α missense mutations were not available for screening.\",\n \"None of the mutations in the glucokinase and HNF-1α genes were found in 100 healthy controls.\",\n \"Analysis of the promoter region and exons 1a, 2-10 of the HNF-4α gene (Furuta, et al (1997) Diabetes 46: 1652) revealed no obvious diabetes-associated mutations.\",\n \"Three patients were heterozygous for a previously described amino acid polymorphism, T/1130 (Yamagata et al, (1997) Nature 384: 458).\",\n \"Two patients were heterozygous for a silent mutation in the codon for L211, and one patient was heterozygous for a silent mutation in the codon for P441 (Table 3).\",\n \"There were two polymorphisms in the intron-upstream of exon 2 (intron 1B) and a G→A substitution in the promoter was found in the heterozygous state in one patient.\",\n \"The G→A substitution in the promoter at nucleotide-462 was not located in a known cis-acting regulatory region of the gene (Furuta, et al (1997) supra) and its effect on the regulation of expression of HNF-4α remains to be determined.\",\n \"Clinical Features of Patients with MODY or Unknown Etiology The clinical features of the patients with mutations in the glucokinase and HNF-1α genes or with unknown etiology are shown in Table 4.Of the 92 patients, 54 (59%) were non-obese at the time of study.\",\n \"The mean age at diagnosis of the patients with glucokinase mutation-associated diabetes (‘glucokinase diabetes’) was 28 years.\",\n \"All three subjects had mild hyperglycemia and satisfactory glycemic control (fasting glucose ≦7.4 mmol/1; HbA1c, 6.7%; non-diabetic range: 5.1-6.4%).\",\n \"These patients had varving degrees of basal pancreatic β cell secretory function as indicated by their fasting C-peptide levels (0.28-1.60 nmol/1) (Chan, et al (1990) supra).\",\n \"All were treated with diet or oral drugs.\",\n \"No diabetic complications were observed in the three patients with glucokinase mutation (Froguel, et al (1993) supra ; Page, et al (1995) 12: 209; Velho, et al (1997) 40: 217).\",\n \"The mean age at diagnosis of the patients with HNF-1α mutation-associated diabetes was 31 years.\",\n \"Among the four patients (HK30, 54,90 and 92) with missense mutations, all had mild hyperglycemia and satisfactory glycemic control (fasting glucose ≦7.4 mmol/1; HbA1c≦7.1%) but exhibited varying degrees of basal pancreatic β cell secretory function (fasting C-peptide, 0.10-0.49 nmol/1).\",\n \"They did not have diabetic complications and were treated with diet or oral drugs.\",\n \"The subject (HK10) with the splice-site IVS2nt-1G→A mutation was not overweight when diagnosed at the age of 19 years (Fajans (1990) Diabetes Care 13: 49-64) and presented with proliferative retinopathy and clinical proteinuria.\",\n \"She was treated with insulin continuously for three months after the diagnosis.\",\n \"She eventually developed neuropathy and renal failure.\",\n \"TABLE 1 Mutations and polymorphisms in the glucokinase gene in Chinese subjects with early-onset Type 2 diabetes mellitus Subject Location Codon/nt Nucleotide change Designation Frequency Mutations HK84 Exon 3 110 ATC (Ile)→ACC (Thr) I110T HK38 Exon 3 119 GCT(Ala)→GAT(Asp) A1I9D HK15 Exon 9 385 GGG(Gly)→GTG(Val) G385V Polymorphisms 5′-UT* −213 A→G 5′-UT β − 213 A/G A 0.96, G 0.04 5′-UT −84 C→G 5′-UT β − 84 C/G C 0.94, G 0.06 Intron 1c nt − 13 C→G IVS1nt − 13 C/G C 0.99, C 0.01 Intron 9 nt + 8 C→T IVS9nt + 8C/T C 0.50, T 0.50 Intron 9* nt + 49 G→A IVS9nt + 49G/A G 0.99, A 0.01 nt indicates the nucleotide location relative to the first nucleotide of codon 1 (ATG) for polymorphisms in the 5′-untranslated region (5′-UT) of the β cell specific exon 1 α/1 β, and splice donor (+) or acceptor site (−).\",\n \"Intron 1c is the intron between exon 1c, which encodes the amino terminal 14 amino acids of the minor liver isoform of glucokinase, and exon 2 (Velho, et al (1996) 19: 915).\",\n \"The asterisks indicate polymorphisms that were reported by Ng, et al (Diabetic Med (1999) 16: 956) and that have not been reported in studies of other populations (Veiga-deCunha, et al (1996) J Biol Chem 271: 6292; Zhang, et al (1995) 38: 1055).\",\n \"TABLE 2 Mutations and polymorphisms in the HNF-1 α gene in Chinese subjects with early-onset Type 2 diabetes mellitus Subject Location Codon/n Nucleotide change Designation Frequency Mutations HK90 Exon 1 20 GGG (Gly)→AGG (Arg) G20R HK10 Intron 2/ nt − 1 AG→AA at splice IVS2nt − 1G→A Exon 3 acceptor site HK54 Exon 3 203 CGT (Arg)→CAT (His) R203H HK30 Exon 6 432 TCC (Ser)→TGC (Cys) S432C HK92 Exon 10 618 ATC (Ile)→ATG (Met) I618M Silent mutations/polymorphisms Exon 1 17 CTC (Leu)→CTG (Leu) L17C/G C 0.63, C 0.37 Exon 1 27 ATC (Ile)→CTC (Leu) I/L27 A 0.57, C 0.43 Intron 1 nt − 42 G→A IVS1nt − 42G/A C 0.58, A 0.42 Intron 2* nt + 53 C→G IVS2nt + 53C/G C 0.99, C 0.01 Intron 2 nt − 51 T→A IVS2nt − 51T/A T 0.77, A 0.23 Intron 2 nt − 23 C→T IVS2nt − 23C/T C 0.48, T 0.52 Intron 5 nt + 9 C→G IVS5nt + 9C/G C 0.98, G 0.02 Intron 5 nt − 42 G→T IVS5nt − 42G/T G 0.87, T 0.13 Intron 6* nt + 26 C→T IVS6nt + 26C/T C 0.99, T 0.01 Exon 7 459 CTG (Leu)→TTG(Leu) L459C/T C 0.48, T 0.52 Exon 7 459 CTG (Leu)→CTA (Leu) L459G/A C 0.99, A 0.01 Exon 7 487 AGC (Ser)→AAC (Asn) S/N487 G 0.48, A 0.52 Intron 7 nt + 7 G→A IVS7nt + 7G/A G 0.48, A 0.52 Exon 8* 531 AGC (Ser)→AGT (Ser) S531 C/T C 0.99, T 0.01 Intron 9 nt − 24 T→C IVS9nt − 24T/C T 0.48, C 0.52 nt indicates the nucleotide location relative to the splice donor (+) or acceptor site (−).\",\n \"The asterisks indicate polymorphisms reported by Ng, et al (Diabetic Med (1999) 16: 956) and that have not been reported in studies of other populations.\",\n \"TABLE 3 Mutations and polymorphisms in the HNF-4 α gene in Chinese subjects with early-onset Type 2 diabetes mellitus Location Codon/nt Nucleotide change Designation Frequency Silent mutations/ polymorphisms Promoter* nt-462 G→A Ptr-462G/A G 099, A 0.01 Intron lB nt-38 C→T IVS1nt-38C/T C 0.80, T 0.20 Intron lB nt-5 C→T IVS1nt-5C/T C 0.79, T 0.21 Exon 4 130 ACT (Thr)→ATT (Ile) T/I130 C 0.98, T 0.02 Exon 6* 211 CTC (Leu)→CTT (Leu) L211C/T C 0.99, T 0.01 Exon.\",\n \"10* 441 CCG (Pro)→CCA (Pro) P441G/A G 0.99, A 0.01 G 0.99, A 0.01 nt indicates the nucleotide location relative to the first nucleoride of codon 1 (ATG) for polymorphisms in the promoter region and splice donor (+) or acceptor site (−).\",\n \"The sequence context of the nt-464 polymorphism is GATA(G/A)TATC.\",\n \"The asterisks indicate polymorphisms that have not been reported in studies of other populations.\",\n \"TABLE 4 Clinical features of Chinese patients with early-onset Type 2 diabetes of unknown etiology as compared with those with diabetes as a result of mutations in glucokinase and HNF-1 α genes Unknown Glucokinase Etiology diabetes (n = 3) HNF-1 α diabetes (n = 5) (n = 84) HK1 HK38 HK84 HK10 HK30 HK54 HK90 HK92 Age at diagnosis (year) 30 ± 5 18 29 38 19 30 33 36 38 Interval since diagnosis (year) 3 (0-16) 15 2 1 9 3 6 0 0 Sex(M/F)(%) 32/68 F F F F M F M M Family history(fa/mot/sib)(%) 45/63/25 fa mot, sib mot gparent, fa fa, mot mot, sib mot uncle, mot, sib BMI (kg/m2) 26 ± 5 nd* 15 28 26 23 20 20 29 HbA1c(%) 7.5 ± 1.9 6.7 6.0 6.6 8.7 7.1 6.0 6.9 6.1 Fasting glucose (mmol/l) 8.5 ± 3.3 7.2 7.4 6.6 13.9 7.4 4.9 4.9 6.6 Fasting C-peptide (nmol/l) 0.43 (0.03-4.96) nd 1.60 0.28 0.47 0.49 0.11 0.16 0.10 Treatment (D/O/I)(%) 49/43/8 O O D I O O D O gparent, grandparent affected; uncle, uncle affected; fa, father affected; mot, mother affected; sib, siblings affected; D, diet; O, oral drugs; I, insulin; nd, not done.\",\n \"Data are expressed as mean ± SD median (range) or n. *BMI was not measured because this patient had spinomuscular atrophy.\",\n \"The mean age at diagnosis of the 84 patients with unknown etiology was 30 years, similar to those with glucokinase or HNF-α gene mutations.\",\n \"Large variations in the degree of hyperglycemia (fasting glucose 8.5±3.3 mmol/1) and basal pancreatic β cell secretory function (fasting C-peptide 0.03-4.96 nmol/1) were observed.\",\n \"Most of these pateints were treated with oral drugs or diet (92%).\",\n \"EXAMPLE 2 Mitochondrial DNA A3243G Mutation in Patients with Early-or Late-Onset Type 2 Diabetes Mellitus in Hong Kong Chinese This example illustrates the prevalence of the mitochondrial DNA A3243G mutation in the Hong Kong Chinese population as represented by a large cohort of type 2 diabetic patients with differing ages of diagnosis and clinical phenotypes.\",\n \"Experimental Design and Methods Subjects The study group consisted of 906 unrelated type 2 diabetic patients diagnosed according to the 1985 WHO criteria (World Health Organization, 1985).\",\n \"This cohort included four groups of patients selected according to the age of diagnosis and the presence or absence of family history of diabetes.\",\n \"Groups 1 and 2 consisted of 219 and 128 patients, respectively, with an early age of diagnosis (≦40 years) and with (Group 1) or without (Group 2) a family history of diabetes.\",\n \"Groups 3 and 4 consisted of 211 and 348 patients, respectively, with an older age of diagnosis (>40 years) and with (Group 3) or without (Group 4) a family history of diabetes.\",\n \"Patients in each of these groups were randomly selected from a cohort recruited in the Diabetes and Endocrine Centre of the Prince of Wales Hospital, which has a catchment of 1.2 million population in Hong Kong.\",\n \"All the patients underwent a structured assessment based on the Europe DiabCare Protocol (Piwernetz, et al (1993) Diabetic Med 10: 371; Chan, et al (1997) Hosp Auth Qual Bull 2: 3).\",\n \"Family members of mt3243 mutation carriers, if available, were recruited and underwent a 75 grain oral glucose tolerance test (OTT) Two hundred and thirteen healthy Chinese without a history of diabetes were recruited as control subjects amongst hospital staff and students.\",\n \"The present study group included 75 early onset type 2 diabetic (two of whom had an mt3243 mutation) and 95 control subjects who were included in a previous report (see Smith, et al (1997) Diabetic Med 14: 1026).\",\n \"Informed consent was obtained from each subject for a blood sample to be taken for DNA isolation and measurement of clinical partners.\",\n \"This study was approved by the Clinical Research Ethics Committee of The Chinese University of Hong Kong.\",\n \"Mt3243 Mutation Analysis Leukocyte DNA was extracted by standard methods involving proteinase K and phenol/chloroform (Sambrook, et al (1989) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York, incorporated herein by reference).\",\n \"The mt3243 genotype was determined by polymerase chain reaction (PCR) amplification and ApaI digestion as described (Smith, et al, (1997) supra).\",\n \"In brief, the DNA region spanning nucleotide 3029 and 3456 was amplified by PCR and labelled with α32PDATP at the last cycle.\",\n \"This method prevents the underestimation of the proportion of mutant mtDNA as a consequence of heteroduplex formation during the PCR (Schoffner, et al (1990) Cell 61: 931).\",\n \"The PCR products were then digested with ApaI (Gibco BRL, Gaithersburg, Md., USA) for 2 h at 30 C. Digested PCR products were electrophoresed on 8% denaturing polyacrylamide gels and visualized by autoradiography.\",\n \"The presence of mt3243 led to the cleavage of the 427 bp product into 213 and 214 bp fragments.\",\n \"Standards containing 0-100% mutant mt3243 (made by mixing a cloned DNA carrying no mt3243 mutation and another cloned DNA carrying >99% mutant mt3243 in different proportions, kindly given by Dr J. van den Ouweland, Leiden University) were also included in the assay.\",\n \"The 100% mutant DNA was used as a positive control to evaluate completeness of PCR product digestion.\",\n \"The intensity of bands was quantified by a Bio-Rad Model GS-670 imaging densitometer and a Molecular Analyst software (version 1.3) (Bio-Rad, Hercules, Calif., USA).\",\n \"The proportion of mt3243 in a sample was calculated by dividing the intensity at mutant bands (213 and 214 bp) by the total intensity of both wild-type and mutant bands.\",\n \"Clinical Studies All patients underwent a structured assessment including documentation of family history, age of diagnosis, body mass index (BMI) and waist-to-hip ratio.\",\n \"Audiometry was performed by a technician at the otolaryngology department to assess the sensorineural status in subjects carrying the mt3243 mutation.\",\n \"A fasting blood sample was taken for the measurement of glucose, C-peptide, insulin and glycosylated haemoglobin (HbA1c).\",\n \"Insulin resistance (IR) was estimated using the homeostasis model assessment (HOMA) where IR=fasting insulin×fasting glucose/22.5 (Matthews et al, (1985) Diabetologia 28: 412).\",\n \"Obesity was defined as a BMI≧27 kg/m2 in men and ≧25 kg/m2 in women (National Diabetes Data Group, 1979).\",\n \"The basal pancreatic β-cell reserve was also assessed by plasma fasting C-peptide level.\",\n \"Patients with a C-peptide level≦0.2 nmol/l were considered to be insulin deficient (Service et al,.\",\n \"(1997) Diabetes Care 20: 198).\",\n \"Biochemical Assays Plasma glucose was measured by a glucose oxidase method (Diagnostic Chemicals, Charlottetown, PEI, Canada).\",\n \"HbA1c was measured by gel electrophoresis (Ciba Coming Diagnostics Corp, Palo Alto, Calif. USA) C-peptide was measured by radioimmunoassay (Novo-Nordisk, Copenhagen, Denmark) with an intra-assay coefficient of variance (CV) of 3.4% and interassay CV of 9.6%.\",\n \"Insulin was measured by radioimmunoassay (Pharmacia, Uppsala, Sweden) with in intra-assay CV of 6% and interassay CV of 13.8%.\",\n \"Statistical Analysis Data are expressed as mean ±SD or median (range) as appropriate.\",\n \"The X2 test was used for analysing categorical data.\",\n \"Spearman correlation was used for measurement of association between variables.\",\n \"All statistics was performed with the Statistical Package for Social Sciences (SPSS) for Windows, version 6.1.A P-value<0.05 was considered as significant.\",\n \"Results The clinical details of the 906 type 2 diabetic patients are shown in Table 5.A significantly higher prevalence of maternal over paternal history of diabetes was found in both early- (Group 1) and late-onset (Group 3) diabetic patients with a positive family history (Table 5).\",\n \"Amongst the 906 type 2 diabetic patients, in addition to the two patients reported previously (Smith, et al (1997) supra), three more patients carrying the mt3243 mutation were identified.\",\n \"In Group 1, this mutation was found in 1.8% of (four of 219) early onset patients with a positive family history.\",\n \"This prevalence increased to 3% (four of 133) if only those with a positive maternal family history were considered.\",\n \"In addition, one of the 348 late-onset patients without a family history (Group 4) was found to have this mutation (0.3%).\",\n \"None of the 128 early onset patients who had no family history (Group 2) or 211 late-onset patients with a positive family history (Group 3) or 213 control subjects had the mutation.\",\n \"Amongst the five probands with the mt3243 mutation, three families were recruited (FIG.\",\n \"8).\",\n \"In family A, family members with diabetes or IGT were identified but none of them carried the mutation.\",\n \"In Family E, two more subjects were found to have the mutation of whom one had diabetes.\",\n \"The clinical and biochemical characteristics of subjects carrying the mt3243 mutation are summarized in Table 6.The percentage of mt3243 varied from 1% to 14% (Table 6).\",\n \"There was no correlation of heteroplasmy level of mutation with levels of HbA1c fasting plasma glucose, C-peptide, insulin, insulin resistance or the presence of sensorineural impairment (P>0.05) (Table 6).\",\n \"Families A and B These two families have been reported in a previous study (Smith, et al, (1997) supra).\",\n \"The 37-year-old proband (II 4) in family A had been treated with oral drugs since diagnosed at the age of 32 years.\",\n \"The mother (I-2) and two siblings (II-1 and II-3) were diabetic while the father (I-1) and one sister (II-2) had IGT.\",\n \"However, none of the family members had the mt3243 mutation although the mother had a history of hearing loss.\",\n \"The proband of family B was diagnosed as having diabetes at the age of 22 years and had been treated with oral drugs.\",\n \"The mother was diabetic and deaf but was not available for screening.\",\n \"Family C The 38-year-old proband (III-3) was diagnosed with diabetes at the age of 31 years.\",\n \"She was treated with diet and oral drugs for 6 years before being changed to insulin therapy.\",\n \"Audiometry revealed bilateral high tone sensorineural impairment.\",\n \"The father (II-1) had normal glucose tolerance and did not have the mt3243 mutation.\",\n \"The older sister (III-2) and the mother (II-2) both developed diabetes at about 40 years of age and the grandmother had diabetes at the age of 50 years.\",\n \"The mother became deaf at the age of 59 years.\",\n \"None of these affected members were available for screening.\",\n \"TABLE 5 The clinical and biochemical features of 902 Chinese patients with type 2 diabetes Group 1 Group 2 Group 3 Group 4 (n = 219) (n = 128) (n = 211) (n = 348) Age of diagnosis (year) 32 ± 6 32 ± 7 52 ± 8 57 ± 9 Duration of disease (years) 4 (0-31) 2 (0-41) 5 (0-24) 4 (0-26) Sex (M/F) (%) 36/64 36/64 44/56 41/59 Family history of diabetes Father (%) 45* 0 21** 0 Mother(%) 61 0 38 0 Siblings(%) 35 0 57 0 At least 1 parent and 1 sibling(%) 26 0 19 0 Body mass index (kg/m2) 25.7 ± 4.8 24.9 ± 4.4 24.4 ± 3.8 24.3 ± 3.8 HbAlc (%) 7.3 (4.1-15.3) 7.1 (3.8-16.8) 7.7 (4.0-16.0) 7.6 (4.2-19.7) Fasting plasma glucose (nmol/l) 7.4 (4.4-23.0) 7.7 (2.8-21.4) 7.8 (3.9-34.0) 8.1 (3.0-24.5) Fasting plasma C-peptide (nmol/l) 0.47 (0.03-4.96) 0.56 (0.09-1.62) 0.57 (0.01-9.40) 0.51 (0.01-8.22) Insulin deficiency (%)† 16 11 12 16 Insulin treatment(%) 11 13 9 11 Mean ± SD or median (range).\",\n \"Group 1: early onset (40 years) patients with a family history of diabetes; Group 2: early onset patients without a family history of diabetes; Group 3: late-onset patient (>40 years) with a family history of diabetes; Group 4: late-onset patients without a family history of diabetes.\",\n \"†Insulin deficiency defined as fasting plasma C-peptide 0.2 nmol/l (Service, et al (1997) supra); *P < 0.005 and **P < 0.0001 for comparison between prevalence rates of paternal vs. maternal family histories.\",\n \"TABLE 6 The clinical and biochemical characteristics of Chinese subjects carrying the mt3243 mutation in the mitochondrial tRNALeu gene Family A Family B Family C Family D Family E proband proband proband proband proband II-2 II-4 Age of diagnosis (yr) 32 22 31 70 33 38 — Duration of disease (yr) 2 1 7 9 2 1 — Sex F M F M M F F Body mass index (kg/m2) 18.2 22.4 18.6 25.3 27.6 21.6 19.6 Waist-to-hip ratio 0.79 0.81 0.75 0.90 0.86 0.79 0.75 HbA1c (%) 5 7.7 6.8 5.9 11.3 7.5 4.3 Fasting glucose(mmol/l) 5.3 11.6 9.8 5.8 15.7 7.1 4.4 Fasting C-peptide (nmol/l) 0.43 0.23 0.30 0.7 0.72 0.83 0.4 Fasting insulin (mIU/I) 13.4 26.0 ND ND 20.6 16.8 13.2 Insulin resistance* 3.2 13.4 5.5 ND 14.4 5.3 2.6 Treatment Oral drugs Oral drugs insulin Oral drugs Oral drugs Oral drugs — Audiogram Normal Normal High tone High tone Normal ND ND impairment Impairment Mt3243 level (%) 13 1 14 1 5 9 4 *HOMA method, ND, not determined Family D The 79-year-old proband was diagnosed with diabetes at the age of 70 years.\",\n \"An audiometry test revealed high tone sensorineural impairment.\",\n \"Neither of the parents and nor any siblings were available for screening or known to have diabetes.\",\n \"Family E The 35-year-old proband (II-3) was diagnosed with diabetes at the age of 33 years and was treated with diet.\",\n \"One of the sisters (II-4) had normal glucose tolerance while two sisters (II-1 and II-2) were diagnosed to have diabetes at the age of 30 and 38 years, respectively.\",\n \"The father (I-1) and mother (I-2) also had diabetes at the age of 50 and 35 years, respectively.\",\n \"All the diabetic and nondiabetic sisters who came for screening had the mt3243 mutation.\",\n \"One of the diabetic sisters (II-1) had high tone sensorineural impairment whereas the audiogram of the proband was normal.\",\n \"EXAMPLE 3 The Role of the Amylin Gene S20G Mutation in Early Onset Type 2 Diabetes and in the Regulation of Cholesterol Metabolism in Chinese This example illustrates the distribution of the amylin gene S20G mutation in Hong Kong Chinese with or without Type 2 diabetes, and its influences on β-cell function and metabolic profiles.\",\n \"The data are consistent with the conclusion that the S20G mutation in the amylin gene may contribute to early occurrence of Type 2 diabetes, and that it may also influence lipid metabolism in the Chinese population.\",\n \"Experimental Design and Methods Subjects The study protocol was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong.\",\n \"Informed consent was obtained from each of the participants.\",\n \"For the study, 227 early-and 235 late-onset Type 2 diabetic patients (defined as age at diagnosis ≦40 and >40 years respectively), as well as 126 non-diabetic subjects (defined as fasting plasma glucose <6 mmol/1), were consecutively recruited at the Diabetes Centre of the Prince of Wales Hospital.\",\n \"Type 2 diabetes was asscertained according to the World Health Organisation criteria (Anonymous (1997) Diabetes Care 20: 1183).\",\n \"None of the patients had typical presentations of Type 1 diabetes, such as acute symptoms and heavy ketonuria (>3+), history of diabetic ketoacidosis or continuous need for insulin treatment within I year of diagnosis.\",\n \"Patients who had anti-glutamic acid decarboxylase autoantibody (Ko, et al (1998) Ann Clin Biochem 35: 761) diabetes-causing mutations in the glucokinase and hepatonuclear factor-1α and 4-α genes (Ng et al (1999) Diabetic Medicine 16: 956) were excluded from the study.\",\n \"Clinical and Biochemical Measurements Patients fasted at least 8 hours prior to their clinical examinations.\",\n \"Blood pressures were taken, after they remained sitting for at least for 5 min using a standard mercury sphygmomanometer.\",\n \"Body height and weight, and waist and hip circumferences were taken while the patients were standing in light clothing but wearing no shoes.\",\n \"Measurements of fasting plasma glucose and lipids were performed by routine assays in the Department of Chemical Pathology at the Prince of Wales Hospital.\",\n \"Levels of total cholesterol and triglyceride were assayed enzymatically with commercial reagents(Centrichem, chemistry system, Baker Instrument Co., Allentown, Pa.).\",\n \"HDL-cholesterol was determined after fractional precipitation with dextran sulfate-MgCl2 and LDL-cholesterol, calculated by the Friedewald's equation (Friedewald et al (1972) Clin Chem 18:499).\",\n \"HbA1c was measured using an automated ion-exchange chromatographic method (BioRad, Hercules, Calif., USA; normal range: 5.1-6.4%).\",\n \"Plasma levels of C-peptide were measured by radioimmunoassays using commercial kits (#7350104 and #141 respectively, Novo Nordisk, Denmark).\",\n \"The detection range was from 0.01 to 1.0 pmol/1.Insulin deficiency was defined as fasting plasma C-peptide level <0.2 pmol/1 (Service et al (1997) Diabetes Care 21: 987).\",\n \"Mutation Detection The S20G mutation creates a MSP I restriction fragment length polymorphism (RFLP), which can be detected using PCR-RFLP analysis (Sakagashira et al (1996) Diabetes 45: 1279).\",\n \"Briefly, DNA fragments spanning the mutation site were amplified by PCR using the primers 5′-TCACATTTGTTCCATGTTAC-3′ (SEQ ID NO: 30) and 5′-CAATAACTATAGAGTTACATTG-3′ (SEQ ID NO: 31), at the annealing temperature of 56° C. Each of the PCR products was then digested overnight with 5 units of the restriction enzyme MSP I (#R6401, Promega, Wis., USA) at 37° C. Alleles were separated on 2.5% agarose gel.\",\n \"The wild-type allele showed a 239 bp product whereas the mutant allele showed 99 and 140 bp products.\",\n \"Statistical Analysis Continuous variants were expressed as mean ±SD.\",\n \"Chi-square test was used for the analysis of proportions.\",\n \"Differences between continuous variables were analysed by the student's t-test using the statistical package for social sciences (SPSS Inc., Chicago, USA).\",\n \"ap value <0.05 was considered to be statistically significant.\",\n \"Results Table 7 summaries the demographic data of the subjects involved in the study.6 early-and 1 late-onset patients heterozygous for the amylin S20G mutation (2.6% vs 0.4%, p=0.055) were identified.\",\n \"None of the non-diabetic subjects had the S20G mutation (Table 7).\",\n \"In the early-onset group, 5 out of the 6 mutation-carrying patients had satisfactory glycemic control with diet and/or oral drug medications, and had fasting plasma C-peptide concentrations of greater than 0.2 pmol/1 (Table 8).\",\n \"Moreover, the mutation carriers had lower total cholesterol (4.3±0.9 vs 5.3 1.1, p=0.02) and LDL-cholesterol (2.3±0.7 vs 3.2±0.9, p=0.01) (Table 9) than those without the mutation.\",\n \"The patients with or without the S20G mutation were of a comparable age (34±6 vs 35±8, p>0.05).\",\n \"TABLE 7 Clinical characteristics of the early and late-onset patients as well as non-diabetic subjects, and the distribution of the amylin gene S20G mutation.\",\n \"Type 2 diabetes Control subjects Early-onset Late-onset Clinical characteristics n 126 227 235 Age (years) 34.9 ± 10.4 36.8 ± 6.7 59.4 ± 10.1 Sex ratio (M/F) 1:1.75 1:1.97 1:1.33 Age of diagnosis (years) NA 31.7 ± 4.6 54.3 ± 9.8 Body mass index (kg/m2) 22.3 ± 3.4 25.1 ± 4.5 24.2 ± 3.6 Waist to hip ratio 0.77 ± 0.05 0.85 ± 0.07 0.89 ± 0.06 Systolic blood 114 ± 10 119 ± 17 137 ± 21 pressure (mmHg) Diastolic blood 64 ± 9 76 ± 10 82.0 ± 11 pressure (mmHg) HbAlc (%) — 7.6 ± 2.0 8.1 ± 2.2 Total cholesterol (mmol/l) 4.7 ± 0.9 5.3 ± 1.2 5.6 ± 1.2 HDL-cholesterol (mmol/l) 1.4 ± 0.3 1.3 ± 0.4 1.3 ± 0.4 LDL-cholesterol (mmol/l) 2.9 ± 0.8 3.2 ± 0.9 3.6 ± 1.1 Triglyceride (mmol/l) 0.9 ± 0.5 1.7 ± 1.8 1.8 ± 1.5 Genotypes Wild-type allele 126 221 234 homozygotes Heterozygotes 0 6 1 Mutant allele 0 0 0 homozygote S20G allele 0 2.6‡ 0.4 frequency (%) Mean ± SD; ‡p = 0.055 TABLE 8 Fasting plasma levels of glucose, HbA1c and C-peptide in early-onset Type 2 diabetic patients with an amylin gene S20G mutation C- Onset Duration peptide HbA1c Glucose Patient age (years) Treatment (pmol/l) (%) (mmol/l) Index a 29 1 Diet >1.0 6.2 5.3 Index b 25 18 Oral drugs >1.0 7.2 7.2 Index c 35 3 Diet >1.0 8.2 9.3 Index d 28 2 Diet + oral 0.02 5.4 4.9 Index e 36 1 Diet + oral 0.51 5.8 14.0 Index f 13 13 Insulin — 11.5 7.2 TABLE 9 Comparisons of clinical characteristics and biochemical measurements between early-onset S20G mutation-carrying patients and early-onset patients without the S20G mutation.\",\n \"Patients Age Sex BMI WHR SBP DBP HbA1c Triglyceride Total-C HDL-C LDL-C Index a 30 F 23.2 0.76 114 79 6.2 0.76 3.8 1.04 2.5 Index b 43 F 17.9 0.78 138 74 7.2 0.46 5.8 2.21 3.3 Index c 38 M 28.2 0.88 128 90 8.2 2.90 4.7 1.06 2.3 Index d 30 F 23.2 0.75 105 66 5.4 0.99 4.0 1.37 2.2 Index e 37 F 24.0 0.89 126 80 5.8 2.76 3.3 0.87 1.2 Index f 26 M 29.1 0.89 120 81 11.5 0.59 4.2 1.71 2.2 Mutation + (n = 6) 34 ± 6 — 24 ± 4 0.8 ± 0.1 122 ± 16 78 ± 8 7.4 ± 2.0 1.4 ± 1.1 4.3 ± 0.9 1.3 ± 0.5 2.3 ± 0.7 Mutation − (n = 221 35 ± 8 — 25 ± 4 0.9 ± 0.1 119 ± 17 76 ± 10 7.6 ± 2.3 1.7 ± 2.5 5.3 ± 1.1* 1.3 ± 0.4 3.2 ± 0.9** Mean ± SD; *p = 0.02; **p = 0.01 BMI, body mass index (kg/m2); WHR, waist to hip ratio; SBP, systolic blood pressure (mmHg); DBP, diastolic blood pressure (mmHg); Total-C, total cholesterol (mmol/l); HDL-C, HDL-cholesterol (mmol/l); LDL-cholesterol (mmol/l).\",\n \"The genetic association between the S20G mutation and early-onset Type 2 diabetes (Table 7 ; Sakagashira et al (1996) supra) is consistent with the physiological data that amylin may play a role in the pathogenesis of the disease (Cooper (1994) Proc Natl Acad Sci 84: 8628).\",\n \"Early onset of Type 2 diabetes is in fact not uncommon (Rosenbloom et al (1999) Diabetes Care 22: 345), although type 2 diabetes is classically a late-onset disease.\",\n \"However, early-onset patients appear to be heterogenous in etiology.\",\n \"In Hong Kong Chinese, in particular, maturity-onset Type 2 diabetes of the young as well as atypical autoimmune diabetes are present, but accounting for only a small proportion of the overall early-onset population (Ng et al (1999) supra; Ko et al (1998) supra).\",\n \"The S20G mutation may also explain some of the early-onset cases.\",\n \"The S20G mutation carriers usually did not require insulin for glycemic control, and did not appear to be insulin deficient (Table 8).\",\n \"These findings are different from previous observations that the S20G mutation may be associated with poor glycemic control as well as β cell dysfunction (Sakagashira et al (1996) supra; Chuang et al (1998) supra).\",\n \"The reported Japanese S20G carriers (Sakagashira et al (1996) supra) had an average diabetes duration of approximately 20 years at the time they were tested.\",\n \"That they commonly required insulin treatment may be due to the deterioration in glycemic control during their long diabetes course, not necessary the presence of the mutation.\",\n \"Moreover, the mutation appears to be associated with lower plasma levels of total cholesterol and LDL-cholesterol (Table 9).\",\n \"This is in keeping with the recent finding that pramlintide (a synthetic human amylin analog) was able to lowers plasma levels of total cholesterol and LDL-cholesterol in Type 2 diabetic patients (Thompson et al (1998) Diabetes Care 21: 987).\",\n \"Few studies to date have been focused on the relationships between amylin action and lipid profiles These data and those from Thompson and co-workers are consistent with the conclusion that amylin may also play a role in the regulation of cholesterol metabolism.\",\n \"EXAMPLE 4 The Significant Roles of Genetics and Obesity in Familial Early-Onset Type 2 Diabetes in Chinese Patients This example illustrates the prevalence of known molecular defects in separate cohorts of Chinese patients with early-and late-onset familial Type 2 diabetes.\",\n \"The genes studied are those that have been found to be associated with diabetes and which may contribute to early onset of the disease under gene-gene and gene-environmental influences, including glucokinase (MODY2), HNF-1α (MODY3), and the A3243G mutation in the mitochondrial DNA coding for tRNALeu(UUR) (mt3243) that has been associated with a form of diabetes characterized by maternal inheritance and deafness (van den Ouweland, et al (1992) Nature Genet.\",\n \"1: 368).\",\n \"Experimental Design and Methods Subjects The Prince of Wales Hospital (PWH) is a regional teaching hospital in Hong Kong.\",\n \"Its catchment area has a population of 1.2 million, accounting for 20% of the total population in Hong Kong.\",\n \"There is a lack of long term health care programs in Hong Kong, and medical insurance is not widely available.\",\n \"Many patients with chronic diseases such as diabetes are managed in public hospitals or clinics where they pay only a nominal fee.\",\n \"Hence, except for high social classes, the patients are largely representative of the diabetic population in Hong Kong.\",\n \"Since 1995, all patients attending the diabetes clinic of the PWH have been entered into the PWH Diabetes Registry after undergoing a structured assessment (Piwemetz, et al (1993) Diabetic Med 10:371; Chan, et al (1997) Hosp Auth Qual Bull 2:3).\",\n \"During the study period, a separate cohort of 150 young patients with early-onset diabetes (age ≦40 years and age at diagnosis ≦35 years) who underwent the structured assessment were recruited consecutively from the diabetes clinics at the PWH to form the Young Chinese Diabetes Database (Ko, et al (1998) Ann Clin Biochem 35: 761).\",\n \"The 150 cases in the Young Chinese Diabetes Database, 92 and 53 patients, respectively, were selected for the present study as they satisfied the following criteria: All these 145 young patients had early-onset (current age and age at diagnosis ≦40 years) Type 2 diabetes (1985 WHO criteria, Geneva) and a positive family history for diabetes (at least 1 first degree relative with diabetes).\",\n \"Patients with classical Type 1 diabetes (acute ketotic presentation or continuous requirement of insulin within 1 year of diagnosis) were excluded from the study.\",\n \"The prevalence of anti-GAD (Ko, et al (1998) supra), mt3243 (Smith, et al (1997) Diabetic Med 14: 1026; Ng, et al (2000) 52: 557) and amylin gene mutations (Lee, et al (2001) J. Endocrinol) amongst patients from the Young Chinese Diabetes Database has been reported.\",\n \"Additionally, the prevalence of mt3243 (Ng, et al (2000) supra), amylin (Lee, et al (2001) supra), glucokinase, HNF-1α and HNF-4α gene mutations (Ng, et al(1999) Diabetic Med 16:956) in a separate cohort from the PWH Diabetes Registry has been reported.\",\n \"In this study, screening for glucokinase and HNF-1α gene mutations was extended to the 53 patients from the Young Chinese Diabetes Database.\",\n \"The HNF-4α gene was not screened in this cohort due to the expected low frequency of mutations.\",\n \"(None were found in the 92 patients from the PWH Diabetes Registry (Ng, et al (1999) supra)).\",\n \"Screening for anti-GAD was extended to the 92 patients from the PWH Diabetes Registry.\",\n \"Nineteen (13%) of these 145 young patients with familial diabetes met the minimal criteria for MODY (age at diagnosis ≦25 years and presence of diabetes in two consecutive generations).\",\n \"Altogether 10 out of 20 families with probands carrying putative diabetogenic gene mutations were recruited for a 75-gram OGTT and clinical assessment.\",\n \"The 1999 WHO classification was used to define the glycemic status of the family members (WHO, Geneva, 1999).\",\n \"For comparison of clinical characteristics of the early-onset patients, 290 sex-matched patients with late-onset diabetes (age at diagnosis >40 years) and a positive family history of diabetes were randomly selected from the current 1800 cases in the PWH Diabetes Registry.\",\n \"One hundred healthy Chinese (age 33±10 years, 40 males and 60 females) were selected as control subjects from hospital staff and students for screening for the gene variants identified in the study patients.\",\n \"Informed consent was obtained from each subject for a blood sample to be taken for DNA extraction and measurement of biochemical indices.\",\n \"This study was approved by the Clinical Research Ethics Committee of The Chinese University of Hong Kong.\",\n \"Clinical Studies All patients underwent a structured assessment based on the Europe DiabCare Protocol.\",\n \"They had documentation of their family history of diabetes, age at diagnosis and anthropometric indices (Piwemetz, et al (1993) supra; Chan, et al (1997) supra).\",\n \"Body mass index (BMI) was used as an index of general obesity.\",\n \"Waist circumference, which is highly correlated in Chinese with visceral fat accumulation measured by magnetic resonance imaging (Anderson, et al (1997) Diabetes Care 20: 1854), was used as an index of central obesity.\",\n \"After an overnight fast, venous blood was sampled for measurement of plasma glucose, insulin, HbA1c, total cholesterol (TC), HDL-C, LDL-C (calculated), triglyceride (TG) and anti-GAD.\",\n \"A morning spot urine sample was collected for assessment of albuminuria.\",\n \"Retinopathy and sensory neuropathy were assessed as previously described (Ko, et al (1999) J Diabetes Complications 13: 300).\",\n \"General obesity was defined as a BMI≧25 kg/m2 using the recent Asian criteria (WHO, Western Pacific Region, 2000).\",\n \"Albuminuria was defined as an albumin: creatinine ratio (ACR)≧3.5 mg/mmol in a spot urine sample (Schwab, et al (1992) Diabetes Care 15: 1581).\",\n \"The HOMA IR index (fasting plasma insulin×glucose/22.5) derived from the HOMA equation was used to assess insulin resistance (Matthews, et al (1985) Diabetologia 28: 412).\",\n \"Biochemical Assays Plasma glucose, HbA1c, lipids, urinary albumin and creatinine were measured by routine assays in the Department of Chemical Pathology at the PWH (see Chan, et al (1996) Diabetic Med 13:150).\",\n \"Plasma insulin was measured in non-insulin treated patients by a radioimmunoassay (Pharmacia, Sweden) with intra-and inter-assay CVs of 6% and 13.8%, respectively.\",\n \"Anti-GAD was measured by a radioimmunoprecipitation assay (Chen, et al (1993) Pediatr Res 34: 785).\",\n \"The upper normal limit of 18 units, is applicable to Asian and European subjects (Tuomi, et al (1995) Clin Immunol Immunopath 74: 202, Chen, et al (1993) supra).\",\n \"Genetic Analysis The minimal promoter regions and exons of the glucokinase (β-cell form), HNF-1α and HNF-4α (HNF-4α 2 form) genes were screened for mutations by direct sequencing of PCR products (see Froguel, et al (1993) N Engl J Med 328: 697; Yamagata, et al (1996) Nature 384: 455; Yamagata, et al (1996) Nature 384: 458).\",\n \"One previously unreported mutation in HNF-1α (A116V) and two previously unreported mutations in glucokinase (V101M and Q239R) were identified in this study.\",\n \"HNF-1α A116V was screened by using the forward primer 5′-CATGCACAGTCCCCACCCTCA-3′ (SEQ ID NO:34) and reverse primer 5′-TCCCACTG ACTTCCTTTCC-3′ (SEQ ID NO:35) for PCR amplification followed by digestion with HphI.\",\n \"The wild-type allele showed 44 and 397 bp products whereas the mutant allele showed 44, 136 and 261 bp products.\",\n \"Glucokinase V101M was screened by using the forward primer 5′-GTCCCTGA-GGCTGACACACTT-3′ (SEQ ID NO: 24) and reverse primer 5′-AGCTGGGCCCTGAGATCC-TGCA-3′ (SEQ ID NO: 25) for PCR amplification followed by digestion with Hsp9211.The wild-type allele showed 20, 56 and 174 bp products whereas the mutant allele showed 20,42,56, and 132 bp products.\",\n \"Glucokinase Q239R was screened by using the forward primer 5′-AGGAACC-AGGCCCTACTCCG-3′ (SEQ ID NO: 36) and reverse primer 5′-TACTCCAGCAGGAACTC-GTCC-3′ (SEQ ID NO: 37) for PCR amplification followed by digestion with Acil.\",\n \"The wild-type allele showed 70 and 134 bp products whereas the mutant allele showed 33,70 and 101 bp products.\",\n \"The occurrence of putative mutations in family members of the probands (Table 10) and control subjects was determined by PCR-RFLP.\",\n \"Mt3243A→G and amylin gene S20G mutations were determined by PCR-RFLP as described (Sakagashira, et al (1996) Diabetes 45: 1279; Smith, et al (1997) supra).\",\n \"Statistical Analysis Normally distributed data are expressed as mean ± SD.\",\n \"Data with skewed distributions were normalised by logarithmic transformation.\",\n \"The resultant means were antilogarithmically transformed and expressed as geometric mean together with 25 and 75 percentiles.\",\n \"Chi square test and Student's unpaired t tests were used for between-group comparisons.\",\n \"A p value<0.05 (2-tailed) was considered to be significant.\",\n \"All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS for Windows, version 9.0).\",\n \"Results Prevalence of Putative Gene Mutations and Anti-Gad in Patients with Familial Early-Onset Diabetes Amongst the 145 patients with familial early-onset diabetes, there were 20 (14%) with putative mutations, involving the HNF-1α gene, 7 (5%), glucokinase gene, 6 (4%), mt3243,4 (3%), and amylin S20G, 3 (2%).\",\n \"Anti-GAD was positive in 6 (4%).\",\n \"No mutation in the HNF-4α gene was found in the 92 patients from the PWH Diabetes Registry.\",\n \"All mutations identified in the HNF-1α and glucokinase genes were previously unreported (Table 10).\",\n \"The HNF-1α G20R and glucokinase Q239R mutations were found in 4 unrelated patients.\",\n \"None of these mutations were found in 100 healthy control subjects.\",\n \"Family Cosegregation Study of Gene Mutations Amongst the 20 patients carrying putative gene mutations, 10 families were recruited for cosegregation study (FIG.\",\n \"9).\",\n \"Cosegregation of a mutation with clinical diabetes or glucose intolerance were observed in 4 families: HK10 with HNF-1α IVS2nt-1G→A, YDM142 with glucokinase V101M, HK84 with glucokinase I110T and HK50 with mt3243.Segregation was inconclusive in the other 6 families.\",\n \"For the families HK54 with HNF-1α R203H, YDM83 with mt3243 and CX216 with amylin S20G mutations, only the probands, and none of the diabetic or non-diabetic family members who presented for screening, carried the gene mutations.\",\n \"For YDM67 with glucokinase Q239R, HK61 with mt3243 and YDM99 with amylin S20G mutations, the mutations were found in both diabetic and non-diabetic family members.\",\n \"Amongst the 3 families with glucokinase mutations, all mutation carriers from the families YDM142 and HK84 had higher fasting plasma glucose concentrations (5.8-8.9 mmol/1) than those with no mutation (4.2-5.3 mmol/1).\",\n \"On the other hand, the 4 mutation-carrying siblings of proband YDM67 had a normal fasting plasma glucose concentration (4.0-5.6 mmol/1) irrespective of their glycemic status.\",\n \"TABLE 10 Mutations in the HNF-1 α and glucokinase genes in Chinese subjects with early-onset diabetes mellitus Subject Location Codon/nt Nucleotide change Designation HNF-1 αmutation HK90*, YDM42† Exon 1 20 GGG (GLy)→AGG (Arg) G20R YDM20† Exon 2 116 GCG (Ala)→GTG(Val) A116V HK10* Intron2/Exon3 nt-1 AG→AA at splice acceptor site IVS2nt-1G→A HK54* Exon 3 203 CGT (Arg)→CAT (His) R203H HK30* Exon 6 432 TCC (Ser) →TGC (Cys) S432C HK92* Exon 10 618 ATC (Ile)→ATG (Met) 1618M Glucokinase mutation YDM142† Exon 3 101 GTG (Val)→ATG (Met) V101M HK84* Exon 3 110 ATC (Ile)→ACC (Thr) I110T HK38* Exon 3 119 GCT (Ala)→GAT (Asp) A119D YDM67†, YDM144† Exon 7 239 CAG (Gln)→CGG (Arg) Q239R HK15* Exon 9 385 GGG (Gly)→GTG(Val) G385V *reported in previous studies (Ng, et al (1999) Diabetic Med 16: 956; Ng, et al (2000) Diabetologia 43: 816) †newly found in the present study Clinical Characteristics of Patients with Familial Early-Onset Diabetes of Unknown Cause Compared with Familial Late-Onset Diabetes Although 26 of the patients with early-onset diabetes carried putative gene mutations associated with diabetes or the autoimmune indicator, anti-GAD antibodies, the causes of diabetes in the other 119 patientsremain to be determined.\",\n \"These young patients with diabetes of unknown cause (age at diagnosis 30±6 years) differed clinically from the 290 late-onset patients (age at diagnosis 52±8 years) (Table 11).\",\n \"Thus, despite a positive family history of diabetes in all patients in both groups, those with early-onset diabetes more frequently had a father with diabetes (39% vs. 22%) and a mother with diabetes (63% vs. 41%), but less frequently a sibling with diabetes (30% vs. 53%) (p<0.001).\",\n \"The early-onset patients had a higher BMI but lower BP and increased prevalence of retinopathy and neuropathy as compared to the late-onset patients.\",\n \"The early-onset patients had better glycemic control (glucose and HbA1c) as well as higher fasting insulin concentrations than the late-onset patients.\",\n \"Notwithstanding similar mean disease duration of only 4 years, both the early-and late-onset patients had a disproportionately high prevalence of albuminuria, 40% and 38%, respectively, as compared with the prevalence rates of other microangiopathic complications.\",\n \"Insulin resistance, as assessed by the HOMA IR index, was similar between the two groups of non-insulin treated patients.\",\n \"The proportion of patients treated with insulin was similar in both groups (8% vs. 7%) but fewer patients with early-onset diabetes were treated with oral drugs (33% vs. 61%, p<0.001) as compared to the late-onset group.\",\n \"Clinical Characteristics of the Patients with Familial Early-Onset Diabetes of Unknown cause and Familial Late-Onset Diabetes Classified According to Obesity Index Due to the high prevalence of general obesity in both early-onset patients of unknown cause and late-onset patients (55% and 46%, respectively), the association of obesity with cardiovascular risk factors and complications in these patients was further analyzed (Table 11).\",\n \"Amongst the early-onset patients, the obese patients had worse glycemic control (HbA1c) as well as a higher systolic BP, a more adverse lipid profile (higher TG, lower HDL-C and higher TC/HDL-C), and higher fasting insulin than the non-obese patients.\",\n \"They were also more insulin resistant (HOMA IR index) and had a higher prevalence of retinopathy and albuminuria than the non-obese patients.\",\n \"Amongst the late-onset patients, the obese patients had better glycemic control (glucose and HbA1c) than the non-obese patients.\",\n \"However, they had a higher systolic and diastolic BP, and a higher fasting insulin than the non-obese patients.\",\n \"The degree of insulin resistance and prevalence of complications were similar in the two groups.\",\n \"TABLE 11 Comparison of clinical features of Chinese patients with familial Type 2 diabetes according to age of diagnosis of diabetes and obesity status Early-onset Late-onset Early-onset patients Late-onset non-obese Early-onset non-obese Late-onset with unknown etiology patients patients obese patients patients obese patients N 119 290 54 65 156 134 Sex Male (%) 37 (31) 98 (34) 12 (22) 25 (38) 56 (36) 42 (31) Female (%) 82 (69) 192 (66) 42 (78) 40 (62) 100 (64) 92 (69) Current age (yr) 34 ± 5 56 ± 9‡ 34 ± 5 33 ± 5 56 ± 10 55 ± 9 Age at diagnosis (yr) 30 ± 6 52 ± 8‡ 31 ± 5 29 ± 6 52 ± 8 52 ± 8 Duration of disease (yr) 4.0 ± 3.9 4.0 ± 4.2 3.9 ± 3.8 4.2 ± 4.0 4.5 ± 4.4 3.5 ± 3.9† Family history Father 46 (39) 64 (22)‡ 21 (39) 27 (42) 28 (18) 36 (27) Mother 75 (63) 119 (41)‡ 38 (70) 39 (60) 65 (42) 54 (40) Sibling 36 (30) 154 (53)‡ 15 (28) 20 (31) 85 (54) 69 (51) BMI (kg/m2) 26.2 ± 4.7 25.0 ± 3.7† 22.3 ˜ 1.8 29.5 ± 3.8‡ 22.4 ± 1.8 28.0 ± 3.1‡ Waist circumference (cm) Male 90 ± 11 87 ± 9 78 ± 6 95 ± 9‡ 81 ± 6 94 ± 7‡ Female 81 ± 11 83 ± 9 74 ± 5 89 ± 10‡ 78 ± 6 89 ± 8‡ Systolic BP (mmHg) 117 ± 14 136 ± 22‡ 114 ± 13 120 ± 14† 134 ± 23 139 ˜ 21† Diastolic BP (mmHg) 75 ± 9 83 ± 11‡ 74 ± 9 77 ± 10 80 ± 11 86 ± 12‡ Triglyceride (mmol/l) 1.4 (0.9-2.0) 1.4 (1.0-2.0) 1.0 (0.7-1.5) 1.7 (1.0-2.4)‡ 1.4 (0.9-1.9) 1.6 (1.1-2.1) Total cholesterol (mmol/l) 5.3 ± 1.2 5.6 ± 1.3 5.1 ± 1.0 5.4 ± 1.4 5.6 ± 1.3 5.5 ± 1.2 HDL-C (mmol/l) 1.2 ± 0.3 1.2 ± 0.3 1.3 ± 0.3 1.1 ± 0.3‡ 1.3 ± 0.4 1.2 ± 0.3 TC/HDL-C 4.7 ± 1.8 4.7 ± 1.5 4.0 ± 1.0 5.3 ± 2.2‡ 4.7 ± 1.7 4.7 ± 1.3 LDL-C (mmol/l) 3.3 ± 0.9 3.5 ± 1.0 3.2 ± 0.8 3.4 ± 1.0 3.5 ± 1.0 3.5 ± 1.0 Fasting glucose (mmol/l) 8.2 ± 3.1 9.1 ± 3.6† 7.6 ± 2.8 8.7 ± 3.3 9.6 ± 3.8 8.6 ± 3.3† HbA1c (%) 7.5 ± 1.8 8.0 ± 1.9† 7.1 ± 1.8 7.9 ± 1.8† 8.2 ± 2.1 7.7 ± 1.5† Fasting insulin (pmol/l)* 105 (72-164) 87 (51-146)† 89 (60-149) 120 (78-179)† 76 (43-129) 99 (57-157)† HOMA IR index* 34.7 (22.9-55.8) 33.1 (19.2-62.8) 27.5 (16.0-46.7) 42.7 (27.6-58.2)† 29.9 (14.7-61.9) 36.4 (19.7-64.2) Urinary albumin creatinine 2.6 (0.7-6.1) 2.8 (0.8-7.1) 1.2 (0.6-1.9) 5.1 (0.9-24.1)‡ 2.6 (0.9-5.8) 3.2 (0.8-8.4) ratio (mg/mmol) Treatment (%) Diet 71 (60) 93 (32) 35 (65) 36 (55) 55 (35) 38 (28) Oral drugs 39 (33) 176 (61)‡ 17 (31) 22 (34) 89 (57) 87 (65) Insulin 9 (8) 21 (7) 2 (4) 7 (11) 12 (8) 9 (7) Retinopathy (%) 10 (8) 62 (21)† 1 (2) 9 (14)† 38 (24) 24 (18) Albuminuria (%) 48 (40) 110 (38) 8 (15) 40 (62)‡ 53 (34) 57 (43) Neuropathy (%) 4 (3) 29 (10)† 2 (4) 2 (3) 13 (8) 16 (12) Data are compared between early- and late-onset patients, between early-onset non-obese and obese patients, and between late-onset non-obese and obese patients Data are expressed as n (%), mean ± SD or geometric mean (25 and 75 percentiles) *only measured in patients not treated with insulin †p < 0.05 ‡p < 0.001 EXAMPLE 5 An Illustration of a Chinese Family with Hepatocyte Nuclear Factor-1α Diabetes (MODY3) that Emphasizes the Need for Early Diagnosis and Appropriate Treatment This example reports the clinical course of HNF-1α diabetes/MODY 3 in a Chinese family with early-onset diabetes and severe complications (FIG.\",\n \"10) (Chan, et al (1990) Diabetic Medicine 7: 211).\",\n \"This family highlights the importance of early diagnosis and prompt treatment in the improvement of clinical outcome even in genetically susceptible subjects.\",\n \"Three family members in the proband's family had severe diabetic complications when they were referred for treatment.\",\n \"The proband (III-5), 19 years of age, had severe proliferative retinopathy, heavy proteinuria (1.4 g protein a day) and necrobiosis lipoidica.\",\n \"She had been diagnosed with Type 2 (non-insulin-dependent) diabetes mellitus 3 months earlier and was treated with glibenclamide.\",\n \"Retinal photocoagulation treatment was initiated and she was started on insulin and an ACE inhibitor.\",\n \"She subsequently developed hypertension and progressed to end-stage renal disease requiring dialysis by the age of 30 years.\",\n \"Her mean HbA1c was 8.0% over the years.\",\n \"She is currently receiving 42 units of insulin.\",\n \"Her older sister (III-2) had a vitreous haemorrhage and had been treated with insulin since diagnosis at the age of 24 years.\",\n \"She became blind and had nephropathy (0.8 g protein a day) 2 years later.\",\n \"She is currently treated with insulin (16 units) and an ACE inhibitor, and has a mean HbA , of 6.4%.\",\n \"The subject's mother (II-3) had a glycosuria complicated pregnancy when she was 33 years old.\",\n \"She was diagnosed to have Type 2 diabetes at the age of 38 years and was then treated with glibenclamide for 10 years.\",\n \"At the time of the study she had proliferative retinopathy, nephropathy, peripheral neuropathy, necrobiosis lipoidica, hypertension and cataracts.\",\n \"Insulin treatment (20 units) was commenced and her HbA1c was reduced from 17.2% to 9.2% within 8 months.\",\n \"Two months later, she had a myocardial infarction followed by progressive deterioration of cardiac and renal functions.\",\n \"She died of pulmonary edema and septicaemia with a gangrenous foot at the age of 52 years.\",\n \"The fourth daughter (III-6) had been treated with insulin since her incidental diagnosis of diabetes at the age of 12 years after a nasal polypectomy.\",\n \"She is currently receiving 68 units of insulin, and has mean HbA1c of 8.8%.\",\n \"Two other family members underwent screening by OGTT.\",\n \"The second daughter (III-3) has fluctuated between having normal glucose tolerance and IGT over the last 11 years.\",\n \"A brother (III-7) had overt diabetes on screening with an initial HbA1c of 10.5%.\",\n \"Insulin was started after 3 months of dietary treatment.\",\n \"He is currently receiving 26 units of insulin, with a mean HbA1c of 5.3%.\",\n \"One maternal uncle (II-4) was diagnosed with diabetes and hyperlipidnemia with thirst and polyuria at the age of 39 years.\",\n \"He has been treated with oral drugs since diagnosis, and has mean HbA1c of 8.4%.\",\n \"His children were not available for detailed genetic testing and clinical assessment.\",\n \"The affected members II-4, III-6 and III-7 (FIG.\",\n \"10) have remained free of complications despite all having had diabetes for more than 10 years.\",\n \"The father was also diagnosed with IGT.\",\n \"He was non-obese and had hyperlipidaemia.\",\n \"Sequencing of the HNF-1α gene in this family showed a novel splice acceptor site mutation (AG→AA) in intron 2 (IVS2nt-1G→A) which cosegregated with diabetes (FIG.\",\n \"10) (Ng, et al (1999) Diabetic Medicine 16: 956).\",\n \"This mutation is expected to produce a nonfunctional mRNA.\",\n \"All the diabetic members, including the maternal uncle, (II4, III-2, III-5, III-6 and III-7) were heterozygous for this mutation but the father (II-2) and the daughter (III-3) with IGT did not have the mutation.\",\n \"Thus it is very likely that the mother (II-3) for whom no DNA sample was available also carried this mutation.\",\n \"As with other patients with HNF-1α diabetes (Byrne, et al (1996) Diabetes 45: 1503), most affected family members exhibited defective pancreatic beta-cell function as assessed by the glucagon stimulation test.\",\n \"The mother and all the affected siblings, except subject III-2, were insulin deficient based on a definition of post-glucagon (1 mg intravenously) stimulated plasma C peptide at 6 min of less than 0.6 nmol/1 (0.24-0.55 nmol/I respectively) (Service, et al (1997) Diabetes Care 20: 198).\",\n \"The brother, III-7, who was diagnosed with diabetes by OGTT was also insulin deficient.\",\n \"All the HNF-1α mutant carriers, except II-4, required insulin treatment for glycemic control.\",\n \"Although all affected family members carried the same HNF-1α gene mutation, their clinical courses have varied tremendously.\",\n \"Severe complications were present in those family members whose diagnosis was delayed and who presumably had poor glycemic control before diagnosis (II-3, III-2 and III-5).\",\n \"Complications were, however, absent in the uncle (II-4) and the younger siblings (III-6 and III-7) despite now having had diabetes for more than 10 years (FIG.\",\n \"10), who were promptly diagnosed and received treatment.\",\n \"This is in accordance with a recent report suggesting that poor glycemic control is associated with a twofold to threefold increased risk among MODY3 patients of developing microalbuminuria and retinopathy, respectively (Isomaa, et al (1998) Diabetologia 41: 467).\",\n \"It is noteworthy that both maternal grandparents (I-3 and I-4) were diagnosed with diabetes diagnosed in their late 50 s. The effect of this bilineality on the natural course of HNF-1α diabetes in this family is uncertain.\",\n \"It is, however, possible the non-MODY maternal grandparent transmitted a modifier gene affecting the age at onset or severity of the diabetes in carriers with the HNF-1α amutation.\",\n \"The age at diagnosis of diabetes in this family was increasingly younger with successive generations despite all carriers being relatively non-obese.\",\n \"This earlier diagnosis could be due to ascertainment bias or, more likely, an epiphenomenon due to increasing westernisation of the Hong Kong lifestyle with increased intake of high fat food and decreased physical activity (Chan and Cockram (1997) Diabetes Care 20:1785).\",\n \"This highlights the important influence of environment interacting with genetics in the natural course of HNF-1α diabetes.\",\n \"In conclusion, this report emphasizes the need for early diagnosis by glucose tolerance testing or genetic screening, and appropriate treatment in patients who have a strong family history of diabetes, especially those with early onset disease and insulin deficiency.\",\n \"The data presented in Examples 1-5 demonstrate a combination of genetic mutations that are uniquely associated with the increased risk of a Chinese individual to develop type 2 diabetes.\",\n \"The mutations are exemplified by, but are not limited to G20R, A116V, IVS2nt→GA, R203H, S432C and I618M of HNF-1α; V101M, I110T, A119D, Q239R and G385V of glucokinase; S20G of amylin; and A3243G of mitochondrial tRNALeu(UUR).\",\n \"Mutations correlative with a genetic predisposition of a Chinese individual to develop type 2 diabetes are efficiently identified in Chinese families with a positive family history of the disease, but find use in screening any Chinese individual that is asymptomatic but at risk of developing diabetes.\",\n \"Methods for identification of a combination of at least two genetic mutations correlative with type 2 diabetes in a Chinese individual offers an important tool for clinicians, not only to initiate prophylactic therapies before the onset of overt diabetic symptoms, but also to design therapies that are directed to the specific etiology of the disease in each individual.\",\n \"All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains.\",\n \"All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporate by reference.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471446"}}},{"rowIdx":594,"cells":{"text":{"kind":"list like","value":[["Propellant powder charge for barrel weapon","The present invention relates to a method for producing propellant powder charges intended for heavy barrel weapons, such as canons and howitzers, and with a high degree of filling and a high energy content, and also to propellant powder charges produced in accordance with this method.","The charges according to the invention are distinguished by the fact that 70 to 95% of their weight consists of a more coarsely grained powder and 30 to 5% consists of a granular powder with a smaller grain size.","These two powders have the same of different chemical composition, and one or more of them can be surface-inhibited."],["1-7.(Cancelled).","8.A method for producing progressive propellant powder charges suitable for use in barrel weapons having a high degree of filling or loading density and thus also a high energy content at which the charge is produced, the method comprising: mixing at least two types of granular powder of different grain size, of which at least one of the at least two powder types with a largest grain size and a larger number of internal priming channels has a progressive burn characteristic, wherein, as subsidiary components of an actual charge, granular powders are chosen having size, geometric shape, and quantities adapted to give a smallest possible empty spaces between the powder grains.","9.The method of claim 1, wherein a starting material chosen for the charge comprises at least two types of powder whose mutual progressivity has been optimized for a particular purpose.","10.The method of claim 1, wherein a starting material chosen for the charge comprises granular powder having a same or different chemical composition, but with different grain size and a different number of internal priming channels.","11.The method of claim 1, wherein, when choosing a starting material, powder types are chosen of which one or more are surface-inhibited.","12.The method of claim 1, wherein, of the powder types included in a finished charge, all the types are introduced contemporaneously with each other and, at the same time a casing or cartridge to which the powder is added and in which the charge is to be stored prior to use is subjected to vibrations which improve a degree of packing of the charge.","13.Propellant powder charge suitable for use in barrel weapons and having a high degree of filling or loading density and with a high energy content, the power charge comprising: at least two different types of granular powder of which at least one type has a progressive burn characteristic and in which one or more of these powders is surface-inhibited, wherein burn characteristics of each of these different powders are adapted to one another while their mutual grain sizes and quantities of each powder included in a charge are adapted to one another such that an empty space remaining between the powder grains is minimized.","14.The propellant powder charge of claim 13, wherein between 70-95% of a powder charge weight comprises relatively coarsely grained powder, and wherein 30-5% of the weight comprises a relatively more finely grained powder, wherein these two powders have a same or different chemical composition and at least one of the two powders is progressive and one or more of the two powders is surface-inhibited.","15.The propellant powder charge of claim 14, wherein between 75 to 85% of of a powder charge weight comprises relatively coarsely grained powder.","16.The propellant powder charge of claim 14, wherein the coarsely grained powder is either 19-hole or 37-hole powder cut into short sections.","17.The propellant powder charge of claim 14, wherein the finely grained powder is either 1-hole or 7-hole powder."],["The present invention relates to a propellant powder charge with high progressivity and an extremely high degree of filling or loading density, intended primarily for large-bore barrel weapons.","In what today is the most common way of producing progressive propellant powder charges intended for large-bore barrel weapons, use is made primarily of what is known as granular holed powder, i.e.","powder produced by extrusion in a matrix, cut up into short rods or cylinders, with one, seven, nineteen or thirty seven longitudinal priming channels.","Because of its geometric configurations throughout its burn time, except during the absolutely final stage, multiple-hole powder of this kind has good progressive burn properties by virtue of the fact that as they are primed they burn from all the surfaces available for priming, i.e.","from the outsides of the grains and from the insides of the priming channels, and, from these surfaces thereby primed, the powder will burn towards other primed surfaces during a successive increase of the burn area, and the gas release thereby also increases.","In order to launch a defined missile from a defined barrel weapon with a predetermined Vo, i.e.","with a defined launch velocity immediately outside the barrel mouth, the propellant powder has to be able to deliver a certain additional amount of energy.","This additional energy, which can be calculated theoretically with great accuracy, must be delivered during the missile's travel through the barrel.","This in turn means that the propellant powder must have time to burn out during the time the missile is on its way through the barrel.","The period of time which the propellant powder should then have to burn out and which is thus the same as the time the missile has to pass through the barrel can be called the “burn time”.","If at the same time the length which a powder with the chemical composition in question is able to burn is also called the “burn time”, this means that the distance between two adjacent priming channels in a multiple-hole powder which has the desired burn time will correspond to twice the burn length which the burn time in question permits.","The distance from respective priming channels to the outer side of the granular powder in which the priming channel is formed must be the same size, provided that the powder grains have not been surface-treated with a burn inhibitor, which is sometimes done in order to increase the powder's progressivity.","In summary, it is readily possible to produce multiple-hole powders with different progressivity using different numbers of priming channels, the individual powder grains being given a greater inherent volume depending on the number of priming channels.","The progressivity of the powder can then in turn be accentuated by surface treatment with a suitable substance which is more difficult to ignite, but burnable.","The main trends in modern-day arterillery technology are to attempt by all possible means to increase the artillery's range of fire and its firing speed.","In view of the refined methods which have been developed in recent years, and which allow an opponent to determine quickly and with very great accuracy from where he has been shot at, it is thus also necessary to rapidly change the site of firing each time the barrel has been opened.","Increased firing speed is mainly achieved by the introduction of mechanized loading systems, and these will not be discussed in any detail here, nor will the tactical need for rapidly changing the firing positions.","By contrast, it has been found that older artillery pieces too which are in good condition can be given much longer ranges of fire by means of newly developed and high-energy powder charges and possibly also newly developed shells.","However, a problem in this context, which is often more serious than the strength limits of barrel and mechanism, is that the space in the rear part of the barrel, i.e.","its chamber position, which is available for the actual propellant charge is too small to accommodate a conventionally configured propellant powder charge with sufficient energy content for this desired increase in the range of fire.","The present invention now relates to a method using holed or unholed granular powder to produce progressive propellant powder charges with a higher degree of filling or loading density and thus also loading weights than has previously been possible.","The invention also includes propellant powder charges produced in accordance with said method.","The starting point for the method according to the invention is that, between the grains of a granular powder introduced into a container without any kind of organized order, there will automatically be a very large number of greater or lesser empty volumes which are many but small in the case of a finely grained powder and which are fewer but also larger in a coarser-grained powder.","Our solution to the problem now is so simple that it is astonishing that it has not been done earlier.","According to the underlying principle of the present invention, we in fact mix two or more different sorts of granular powder in proportions adapted for the particular purpose, where the more finely grained powder is able to fill the otherwise empty space between the larger powder grains.","By suitable choice of powder types and suitable proportions between them, it is thus possible to produce charges which have loading densities lying very near to or higher than those one would have obtained if the powder grains had been rammed in manually for the best degree of packing, and the latter method is entirely excluded for practical use.","The only additional measure which may be required in connection with the present invention is that the charges are vibrated during powder filling, which is also preferably done simultaneously with the two powder types.","One or more of the powder types used can additionally be surface-treated or surface-inhibited in order to further control the progressivity.","The present invention thus makes it possible to produce propellant powder charges with a high loading density and exactly the high energy content which is required for the case in question.","A propellant powder charge produced according to the invention can thus contain 70-95% by weight of a coarsely grained multiple-hole powder, for example a 19-hole or 37-hole powder, and 30-5% by weight of a smaller multiple-hole powder, for example a 1-hole powder or 7-holed powder, and, in view of the desired final result, the powders can each have the same or different chemcial composition and be surface-inhibited (surface-treated with suitable burn inhibitors) or not surface-inhibited.","The percentages shown above apply to the outer limits characterizing the invention, whereas, when in practice calculating the charges of the type characterizing the invention, they will in most cases be in the range of 75-85% by weight for the coarser multiple-hole powder and 25-15% by weight for the multiple-hole and more finely grained powder or powders.","The nearest prior art we are aware of is the charge which is described in U.S. Pat.","No.","4,519,855 which describes a propellant powder charge for ballistic ammunition comprising a first powder component consisting of large balls or spheres easily fragmentable into small grains of a first powder component surrounded by a second powder component in the form of a conventionally granular powder which fills the space between the large spheres of powder.","Here too the aim has been to produce a powder charge with the highest possible degree of filling taking into consideration the powder types used, but the progressivity of the charges obtained by this method will be based primarily on the large spheres of powder bursting apart during combustion of the powder and thereafter burning like conventionally granular powder, whereas the progressivity for our specific charge is based entirely on the original geometrical shape of the powder grains used.","The present invention thus relates to a method in which granular powder is used to produce propellant powder charges with high degree of filling or loading density and a high energy content per charge.","The invention also covers the charge produced by this method.","The invention also makes it possible to produce charges with precisely controlled progressive characteristics by virtue of the fact that it can be built up from different quantities of different powders which have radically different progressive characteristics.","The invention is thus based entirely on combining two or more types of granular powder having such geometric external shapes and grain sizes that the powder with the smaller grain size will in the best possible way fill the empty space between the larger grains.","The invention has in all its features been defined in the attached patent claims and it can be illustrated by the following example.","EXAMPLE From a nitrocellulose powder of standard quality which we have used for many years for producing artillery powder, we produced on the one hand a 19-hole powder with grain size of 17×17 mm and a 1-hole powder with a grain size of 5×5 mm.","Of these powders, we mixed 2.3 kg of the 19-hole powder with 0.5 kg of the 1-hole powder and thus obtained a charge with an energy content corresponding to 122% of the energy content in a standard charge which we had previously produced and which was made only from 19-hole powder.","Both the charge types are held in the same volume.","The invention thus makes it possible to achieve considerable advantages by very small means."]],"string":"[\n [\n \"Propellant powder charge for barrel weapon\",\n \"The present invention relates to a method for producing propellant powder charges intended for heavy barrel weapons, such as canons and howitzers, and with a high degree of filling and a high energy content, and also to propellant powder charges produced in accordance with this method.\",\n \"The charges according to the invention are distinguished by the fact that 70 to 95% of their weight consists of a more coarsely grained powder and 30 to 5% consists of a granular powder with a smaller grain size.\",\n \"These two powders have the same of different chemical composition, and one or more of them can be surface-inhibited.\"\n ],\n [\n \"1-7.(Cancelled).\",\n \"8.A method for producing progressive propellant powder charges suitable for use in barrel weapons having a high degree of filling or loading density and thus also a high energy content at which the charge is produced, the method comprising: mixing at least two types of granular powder of different grain size, of which at least one of the at least two powder types with a largest grain size and a larger number of internal priming channels has a progressive burn characteristic, wherein, as subsidiary components of an actual charge, granular powders are chosen having size, geometric shape, and quantities adapted to give a smallest possible empty spaces between the powder grains.\",\n \"9.The method of claim 1, wherein a starting material chosen for the charge comprises at least two types of powder whose mutual progressivity has been optimized for a particular purpose.\",\n \"10.The method of claim 1, wherein a starting material chosen for the charge comprises granular powder having a same or different chemical composition, but with different grain size and a different number of internal priming channels.\",\n \"11.The method of claim 1, wherein, when choosing a starting material, powder types are chosen of which one or more are surface-inhibited.\",\n \"12.The method of claim 1, wherein, of the powder types included in a finished charge, all the types are introduced contemporaneously with each other and, at the same time a casing or cartridge to which the powder is added and in which the charge is to be stored prior to use is subjected to vibrations which improve a degree of packing of the charge.\",\n \"13.Propellant powder charge suitable for use in barrel weapons and having a high degree of filling or loading density and with a high energy content, the power charge comprising: at least two different types of granular powder of which at least one type has a progressive burn characteristic and in which one or more of these powders is surface-inhibited, wherein burn characteristics of each of these different powders are adapted to one another while their mutual grain sizes and quantities of each powder included in a charge are adapted to one another such that an empty space remaining between the powder grains is minimized.\",\n \"14.The propellant powder charge of claim 13, wherein between 70-95% of a powder charge weight comprises relatively coarsely grained powder, and wherein 30-5% of the weight comprises a relatively more finely grained powder, wherein these two powders have a same or different chemical composition and at least one of the two powders is progressive and one or more of the two powders is surface-inhibited.\",\n \"15.The propellant powder charge of claim 14, wherein between 75 to 85% of of a powder charge weight comprises relatively coarsely grained powder.\",\n \"16.The propellant powder charge of claim 14, wherein the coarsely grained powder is either 19-hole or 37-hole powder cut into short sections.\",\n \"17.The propellant powder charge of claim 14, wherein the finely grained powder is either 1-hole or 7-hole powder.\"\n ],\n [\n \"The present invention relates to a propellant powder charge with high progressivity and an extremely high degree of filling or loading density, intended primarily for large-bore barrel weapons.\",\n \"In what today is the most common way of producing progressive propellant powder charges intended for large-bore barrel weapons, use is made primarily of what is known as granular holed powder, i.e.\",\n \"powder produced by extrusion in a matrix, cut up into short rods or cylinders, with one, seven, nineteen or thirty seven longitudinal priming channels.\",\n \"Because of its geometric configurations throughout its burn time, except during the absolutely final stage, multiple-hole powder of this kind has good progressive burn properties by virtue of the fact that as they are primed they burn from all the surfaces available for priming, i.e.\",\n \"from the outsides of the grains and from the insides of the priming channels, and, from these surfaces thereby primed, the powder will burn towards other primed surfaces during a successive increase of the burn area, and the gas release thereby also increases.\",\n \"In order to launch a defined missile from a defined barrel weapon with a predetermined Vo, i.e.\",\n \"with a defined launch velocity immediately outside the barrel mouth, the propellant powder has to be able to deliver a certain additional amount of energy.\",\n \"This additional energy, which can be calculated theoretically with great accuracy, must be delivered during the missile's travel through the barrel.\",\n \"This in turn means that the propellant powder must have time to burn out during the time the missile is on its way through the barrel.\",\n \"The period of time which the propellant powder should then have to burn out and which is thus the same as the time the missile has to pass through the barrel can be called the “burn time”.\",\n \"If at the same time the length which a powder with the chemical composition in question is able to burn is also called the “burn time”, this means that the distance between two adjacent priming channels in a multiple-hole powder which has the desired burn time will correspond to twice the burn length which the burn time in question permits.\",\n \"The distance from respective priming channels to the outer side of the granular powder in which the priming channel is formed must be the same size, provided that the powder grains have not been surface-treated with a burn inhibitor, which is sometimes done in order to increase the powder's progressivity.\",\n \"In summary, it is readily possible to produce multiple-hole powders with different progressivity using different numbers of priming channels, the individual powder grains being given a greater inherent volume depending on the number of priming channels.\",\n \"The progressivity of the powder can then in turn be accentuated by surface treatment with a suitable substance which is more difficult to ignite, but burnable.\",\n \"The main trends in modern-day arterillery technology are to attempt by all possible means to increase the artillery's range of fire and its firing speed.\",\n \"In view of the refined methods which have been developed in recent years, and which allow an opponent to determine quickly and with very great accuracy from where he has been shot at, it is thus also necessary to rapidly change the site of firing each time the barrel has been opened.\",\n \"Increased firing speed is mainly achieved by the introduction of mechanized loading systems, and these will not be discussed in any detail here, nor will the tactical need for rapidly changing the firing positions.\",\n \"By contrast, it has been found that older artillery pieces too which are in good condition can be given much longer ranges of fire by means of newly developed and high-energy powder charges and possibly also newly developed shells.\",\n \"However, a problem in this context, which is often more serious than the strength limits of barrel and mechanism, is that the space in the rear part of the barrel, i.e.\",\n \"its chamber position, which is available for the actual propellant charge is too small to accommodate a conventionally configured propellant powder charge with sufficient energy content for this desired increase in the range of fire.\",\n \"The present invention now relates to a method using holed or unholed granular powder to produce progressive propellant powder charges with a higher degree of filling or loading density and thus also loading weights than has previously been possible.\",\n \"The invention also includes propellant powder charges produced in accordance with said method.\",\n \"The starting point for the method according to the invention is that, between the grains of a granular powder introduced into a container without any kind of organized order, there will automatically be a very large number of greater or lesser empty volumes which are many but small in the case of a finely grained powder and which are fewer but also larger in a coarser-grained powder.\",\n \"Our solution to the problem now is so simple that it is astonishing that it has not been done earlier.\",\n \"According to the underlying principle of the present invention, we in fact mix two or more different sorts of granular powder in proportions adapted for the particular purpose, where the more finely grained powder is able to fill the otherwise empty space between the larger powder grains.\",\n \"By suitable choice of powder types and suitable proportions between them, it is thus possible to produce charges which have loading densities lying very near to or higher than those one would have obtained if the powder grains had been rammed in manually for the best degree of packing, and the latter method is entirely excluded for practical use.\",\n \"The only additional measure which may be required in connection with the present invention is that the charges are vibrated during powder filling, which is also preferably done simultaneously with the two powder types.\",\n \"One or more of the powder types used can additionally be surface-treated or surface-inhibited in order to further control the progressivity.\",\n \"The present invention thus makes it possible to produce propellant powder charges with a high loading density and exactly the high energy content which is required for the case in question.\",\n \"A propellant powder charge produced according to the invention can thus contain 70-95% by weight of a coarsely grained multiple-hole powder, for example a 19-hole or 37-hole powder, and 30-5% by weight of a smaller multiple-hole powder, for example a 1-hole powder or 7-holed powder, and, in view of the desired final result, the powders can each have the same or different chemcial composition and be surface-inhibited (surface-treated with suitable burn inhibitors) or not surface-inhibited.\",\n \"The percentages shown above apply to the outer limits characterizing the invention, whereas, when in practice calculating the charges of the type characterizing the invention, they will in most cases be in the range of 75-85% by weight for the coarser multiple-hole powder and 25-15% by weight for the multiple-hole and more finely grained powder or powders.\",\n \"The nearest prior art we are aware of is the charge which is described in U.S. Pat.\",\n \"No.\",\n \"4,519,855 which describes a propellant powder charge for ballistic ammunition comprising a first powder component consisting of large balls or spheres easily fragmentable into small grains of a first powder component surrounded by a second powder component in the form of a conventionally granular powder which fills the space between the large spheres of powder.\",\n \"Here too the aim has been to produce a powder charge with the highest possible degree of filling taking into consideration the powder types used, but the progressivity of the charges obtained by this method will be based primarily on the large spheres of powder bursting apart during combustion of the powder and thereafter burning like conventionally granular powder, whereas the progressivity for our specific charge is based entirely on the original geometrical shape of the powder grains used.\",\n \"The present invention thus relates to a method in which granular powder is used to produce propellant powder charges with high degree of filling or loading density and a high energy content per charge.\",\n \"The invention also covers the charge produced by this method.\",\n \"The invention also makes it possible to produce charges with precisely controlled progressive characteristics by virtue of the fact that it can be built up from different quantities of different powders which have radically different progressive characteristics.\",\n \"The invention is thus based entirely on combining two or more types of granular powder having such geometric external shapes and grain sizes that the powder with the smaller grain size will in the best possible way fill the empty space between the larger grains.\",\n \"The invention has in all its features been defined in the attached patent claims and it can be illustrated by the following example.\",\n \"EXAMPLE From a nitrocellulose powder of standard quality which we have used for many years for producing artillery powder, we produced on the one hand a 19-hole powder with grain size of 17×17 mm and a 1-hole powder with a grain size of 5×5 mm.\",\n \"Of these powders, we mixed 2.3 kg of the 19-hole powder with 0.5 kg of the 1-hole powder and thus obtained a charge with an energy content corresponding to 122% of the energy content in a standard charge which we had previously produced and which was made only from 19-hole powder.\",\n \"Both the charge types are held in the same volume.\",\n \"The invention thus makes it possible to achieve considerable advantages by very small means.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471457"}}},{"rowIdx":595,"cells":{"text":{"kind":"list like","value":[["Method of synchronizing fin fold-out on a fin-stabilized artillery shell, and an artillery shell designed in accordance therewith","The present invention relates to a method of as far as possible limiting the yawing motion on the trajectory of an artillery shell(1), provided during the firing phase with a sliding driving band and completely folded-in guide fins (3, 16), which shell, as soon as possible outside the mouth of the barrel of the firing piece, is converted, by fold-out of the guide fins (3, 16), into a fin-stabilized artillery shell, any form of non-uniform fin fold-out being avoided by virtue of all the guide fins (3, 16) being interconnected, by means (18, 19, 20) adapted thereto, to form a system which gives all the fins (3, 16) the same movement pattern and the same fold-out speed in each phase of fin fold-out.","The invention also includes a shell (1) designed in accordance therewith, in which the means for synchronization of fin fold-out consists of a rotatable control ring (19) which is arranged around the axis of the shell and is connected to the rotation spindles (13) of all the fins."],["1-10.","(canceled) 11.A method for firing an artillery shell having a sliding driving band and completely folded-in and interconnected guide fins from a firing piece, the method comprising: firing the artillery shell; converting, as soon as possible outside a mouth of a barrel of the firing piece, the artillery shell by fold-out of the guide fins into a fin-stabilized artillery shell; avoiding any form of non-uniform fin fold-out by interconnecting all of the guide fins; and forming a system which gives all the guide fins a same movement pattern and a same fold-out speed during each of a plurality of fin fold-out phases.","12.The method of claim 1, further comprising: allowing moving of each of the interconnected fins around a respective rotation spindle arranged essentially in a longitudinal direction of the shell from a first, folded-in position in which an active area of a fin in a region of the rotation spindle lies essentially tangential to a shell body, to a second, folded-out position in which the active area is oriented essentially radially relative to the shell body; and interconnecting each of the fins to form a continuous system which assists with braking a fold-out of each of the fins according to a wind load acting on the active area of each fin.","13.The method of claim 1, further comprising: controlling an interaction of a relative fold-out of each of the fins by using a toothed ring connecting the fin spindles and a corresponding toothing on each fin spindle.","14.An artillery shell suitable for firing from a rifled barrel, the shell comprising: a sliding driving band; foldable stabilizing fins which are folded out after firing of the shell and which convert the shell into a fin-stabilized projectile, movement transmission means for interconnecting the foldable stabilizing fins and, when the fin-stabilized projectile is on a firing trajectory, synchronizing and making uniform fold-out movements of the foldable stabilizing fins.","15.The artillery shell of claim 14, wherein each of the fins have an associated active area which is rotatably mounted around an associated spindle arranged essentially in a longitudinal direction of the shell and around which the associated active area rotates from a first, folded-in position, in which said associated active area lies essentially tangential to a shell body and a free outer end thereof is curved in towards the shell body, to a second, folded-out position, in which the associated active area extends essentially radially out from a surface of the shell body, wherein the movement transmission means comprises at least one control ring arranged rotatably around the longitudinal axis of the shell and is connected to spindles of all of the fins so as to control the movement of each of the fins.","16.The artillery shell of claim 15, wherein the at least one control ring has an external toothing while the spindle of each fin has, at a respective place of connection to the control ring, corresponding toothing in engagement with the teeth of the control ring.","17.The artillery shell of claim 15, wherein the control ring comprises external knurling or another friction-increasing surface treatment, and wherein the rotation spindle of each fin has a corresponding friction-increasing surface treatment where the spindles make contact with the control ring.","18.The artillery shell of claim 14, wherein the movement transmission means is located around an exhaust opening of a base bleed unit which is arranged in a same part of the shell as the fins, said fins being mounted concentrically outside the base-bleed unit.","19.The artillery shell of claim 14, wherein an action of forces of air on the fins is augmented by the fins being given a relatively small angle of attack relative to the main axis of the shell.","20.The artillery shell of claim 19, wherein the angle of attack is brought about by the fin, in the folded-out position, being provided with a spiral or propeller twist.","21.The artillery shell of claim 19, wherein the angle of attack is brought about by the fin, in the folded-out position, being provided with a dog-ear design on at least a portion of an outer portion."],["The present invention relates to a method of synchronizing fin fold-out on a long-range artillery shell which is fin-stabilized on its trajectory towards the target and is intended to be fired from a rifled barrel and is to this end provided with a sliding driving band as the main contact surface against the inside of the barrel and also with a number of stabilizing fins which can be folded out after the shell has left the barrel.","The purpose of the sliding driving band is to allow the shell, in spite of the rifling of the barrel, to leave the latter with only low rotation or no rotation at all.","It is particularly characteristic of the method and the shell according to the invention that the stabilizing fins of the shell are interconnected by specially designed movement transmission means which bring about uniform fold-out of all the fins irrespective of how these are loaded during the fold-out phase itself.","Even if the shell should leave the barrel entirely without rotation, the fins arranged around the shell will nevertheless be loaded differently during the fold-out phase by the forces generated by the air flowing past.","This is because it has proved to be impossible to avoid any type of shell being subjected to a certain conical yawing motion on its trajectory, and this yawing motion begins immediately after the shell has left the mouth of the barrel.","The reason why an artillery shell is fin-stabilized instead of being rotation-stabilized may be, for example, that it is desirable to make it guidable on its way towards the target, and it is considerably easier to correct the course of a fin-stabilized shell than of a rotation-stabilized shell, and this is the case irrespective of whether the course correction concerned is intended to be performed by impulse motors, steering rudders or in another manner.","It is a requirement of the shell according to the invention that it should be capable of being given an extra long range.","A method used increasingly in recent years of achieving extremely long ranges even in older barrel-type artillery is the base-bleed technique, which is used in order to eliminate the turbulence and negative pressure which are formed behind the shells flying through the atmosphere and have a braking effect on the shells and shorten their flying distance.","The base-bleed technique is based on arranging a combustion chamber in the rear part of the shell, which chamber is filled with a slow-burning pyrotechnic composition which, while it burns, produces combustion gases which are allowed, in a predetermined quantity, to flow out through an opening in the rear end wall of the shell and there eliminate and fill the abovementioned braking turbulence and negative pressure behind the shell.","When a shell is to be provided with both a base-bleed unit and stabilizing fins, however, it is easy for positioning problems to arise, because the base-bleed unit definitely has to be arranged in the rear part of the shell with at least one gas outflow opening in the rear end wall of the shell, while the fins too ought to be positioned in the rear body of the shell as far away as possible from the centre of gravity of the shell, that is to say fins and base-bleed unit should preferably be arranged within the same part of the shell.","An additional problem is that, in order to allow firing of the shell from a rifled barrel, the fins must be fully folded in inside the minimum diameter of the barrel during firing, at the same time as they must not occupy too great a volume either and thus prevent the use of this space for other purposes such as, therefore, the base-bleed unit or payload.","In a known type of fold-in fin, which takes up little space and can be designed so that, in the folded-in position, the fins can share the rearmost part of the shell with a base-bleed unit, each fin consists of a plate which is fixed to a rotatable spindle arranged in the longitudinal direction of the shell and which, in the folded-out position, will constitute the active area of the fin and, in the folded-in position, is rotated in towards the shell body about its spindle, and is in this position curved in towards the shell body and, until the desired fold-out time, is retained in this position by a protective cover or equivalent.","Previously, such fins were designed with a curved shape following the shell body and they retained this shape in the folded-out position as well, but, in recent years, elastically deformable materials have become available, which have such a good shape memory that it is now possible to produce fins which, even after years of incurvation in the folded-in position, essentially recover their original shape.","It has therefore become possible to use these materials to produce fins which, as soon as they are given the opportunity, tend to recover the shape they were originally given, and this may have been entirely plane or slightly propeller-shaped or designed in another way so as to be provided with a limited angle of attack relative to the air rushing past.","One way, which is relatively simple in terms of manufacture in this context, of giving the fins the desired angle of attack is to provide them with a sharp or gently curved dog-ear design or a few degrees of propeller twist.","All these types of guide fins are presupposed at the same time to have a radial main direction seen in the cross-sectional direction of the shell.","The angles of action relative to the air rushing past the shell which are chiefly of interest in the case of the guide fins for fin-stabilized shells are usually of the order of 1-2°, and corresponding angles of action can of course also be brought about by means of axes of rotation for folding in and folding out the fins which are inclined relative to the longitudinal axis of the shell, but this would as a rule involve more expensive overall solutions.","As an example of the state of the art, WO 98/43037 may be mentioned, in which a fin-stabilized artillery shell with fold-out stabilizing fins of the type described above is disclosed.","In the introduction, it was stated that every type of artillery shell is already subjected to a certain form of conical yawing motion on the trajectory immediately after it has left the mouth of the barrel and that this results in fold-out fins arranged on the shell being subjected to different degrees of loading by the relative wind of the surrounding air, which can moreover, to some extent, be from different directions.","In brief, this means that the various fins on a fin-stabilized artillery shell will be loaded differently during the fold-out phase itself.","In the case of shells provided with sliding driving bands, the centrifugal force acting on the fins is of little importance for fin fold-out.","Instead, the majority of the fold-out force comes from the straightening force of the fin material, that is to say the force which is generated when the elastic deformation of the fin material returns to the original shape the fin was once given.","In their folded-in position, elastically deformed fins of the type concerned here will quite simply spread out by virtue of their own built-in force but, in spite of this, the fold-out function cannot be left entirely to this mechanical energy development, inter alia because it is clearly most marked during the initial introductory phase of fold-out.","For this reason, the fins are normally also provided in the previously indicated manner with a small angle of attack relative to the flying direction of the shell, so that the forces of the air will, above all in the final stage of fold-out, make their contribution to the requisite fin fold-out force.","However, on account of the yawing motion of the shell, the air forces may vary quite considerably in strength and direction between the different sides of the shell because the relative wind against the shell is dependent on the yawing motion of the shell which begins directly outside the mouth of the barrel.","A fin on one side of the shell could therefore, if it were able to define its own fold-out speed, have such a high fold-out speed that its strength is put at risk, while a fin on another side of the shell could at the same time have such a low fold-out speed that it does not completely reach its intended radial position.","Accordingly, the object of the present invention is to eliminate, in a reliable manner, the effects of an otherwise readily occurring incomplete fin fold-out, and this is achieved by fold-out of the fins in relation to one another being synchronized using means adapted thereto.","According to the invention, the fins are therefore to be interconnected in such a manner in relation to one another that they are folded out at the same speed.","The invention therefore concerns a method of forcing the fins most heavily loaded in the fold-out direction to share the fold-out force acting on them with fins which are more lightly loaded in the fold-out direction at the same time as the latter are to force the more heavily loaded fins to slow down their fold-out speed and thus also to reduce the risk of them being overloaded.","The basic principle of the invention is therefore that all the fins are to be connected by means of a common fin fold-out control or synchronizing arrangement which is to be designed in such a manner that it gives all the fins a simultaneously initiated uniform fold-out at the same speed from their initial folded-in position with that part of the fin blade or the active area of the fin which lies closest to the spindle extending tangentially to the immediately adjacent outer side of the shell into a folded-out position in which the fin blades are angled at in principle 90° relative to the folded-in position, in which position the fin blades or the active areas of the fins extend radially out from the shell body.","The invention also includes the fact that the fins should, via the synchronizing arrangement, help one another with fold-out or alternatively brake one another as required.","A direct drive function is therefore, at least in the first place, not intended to be included in the system.","An essential part of fin fold-out is also that the fin plates which constitute the active areas of the fins recover elastically from their incurvation towards the shell body to the finally intended shape they were once given.","Another advantage of the invention is that, in an especially preferred embodiment, it requires very limited extra space and by virtue of this makes it possible to arrange both the fold-out fins and a base-bleed unit within the same part of the shell.","The invention therefore provides a method and an arrangement which guarantee that the fold-out fins on an artillery shell with a sliding driving band fired from a rifled barrel achieve their completely folded-out and locked end position.","It is characteristic of the method and the arrangement according to the invention in this connection that any form of non-uniform fin fold-out and associated negative influence on the flight of the shell will be avoided by virtue of all the guide fins being interconnected by means adapted thereto to form a system which, during the fold-out phase, gives the fins a synchronized movement pattern with simultaneous and uniform fold-out movements.","In order to make it possible to perform such a synchronized fin fold-out function, we have introduced a movement transmission means which connects all the rotation spindles around which the fins have, during the firing phase, been curved in towards the shell body, in which position they have been retained by a special protective cover from the completion of the shell during manufacture until it leaves the mouth of the barrel.","When the shell leaves the mouth of the barrel, the protective cover is torn away from the shell by an inner powder gas pressure which, during the firing phase, is allowed to leak into the cover and which, inside the barrel, is balanced by the powder gas pressure behind the shell.","This is because, when the shell leaves the barrel, this counterpressure ceases very rapidly and, by dimensioning the gas supply to the cover so that it is not possible for its inner overpressure to be eliminated at the same rate as the abrupt reduction in pressure behind the shell takes place, the cover will be thrown off.","As soon as the protective cover has been removed, fin fold-out will begin and, as the method and the arrangement according to the invention are primarily intended for use on shells with sliding driving bands, there is only at the very most a weak centrifugal force available to assist fin fold-out.","The majority of the force necessary for fin fold-out therefore has to be obtained, as already mentioned, from the straightening force built into the fins and also, to some extent, from the relative wind force against the fins of the passing air.","The object of the method and the arrangement according to the invention is therefore to even out this non-uniformity and to give all the fins the same fold-out speed.","According to an especially preferred embodiment, the main means of synchronizing the fin fold-out function consists of a control ring which is arranged concentrically around the longitudinal axis of the shell close to its outer wall, can rotate in a groove adapted thereto and connects the various fin spindles and gives these and the active areas of the fins identical movement patterns.","In its most developed form, the outer surface of the control ring is designed as a toothed ring and each fin spindle is in turn provided with a corresponding toothed segment covering at least a quarter of a turn.","Under certain circumstances, it would probably be possible to replace the toothing with low-cost variants in the form of knurling or another friction-increasing treatment of the outer surface of the control ring and the rotation spindles of the fins.","Another possible but, because it would result in so many small parts, less practical solution would be to use a number of links which interconnect cranks rigidly connected to respective spindles.","The invention is defined in greater detail in the patent claims below and will moreover be described in somewhat greater detail in connection with accompanying figures, in which FIG.","1 shows an oblique projection of an artillery shell while FIG.","2 shows a longitudinal section through the rear part of the shell, FIG.","3 shows the section III-III in FIG.","2 with the fins folded in and covered by a protective cover while FIG.","4 shows the section III-III in FIG.","2 but with the fins folded out, and FIG.","5 shows a detail from FIG.","4 while FIG.","6 shows the rear part of the shell according to FIG.","2 but in an oblique projection.","The shell shown in an oblique projection in FIG.","1 represents an example of how a shell designed according to the invention may appear on its way towards the target.","The shell in question consists of a shell body 1 provided with a groove for a sliding driving band 2 which has already been lost, a number of folded-out fins 3 which are attached to the rear portion 4 of the shell, the connection of which to the shell body 1 is indicated by the join 5.At the front end of the shell, there are four canard rudders 6a, 6b and 7a, 7b which can likewise be folded out and are moreover guidable.","All the fins and rudders are designed in such a manner that they can be kept folded in during the firing phase.","FIG.","2 shows in greater detail how the rear portion 4 is designed.","This portion accordingly comprises an inner cavity 8, in which a base-bleed charge 9 is arranged.","There is also an initiator 10 for the base-bleed charge and a support dome 12 arranged around the outlet 11 thereof.","Each of the fins 3 is attached to a rotatable spindle 13 aligned essentially in the longitudinal direction of the shell.","Each such spindle has a bearing point 14 and, respectively, 15 at each end.","The active areas of the fins, which consist of plane plates as in FIGS.","2-6 in the folded-out position, have been given the general designation 16.In their folded-in position, the active areas 16 of the fins, which can be seen more clearly in FIG.","3, are on the one hand folded down a quarter of a turn around their respective spindles 13 towards the rear body 4 of the shell so that, in the region of their respective spindles 13, they extend essentially tangentially along the rear body 4, and on the other hand curved in at their respective free outer end along this body and moreover covered by a protective cover 17 which is removed as soon as the shell has left the mouth of the barrel.","In order for it to be possible to bring about the synchronization of fold-out of the fins 16 which is characteristic of the invention, the spindles 13 of the fins are, somewhere along their length, in this case at one of their ends, designed with toothed arcs or toothed segments 18 which in turn are all in engagement with an externally toothed control ring 19 characteristic of the invention, which, in a groove 20 adapted thereto inside the rear body 4 close to its outer wall, runs concentrically around the central outlet 21 of the rear body 4 for the base-bleed charge.","Until and when the shell leaves the barrel from which it is fired, the fins will therefore be covered by the cover 17 which, by interaction between powder gases penetrating into the cover and the vacuum directly outside the mouth of the barrel, is pulled off, whereupon fin fold-out begins immediately.","By virtue of the fact that the spindles 13 of all the fins 16, via the toothed arcs 18 and then in turn by the externally toothed control ring or synchronizing means, are interconnected to form a continuous system, all the fins will be folded out at the same speed.","As can be seen from FIGS.","3 and 5 in particular, we have, in the case illustrated, selected a tooth size which, with four teeth for each toothed arc 18 on the spindle 13 of each fin 16, gives a fold-out movement corresponding to a quarter of a turn for the active area 16 of the fin."]],"string":"[\n [\n \"Method of synchronizing fin fold-out on a fin-stabilized artillery shell, and an artillery shell designed in accordance therewith\",\n \"The present invention relates to a method of as far as possible limiting the yawing motion on the trajectory of an artillery shell(1), provided during the firing phase with a sliding driving band and completely folded-in guide fins (3, 16), which shell, as soon as possible outside the mouth of the barrel of the firing piece, is converted, by fold-out of the guide fins (3, 16), into a fin-stabilized artillery shell, any form of non-uniform fin fold-out being avoided by virtue of all the guide fins (3, 16) being interconnected, by means (18, 19, 20) adapted thereto, to form a system which gives all the fins (3, 16) the same movement pattern and the same fold-out speed in each phase of fin fold-out.\",\n \"The invention also includes a shell (1) designed in accordance therewith, in which the means for synchronization of fin fold-out consists of a rotatable control ring (19) which is arranged around the axis of the shell and is connected to the rotation spindles (13) of all the fins.\"\n ],\n [\n \"1-10.\",\n \"(canceled) 11.A method for firing an artillery shell having a sliding driving band and completely folded-in and interconnected guide fins from a firing piece, the method comprising: firing the artillery shell; converting, as soon as possible outside a mouth of a barrel of the firing piece, the artillery shell by fold-out of the guide fins into a fin-stabilized artillery shell; avoiding any form of non-uniform fin fold-out by interconnecting all of the guide fins; and forming a system which gives all the guide fins a same movement pattern and a same fold-out speed during each of a plurality of fin fold-out phases.\",\n \"12.The method of claim 1, further comprising: allowing moving of each of the interconnected fins around a respective rotation spindle arranged essentially in a longitudinal direction of the shell from a first, folded-in position in which an active area of a fin in a region of the rotation spindle lies essentially tangential to a shell body, to a second, folded-out position in which the active area is oriented essentially radially relative to the shell body; and interconnecting each of the fins to form a continuous system which assists with braking a fold-out of each of the fins according to a wind load acting on the active area of each fin.\",\n \"13.The method of claim 1, further comprising: controlling an interaction of a relative fold-out of each of the fins by using a toothed ring connecting the fin spindles and a corresponding toothing on each fin spindle.\",\n \"14.An artillery shell suitable for firing from a rifled barrel, the shell comprising: a sliding driving band; foldable stabilizing fins which are folded out after firing of the shell and which convert the shell into a fin-stabilized projectile, movement transmission means for interconnecting the foldable stabilizing fins and, when the fin-stabilized projectile is on a firing trajectory, synchronizing and making uniform fold-out movements of the foldable stabilizing fins.\",\n \"15.The artillery shell of claim 14, wherein each of the fins have an associated active area which is rotatably mounted around an associated spindle arranged essentially in a longitudinal direction of the shell and around which the associated active area rotates from a first, folded-in position, in which said associated active area lies essentially tangential to a shell body and a free outer end thereof is curved in towards the shell body, to a second, folded-out position, in which the associated active area extends essentially radially out from a surface of the shell body, wherein the movement transmission means comprises at least one control ring arranged rotatably around the longitudinal axis of the shell and is connected to spindles of all of the fins so as to control the movement of each of the fins.\",\n \"16.The artillery shell of claim 15, wherein the at least one control ring has an external toothing while the spindle of each fin has, at a respective place of connection to the control ring, corresponding toothing in engagement with the teeth of the control ring.\",\n \"17.The artillery shell of claim 15, wherein the control ring comprises external knurling or another friction-increasing surface treatment, and wherein the rotation spindle of each fin has a corresponding friction-increasing surface treatment where the spindles make contact with the control ring.\",\n \"18.The artillery shell of claim 14, wherein the movement transmission means is located around an exhaust opening of a base bleed unit which is arranged in a same part of the shell as the fins, said fins being mounted concentrically outside the base-bleed unit.\",\n \"19.The artillery shell of claim 14, wherein an action of forces of air on the fins is augmented by the fins being given a relatively small angle of attack relative to the main axis of the shell.\",\n \"20.The artillery shell of claim 19, wherein the angle of attack is brought about by the fin, in the folded-out position, being provided with a spiral or propeller twist.\",\n \"21.The artillery shell of claim 19, wherein the angle of attack is brought about by the fin, in the folded-out position, being provided with a dog-ear design on at least a portion of an outer portion.\"\n ],\n [\n \"The present invention relates to a method of synchronizing fin fold-out on a long-range artillery shell which is fin-stabilized on its trajectory towards the target and is intended to be fired from a rifled barrel and is to this end provided with a sliding driving band as the main contact surface against the inside of the barrel and also with a number of stabilizing fins which can be folded out after the shell has left the barrel.\",\n \"The purpose of the sliding driving band is to allow the shell, in spite of the rifling of the barrel, to leave the latter with only low rotation or no rotation at all.\",\n \"It is particularly characteristic of the method and the shell according to the invention that the stabilizing fins of the shell are interconnected by specially designed movement transmission means which bring about uniform fold-out of all the fins irrespective of how these are loaded during the fold-out phase itself.\",\n \"Even if the shell should leave the barrel entirely without rotation, the fins arranged around the shell will nevertheless be loaded differently during the fold-out phase by the forces generated by the air flowing past.\",\n \"This is because it has proved to be impossible to avoid any type of shell being subjected to a certain conical yawing motion on its trajectory, and this yawing motion begins immediately after the shell has left the mouth of the barrel.\",\n \"The reason why an artillery shell is fin-stabilized instead of being rotation-stabilized may be, for example, that it is desirable to make it guidable on its way towards the target, and it is considerably easier to correct the course of a fin-stabilized shell than of a rotation-stabilized shell, and this is the case irrespective of whether the course correction concerned is intended to be performed by impulse motors, steering rudders or in another manner.\",\n \"It is a requirement of the shell according to the invention that it should be capable of being given an extra long range.\",\n \"A method used increasingly in recent years of achieving extremely long ranges even in older barrel-type artillery is the base-bleed technique, which is used in order to eliminate the turbulence and negative pressure which are formed behind the shells flying through the atmosphere and have a braking effect on the shells and shorten their flying distance.\",\n \"The base-bleed technique is based on arranging a combustion chamber in the rear part of the shell, which chamber is filled with a slow-burning pyrotechnic composition which, while it burns, produces combustion gases which are allowed, in a predetermined quantity, to flow out through an opening in the rear end wall of the shell and there eliminate and fill the abovementioned braking turbulence and negative pressure behind the shell.\",\n \"When a shell is to be provided with both a base-bleed unit and stabilizing fins, however, it is easy for positioning problems to arise, because the base-bleed unit definitely has to be arranged in the rear part of the shell with at least one gas outflow opening in the rear end wall of the shell, while the fins too ought to be positioned in the rear body of the shell as far away as possible from the centre of gravity of the shell, that is to say fins and base-bleed unit should preferably be arranged within the same part of the shell.\",\n \"An additional problem is that, in order to allow firing of the shell from a rifled barrel, the fins must be fully folded in inside the minimum diameter of the barrel during firing, at the same time as they must not occupy too great a volume either and thus prevent the use of this space for other purposes such as, therefore, the base-bleed unit or payload.\",\n \"In a known type of fold-in fin, which takes up little space and can be designed so that, in the folded-in position, the fins can share the rearmost part of the shell with a base-bleed unit, each fin consists of a plate which is fixed to a rotatable spindle arranged in the longitudinal direction of the shell and which, in the folded-out position, will constitute the active area of the fin and, in the folded-in position, is rotated in towards the shell body about its spindle, and is in this position curved in towards the shell body and, until the desired fold-out time, is retained in this position by a protective cover or equivalent.\",\n \"Previously, such fins were designed with a curved shape following the shell body and they retained this shape in the folded-out position as well, but, in recent years, elastically deformable materials have become available, which have such a good shape memory that it is now possible to produce fins which, even after years of incurvation in the folded-in position, essentially recover their original shape.\",\n \"It has therefore become possible to use these materials to produce fins which, as soon as they are given the opportunity, tend to recover the shape they were originally given, and this may have been entirely plane or slightly propeller-shaped or designed in another way so as to be provided with a limited angle of attack relative to the air rushing past.\",\n \"One way, which is relatively simple in terms of manufacture in this context, of giving the fins the desired angle of attack is to provide them with a sharp or gently curved dog-ear design or a few degrees of propeller twist.\",\n \"All these types of guide fins are presupposed at the same time to have a radial main direction seen in the cross-sectional direction of the shell.\",\n \"The angles of action relative to the air rushing past the shell which are chiefly of interest in the case of the guide fins for fin-stabilized shells are usually of the order of 1-2°, and corresponding angles of action can of course also be brought about by means of axes of rotation for folding in and folding out the fins which are inclined relative to the longitudinal axis of the shell, but this would as a rule involve more expensive overall solutions.\",\n \"As an example of the state of the art, WO 98/43037 may be mentioned, in which a fin-stabilized artillery shell with fold-out stabilizing fins of the type described above is disclosed.\",\n \"In the introduction, it was stated that every type of artillery shell is already subjected to a certain form of conical yawing motion on the trajectory immediately after it has left the mouth of the barrel and that this results in fold-out fins arranged on the shell being subjected to different degrees of loading by the relative wind of the surrounding air, which can moreover, to some extent, be from different directions.\",\n \"In brief, this means that the various fins on a fin-stabilized artillery shell will be loaded differently during the fold-out phase itself.\",\n \"In the case of shells provided with sliding driving bands, the centrifugal force acting on the fins is of little importance for fin fold-out.\",\n \"Instead, the majority of the fold-out force comes from the straightening force of the fin material, that is to say the force which is generated when the elastic deformation of the fin material returns to the original shape the fin was once given.\",\n \"In their folded-in position, elastically deformed fins of the type concerned here will quite simply spread out by virtue of their own built-in force but, in spite of this, the fold-out function cannot be left entirely to this mechanical energy development, inter alia because it is clearly most marked during the initial introductory phase of fold-out.\",\n \"For this reason, the fins are normally also provided in the previously indicated manner with a small angle of attack relative to the flying direction of the shell, so that the forces of the air will, above all in the final stage of fold-out, make their contribution to the requisite fin fold-out force.\",\n \"However, on account of the yawing motion of the shell, the air forces may vary quite considerably in strength and direction between the different sides of the shell because the relative wind against the shell is dependent on the yawing motion of the shell which begins directly outside the mouth of the barrel.\",\n \"A fin on one side of the shell could therefore, if it were able to define its own fold-out speed, have such a high fold-out speed that its strength is put at risk, while a fin on another side of the shell could at the same time have such a low fold-out speed that it does not completely reach its intended radial position.\",\n \"Accordingly, the object of the present invention is to eliminate, in a reliable manner, the effects of an otherwise readily occurring incomplete fin fold-out, and this is achieved by fold-out of the fins in relation to one another being synchronized using means adapted thereto.\",\n \"According to the invention, the fins are therefore to be interconnected in such a manner in relation to one another that they are folded out at the same speed.\",\n \"The invention therefore concerns a method of forcing the fins most heavily loaded in the fold-out direction to share the fold-out force acting on them with fins which are more lightly loaded in the fold-out direction at the same time as the latter are to force the more heavily loaded fins to slow down their fold-out speed and thus also to reduce the risk of them being overloaded.\",\n \"The basic principle of the invention is therefore that all the fins are to be connected by means of a common fin fold-out control or synchronizing arrangement which is to be designed in such a manner that it gives all the fins a simultaneously initiated uniform fold-out at the same speed from their initial folded-in position with that part of the fin blade or the active area of the fin which lies closest to the spindle extending tangentially to the immediately adjacent outer side of the shell into a folded-out position in which the fin blades are angled at in principle 90° relative to the folded-in position, in which position the fin blades or the active areas of the fins extend radially out from the shell body.\",\n \"The invention also includes the fact that the fins should, via the synchronizing arrangement, help one another with fold-out or alternatively brake one another as required.\",\n \"A direct drive function is therefore, at least in the first place, not intended to be included in the system.\",\n \"An essential part of fin fold-out is also that the fin plates which constitute the active areas of the fins recover elastically from their incurvation towards the shell body to the finally intended shape they were once given.\",\n \"Another advantage of the invention is that, in an especially preferred embodiment, it requires very limited extra space and by virtue of this makes it possible to arrange both the fold-out fins and a base-bleed unit within the same part of the shell.\",\n \"The invention therefore provides a method and an arrangement which guarantee that the fold-out fins on an artillery shell with a sliding driving band fired from a rifled barrel achieve their completely folded-out and locked end position.\",\n \"It is characteristic of the method and the arrangement according to the invention in this connection that any form of non-uniform fin fold-out and associated negative influence on the flight of the shell will be avoided by virtue of all the guide fins being interconnected by means adapted thereto to form a system which, during the fold-out phase, gives the fins a synchronized movement pattern with simultaneous and uniform fold-out movements.\",\n \"In order to make it possible to perform such a synchronized fin fold-out function, we have introduced a movement transmission means which connects all the rotation spindles around which the fins have, during the firing phase, been curved in towards the shell body, in which position they have been retained by a special protective cover from the completion of the shell during manufacture until it leaves the mouth of the barrel.\",\n \"When the shell leaves the mouth of the barrel, the protective cover is torn away from the shell by an inner powder gas pressure which, during the firing phase, is allowed to leak into the cover and which, inside the barrel, is balanced by the powder gas pressure behind the shell.\",\n \"This is because, when the shell leaves the barrel, this counterpressure ceases very rapidly and, by dimensioning the gas supply to the cover so that it is not possible for its inner overpressure to be eliminated at the same rate as the abrupt reduction in pressure behind the shell takes place, the cover will be thrown off.\",\n \"As soon as the protective cover has been removed, fin fold-out will begin and, as the method and the arrangement according to the invention are primarily intended for use on shells with sliding driving bands, there is only at the very most a weak centrifugal force available to assist fin fold-out.\",\n \"The majority of the force necessary for fin fold-out therefore has to be obtained, as already mentioned, from the straightening force built into the fins and also, to some extent, from the relative wind force against the fins of the passing air.\",\n \"The object of the method and the arrangement according to the invention is therefore to even out this non-uniformity and to give all the fins the same fold-out speed.\",\n \"According to an especially preferred embodiment, the main means of synchronizing the fin fold-out function consists of a control ring which is arranged concentrically around the longitudinal axis of the shell close to its outer wall, can rotate in a groove adapted thereto and connects the various fin spindles and gives these and the active areas of the fins identical movement patterns.\",\n \"In its most developed form, the outer surface of the control ring is designed as a toothed ring and each fin spindle is in turn provided with a corresponding toothed segment covering at least a quarter of a turn.\",\n \"Under certain circumstances, it would probably be possible to replace the toothing with low-cost variants in the form of knurling or another friction-increasing treatment of the outer surface of the control ring and the rotation spindles of the fins.\",\n \"Another possible but, because it would result in so many small parts, less practical solution would be to use a number of links which interconnect cranks rigidly connected to respective spindles.\",\n \"The invention is defined in greater detail in the patent claims below and will moreover be described in somewhat greater detail in connection with accompanying figures, in which FIG.\",\n \"1 shows an oblique projection of an artillery shell while FIG.\",\n \"2 shows a longitudinal section through the rear part of the shell, FIG.\",\n \"3 shows the section III-III in FIG.\",\n \"2 with the fins folded in and covered by a protective cover while FIG.\",\n \"4 shows the section III-III in FIG.\",\n \"2 but with the fins folded out, and FIG.\",\n \"5 shows a detail from FIG.\",\n \"4 while FIG.\",\n \"6 shows the rear part of the shell according to FIG.\",\n \"2 but in an oblique projection.\",\n \"The shell shown in an oblique projection in FIG.\",\n \"1 represents an example of how a shell designed according to the invention may appear on its way towards the target.\",\n \"The shell in question consists of a shell body 1 provided with a groove for a sliding driving band 2 which has already been lost, a number of folded-out fins 3 which are attached to the rear portion 4 of the shell, the connection of which to the shell body 1 is indicated by the join 5.At the front end of the shell, there are four canard rudders 6a, 6b and 7a, 7b which can likewise be folded out and are moreover guidable.\",\n \"All the fins and rudders are designed in such a manner that they can be kept folded in during the firing phase.\",\n \"FIG.\",\n \"2 shows in greater detail how the rear portion 4 is designed.\",\n \"This portion accordingly comprises an inner cavity 8, in which a base-bleed charge 9 is arranged.\",\n \"There is also an initiator 10 for the base-bleed charge and a support dome 12 arranged around the outlet 11 thereof.\",\n \"Each of the fins 3 is attached to a rotatable spindle 13 aligned essentially in the longitudinal direction of the shell.\",\n \"Each such spindle has a bearing point 14 and, respectively, 15 at each end.\",\n \"The active areas of the fins, which consist of plane plates as in FIGS.\",\n \"2-6 in the folded-out position, have been given the general designation 16.In their folded-in position, the active areas 16 of the fins, which can be seen more clearly in FIG.\",\n \"3, are on the one hand folded down a quarter of a turn around their respective spindles 13 towards the rear body 4 of the shell so that, in the region of their respective spindles 13, they extend essentially tangentially along the rear body 4, and on the other hand curved in at their respective free outer end along this body and moreover covered by a protective cover 17 which is removed as soon as the shell has left the mouth of the barrel.\",\n \"In order for it to be possible to bring about the synchronization of fold-out of the fins 16 which is characteristic of the invention, the spindles 13 of the fins are, somewhere along their length, in this case at one of their ends, designed with toothed arcs or toothed segments 18 which in turn are all in engagement with an externally toothed control ring 19 characteristic of the invention, which, in a groove 20 adapted thereto inside the rear body 4 close to its outer wall, runs concentrically around the central outlet 21 of the rear body 4 for the base-bleed charge.\",\n \"Until and when the shell leaves the barrel from which it is fired, the fins will therefore be covered by the cover 17 which, by interaction between powder gases penetrating into the cover and the vacuum directly outside the mouth of the barrel, is pulled off, whereupon fin fold-out begins immediately.\",\n \"By virtue of the fact that the spindles 13 of all the fins 16, via the toothed arcs 18 and then in turn by the externally toothed control ring or synchronizing means, are interconnected to form a continuous system, all the fins will be folded out at the same speed.\",\n \"As can be seen from FIGS.\",\n \"3 and 5 in particular, we have, in the case illustrated, selected a tooth size which, with four teeth for each toothed arc 18 on the spindle 13 of each fin 16, gives a fold-out movement corresponding to a quarter of a turn for the active area 16 of the fin.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471458"}}},{"rowIdx":596,"cells":{"text":{"kind":"list like","value":[["Method for pumping fluids","The production rate, useful life and operating efficiency of electric submergible pumping systems (ESP) is improved by operating the system so that the motor (220, 320) is cooled by two passes of production.","Production fluids remove waste heat from the motor (220, 320) by passing the fluid in contact with the exterior of the motor (220, 320).","The improved cooling permits the motor to be operated at a lower temperature improving life and efficiency, and/or to be operated at higher power at a similar temperature.","Additionally, oversized and excess equipment is not required, further improving performance and economics for the user.","In another aspect, the invention is a method of pumping fluids using a motor having vortex generators (366) on its exterior surfaces or on the interior surfaces of a surrounding shroud."],["1.A method comprising pumping a fluid with a submergible pumping system including a pump and an electrical motor, wherein the electrical motor has a hollow rotor shaft and said pumping system is adapted to pump at least a portion of said fluid through said hollow rotor shaft, and wherein during operation at least a portion of said fluid passes in fluid contact with exterior portions of the electrical motor and removes heat therefrom.","2.The method of claim 1, wherein the pumping system is submerged in said fluid such that production fluids entering the pumping system pass in fluid contact with exterior portions of the electrical motor and remove heat therefrom before entering the pumping system, and said liquid production fluids then pass through the hollow rotor shaft of the electrical motor as the fluid is pumped, and remove additional heat from the motor.","3.A method for producing fluids from a well having a casing that contains perforations through which production fluids enter the well, comprising (a) positioning an electrical submergible pumping system including an electric motor within said casing such that the electric motor is at or below at least some of said perforations, and (b) pumping production fluids from the well with said pumping system, wherein (1) the pumping system includes a pump, an electrical motor having a hollow rotor shaft and production fluid intakes located below said electrical motor, said pumping system being adapted to pump said production fluids through said hollow rotor shaft of the electrical motor, (2) production fluids entering the well through at least some of the perforations in the casing pass in fluid contact with exterior portions of the electrical motor and remove heat therefrom before entering the production fluid intakes, and (3) production fluids pass through the hollow rotor shaft of the electrical motor as the fluid travels to the wellhead, and remove additional heat from the motor.","4.The method of claim 3 wherein the production fluids pass through the hollow rotor shaft of the electrical motor under conditions of turbulent flow.","5.The method of claim 4 wherein the electrical motor is located between the wellhead and the pump.","6.The method of claim 5 wherein production fluids entering the well through the perforations in the casing travel downward in fluid contact with exterior portions of the electrical motor to an intake, and the liquid production fluids are subsequently pumped upwardly through the hollow rotor shaft of the electrical motor and then to the wellhead.","7.The method of claim 6, wherein the flow of production fluid through the hollow rotor shaft is characterized by a Reynolds number of at least 2300.8.The method of claim 7, wherein the motor contains a motor fluid at a positive pressure to the exterior of the motor, and has seals which allow motor fluid to leak into the production fluid.","9.The method of claim 7, wherein conditions are selected such that heat transfer from at least one of the hollow rotor shaft and the exterior surfaces of the motor to the production fluid is characterized by a Nusselt number of at least 10.10.The method of claim 7 wherein conditions are selected so as to generate a Brinkmann number of less than 2.11.The method of claim 7 wherein conditions are selected so as to generate a Rossby number of more than 0.5.12.The method of claim 4 wherein the pump is located between the wellhead and the electric motor.","13.The method of claim 4 wherein the electrical submergible pumping system contains two pumps, one of which is located above the electric motor and one of which is located below the electric motor.","14.The method of claim 1 wherein (a) the electrical motor has a hollow rotor shaft and the pumping system has a fluid intake below the electrical motor and in liquid communication with said hollow rotor shaft; (b) the pumping system has at least one outlet located at or below the electrical motor, said outlet being in liquid communication with the fluid intake, (c) the pumping system is submerged in a well having a well casing; (d) the cross-section that the electrical motor is such that the electrical motor fits within the well casing and a space is defined between the well casing and the electrical motor; and (e) said pumping system is adapted so that during operation a portion of the fluid entering the fluid intake is pumped through the hollow rotor shaft of the electrical motor, and a portion of the fluid entering the fluid intake is pumped through the outlet and upwardly in fluid contact with the outside of the electrical motor.","15.A method of pumping fluids, comprising (I) positioning a pumping system in a well with the pump being located between the electrical motor and the wellhead; wherein (a) electrical motor has a hollow rotor shaft which is in liquid communication with the pump, (e) the cross-section of the electrical motor is such that the electrical motor fits within the well casing and a space is defined between the well casing and the electrical motor; (f) the pumping system has a first fluid intake in liquid communication with said hollow rotor shaft; (g) the pumping system has a second fluid intake above the motor in liquid communication with the space defined between the well casing and the electrical motor, with the at least one outlet located at or below the electrical motor, said outlet being in liquid communication with the fluid intake; and (II) operating said pumping system under conditions such that during operation a portion of the fluids enter said first fluid intake and is pumped through the hollow rotor shaft of the electrical motor, and a second portion of the fluids are pumped through the space defined between the well casing and the electrical motor and in fluid contact with the exterior of the electrical motor to enter the second fluid intake, and said first and second portions of the fluids are then pumped to the wellhead.","16.A mechanism for providing motive force, the mechanism comprising (I) a power unit including: (a) a housing having an exterior wall; (b) a hollow rotor shaft having a longitudinal axis and opposite ends, the hollow rotor shaft being rotatably mounted within the housing for rotation of the hollow rotor shaft relative to the housing, substantially about the longitudinal axis of the hollow rotor shaft; and (c) a drive system mounted within said housing connected to the hollow rotor shaft for causing rotation of the hollow rotor shaft relative to the housing, wherein the drive system includes a plurality of magnets mounted within the housing, located around the hollow rotor shaft, wherein the magnets create magnetic forces for causing the hollow rotor shaft to rotate relative to the housing; and (II) a pumping unit located below the power unit, wherein (i) the pumping unit includes a longitudinal hollow drive shaft that is in fluid communication with the hollow rotor shaft in the power unit, the hollow drive shaft being rotated substantially about its longitudinal axis when the hollow rotor shaft of the power unit is rotated; (ii) the hollow drive shaft of the pumping unit has a shaft inlet proximate to its bottom portion for allowing fluids being pumped to enter the hollow drive shaft; (iii) at least one impeller mounted to the exterior of the hollow drive shaft of the pumping unit such that when the hollow drive shaft is rotated, the impeller pumps fluids downwardly toward the inlet in the hollow drive shaft; and (iv) the pumping unit has fluid intakes proximate to a topmost portion thereof through which fluids being pumped enter the pumping unit; the fluid intakes, hollow drive shaft, shaft inlet and impellers being adapted such that when the hollow drive shaft is rotated about its longitudinal axis, fluids are pumped into the pumping unit through said fluid intakes, downwardly within the pumping unit to the shaft inlet, and then through the hollow drive shaft of the pumping unit and through the hollow rotor shaft of the power unit.","17.A method of removing a mixture of gaseous and liquid fluids from a well having at least one point where a mixture of gaseous and liquid fluids enter the well, comprising (I) positioning a pumping system in the well above the point or points where the mixture of gaseous and liquid fluids enter the well, wherein: (a) the pumping system includes at least one power unit, at least one pumping unit, and at least one intake through which fluids being pumped enter the pumping system; (b) the power unit includes a hollow rotor shaft in fluid communication said intake, and the pumping system is adapted such that fluids entering the intake are pumped through the hollow rotor shaft of the motor; (c) the power unit and pumping unit are smaller in diameter than the well; (d) the intake is located below at least a portion of the pumping system and is of smaller diameter than at least that portion of the pumping system above the intake; (e) the intake is adapted such that liquid fluids being pumped by the pumping system are caused to flow downwardly through a section of the intake to enter the pumping system; and (II) operating the pumping system such that (i) the gaseous fluids and the liquid fluids separate at a point proximate to the intake and below that portion of the pumping system above the intake, (ii) the gaseous fluids bypass the pumping system and rise to the surface of the well, and (iii) the liquid fluids being pumped flow upwardly to the intake, then downwardly through a section of the intake and then through the hollow rotor shaft of the motor and out of the well.","18.A method of pumping fluids with a submergible pumping system including a pump and an electrical motor, wherein the pumping system is submerged in production fluids such that said fluids flow across vortex generators adapted to impart streamwise vorticity to the production fluids as they pass in fluid contact with the exterior portions of the electrical motor.","19.The method of claim 18 wherein the vortex generators are attached to the exterior portions of the electric motor.","20.The method of claim 18 wherein the vortex generators are attached to the interior surface of a motor shroud."],["The present invention relates to methods for pumping fluids, primarily from wells.","Water, oil and natural gas are commonly produced in wells.","The wells are formed by drilling a hole into a rock formation to the depth where the fluid reservoir lies.","A well casing is inserted into the hole following the drilling.","If natural gas is present in the well, it is readily recovered from the well due to its natural buoyancy, while in most cases water and oil must be pumped to the surface.","Various types of pumping systems are used to produce fluids from wells.","Among these are sucker-rod/beam systems, plunger lift systems, continuous or intermittent gas lift systems, hydraulic reciprocating pumps, progressive cavity pumps and electrical submergible pumps (ESPs).","An ESP includes an electric motor that drives a submergible pump that forces fluids from the reservoir to the surface.","ESPs are often remotely operated in wells commonly at great depths, in harsh environmental conditions.","A challenge in using ESPs is effective heat removal.","Resistance in the electric motor windings generates a significant amount of waste heat in operation, as do mechanical friction and fluid friction.","If this waste heat is not sufficiently removed, the motor temperature can rise significantly.","Increasing the motor temperature leads to a number of problems.","Motor life becomes considerably shorter as temperatures increase.","Motor winding insulation, bearings, seals, and lubrication are all adversely affected by high temperature.","As a result, ESPs commonly are removed from wells more often than desired in order to replace or repair the electric motor.","In the oil market in particular, this results in high maintenance and repair costs as well as significant losses of revenue due to lost production.","Alternatively, to keep motor temperatures within reasonable limits, sometimes the production is reduced to a rate lower than desired, equipment is oversized or excess equipment is used, compromising the efficiency and profitability of the well.","Electrical efficiency also tends to decrease as the temperature rises and additional horsepower is necessary when excess equipment, like gas separators or additional seals, are used.","This results in increased energy costs, as more electrical power is needed to perform a given amount of work.","Sometimes this problem is exacerbated because of particular well conditions or operator choices.","Well conditions such as high gas content, high viscosity production fluids, the formation of emulsions, low flow rates, multiphase flows, steam flood production, deviated and horizontal wells, increasing well depth and other factors can contribute to high motor temperatures.","Some of these conditions make it difficult for a well operator to properly size and operate an ESP.","The production fluid is often used to remove some of the waste heat and provide some temperature control.","In conventional ESP installations, production fluids are drawn or pumped past the motor and remove some heat.","This effect is illustrated in FIGS.","1 and 1A, which represent conventional ESP and so-called “inverted ESP” pumping systems.","In FIG.","1, well 1 has casing 2 that has perforations 7 through which production fluids enter the well.","The ESP system includes pump 3, motor 4 and seal section 5.Production fluids enter the well at a point below or near the bottom of motor 4, pass by motor 4 in the direction indicated by arrows 8, and then enter pump 3 at pump intake 6.The production fluids are forced upwardly by pump 3, exiting at the top of pump 3 and traveling to the surface in the direction indicated by arrow 9.Cable 10 provides electrical power to motor 4 from the surface.","As shown, a packer 16 may be used in the assembly.","In the “inverted ESP” system shown in FIG.","1A, the relative positions of the pump and motor are reversed.","Well 11 has casing 12 and perforations 17 as before.","Production fluids enter pump intake 22 in the direction indicated by arrow 18.The production fluids are pumped upwardly through pump 13, exiting from outlet 21 and then flowing upwardly past seal section 15 and motor 14 in the direction indicated by arrow 19.As before, cable 20 provides electrical power from the surface.","A packer 16 is again illustrated in this assemblage.","In the systems shown in FIGS.","1 and 1A, the production fluids remove some heat as they flow past the motor.","However, the amount of heat that is removed is often insufficient to optimize motor performance and life.","As a result, well operators have attempted in various ways to improve motor performance and life.","One way of accomplishing this is to use special high temperature windings in the motor so that it can better tolerate higher temperatures.","Unfortunately, this does nothing to reduce heat in the bearings, seals, and lubrication, and significantly increases the cost of the motor.","Additionally, running at higher temperatures makes the equipment more prone to scale and corrosion and lowers electrical efficiency.","Another technique is to add a shroud around the motor to increase the fluid velocity.","Except in installations where fluid does not flow across the motor housing, shrouds will not significantly change the character of the flow (laminar or turbulent), are expensive and can be difficult or impossible to install in deviated and slim-hole wells.","Another attempt to improve production using ESPs is described in “Operating Electric Submersible Pumps Below Perforations, J. of Petrol.","Techn., p. 742 (July 1997) and by Sison in “Use of A Motor Cooling By-Pass System in An Electric Submersible Pump to Increase the Economic Life of Gas Wells”, Feb. 28, 2000.In these methods, an ESP system is installed in the well below the perforations.","Production fluids enter the well and the pump intake without passing in fluid contact with the motor.","Without convective cooling, a portion of the production fluids must be drawn off from the pump and recirculated down to the base of the motor and released to remove heat.","This typically requires that a second, recirculation pump be used together with special recirculation tubing, and thus is complex, expensive and less efficient than desired.","It is also difficult to effectively recirculate enough fluid to provide adequate heat removal.","Another approach is the so-called Framco ESP system, which also involves circulating a cooling fluid through the motor from the wellhead.","This is described in “The Framco ESP System: A New Approach to Downhole Electric Submersible Pumping”, presented by Jon A. Svaeren and Frank Mohn at the ESP Workshop, Houston, Tex., Apr.","30-May 2, 1990.Again, the complexity and expense of such a system makes it undesirable.","Another approach is described in U.S. Pat.","Nos.","5,951,262 and 6,000,915 to Hartman.","The Hartman patents describe a pump motor having a hollow rotor shaft that can drive a separate pumping unit.","The pumping unit is in fluid communication with the hollow rotor shaft, so that production fluids are pumped through the hollow rotor shaft of the motor, thereby providing cooling to the interior of the motor and allowing it to operate more efficiently.","Thus, an improved ESP system would be highly desirable, particularly one which enables more effective and efficient heat removal from the electrical motor particularly if this can be achieved without the expense of additional components or unnecessary features.","A further concern in many wells is the separation of liquids and gasses.","This problem is often seen in wells that produce both natural gas and oil.","Pumping a gas/liquid mixture creates inefficiencies that can be avoided if the gasses and liquids can be separated easily before the liquids enter the pump intakes.","It would be desirable to provide a method by which gaseous and liquid fluids can be easily and inexpensively separated before the liquids enter a pumping system.","In a first aspect, this invention is method of pumping a fluid with a submergible pumping system including a pump and an electrical motor, wherein the electrical motor has a hollow rotor shaft and said pumping system is adapted to pump at least a portion of said fluid through said hollow rotor shaft, and wherein at least a portion of said fluid passes in fluid contact with exterior portions of the electrical motor and remove heat from the electrical motor.","In a second aspect, this invention is a method of pumping fluids with a submergible pumping system including a pump and an electrical motor, wherein (a) the electrical motor has a hollow rotor shaft and said pumping system is adapted to pump said fluid through said hollow rotor shaft, and (b) the pumping system is submerged in said fluid such that production fluids entering the pumping system pass in fluid contact with exterior portions of the electrical motor and remove heat therefrom before entering the production fluid intakes, and said production fluids then pass through the hollow rotor shaft of the electrical motor as the fluid is pumped, and remove additional heat from the motor.","In a third aspect, this invention is a method for pumping fluids from a well having a casing that contains perforations through which production fluids enter the well, comprising (a) positioning an electrical submergible pumping system including an electric motor within said casing such that the electric motor is at or below at least some of said perforations, and (b) pumping production fluids from the well with said pumping system, wherein (i) the pumping system includes a pump, an electrical motor having a hollow rotor shaft and production fluid intakes located below said electrical motor, said pumping system being adapted to pump said production fluids through said hollow rotor shaft of the electrical motor, (ii) liquid production fluids entering the well through at least some of the perforations in the well casing come into fluid contact with exterior portions of the electrical motor and remove heat therefrom before entering the production fluid intakes, and (iii) liquid production fluids pass through the hollow rotor shaft of the electrical motor as the fluid travels toward the wellhead, and remove additional heat from the motor.","In a fourth aspect, this invention is a method comprising pumping a fluid with a submergible pumping system including a pump and an electrical motor, wherein (a) the electrical motor has a hollow rotor shaft and the pumping system has a fluid intake below the electrical motor in liquid communication with said hollow rotor shaft; (b) the pumping system has at least one outlet located at or below the electrical motor, said outlet being in liquid communication with the fluid intake; (c) the pumping system is submerged in a well having a well casing; (d) the cross-section of the electrical motor is such that the electrical motor fits within the well casing and a space is defined between the well casing and exterior of the electrical motor; and said pumping system is adapted so that during operation a portion of the fluid entering the fluid intake is pumped through the hollow rotor shaft, and a portion of the fluid entering the fluid intake is pumped through the outlet and upwardly in fluid contact with the of outside of the electrical motor in the space defined between the well casing and the exterior of the electrical motor.","In a fifth aspect, this invention is a method of pumping a fluid from a well having a wellhead and a well casing, using a submergible pumping system including a pump and an electrical motor, comprising (I) positioning a pumping system in a well with the pump being located between the electrical motor and the wellhead; wherein (a) electrical motor has a hollow rotor shaft which is in liquid communication with the pump, (b) the cross-section of the electrical motor is such that the electrical motor fits within the well casing and a space is defined between the well casing and the electrical motor; (c) the pumping system has a first fluid intake in liquid communication with said hollow rotor shaft; (d) the pumping system has a second fluid intake above the motor in liquid communication with the space defined between the well casing and the electrical motor, with the at least one outlet located at or below the electrical motor, said outlet being in liquid communication with the fluid intake; and (II) operating said pumping system under conditions such that during operation a portion of the fluids enter said first fluid intake and is pumped through the hollow rotor shaft of the electrical motor, and a second portion of the fluids are pumped through the space defined between the well casing and the electrical motor and in fluid contact with the exterior of the electrical motor to enter the second fluid intake, and said first and second portions of the fluids are then pumped to the wellhead.","The methods of these aspects of the invention provide several significant benefits.","Production fluids in laminar or turbulent flow that come into fluid contact with the exterior of the electrical motor provide heat removal.","In gassy wells, the increased flow rate made possible by operating the ESP below the perforations may foster turbulent flow in the annulus between the motor housing and the well casing, contributing to a significant increase in heat transfer.","An additional cooling effect is seen when the production fluids pass through the hollow rotor shaft; thus, the production fluids are used to remove heat twice in this invention, once as they pass outside of the motor and a second time as they pass through hollow rotor shaft.","The combined cooling effects help maintain the motor within optimal or desired temperature limits.","This prolongs motor life and reduces maintenance costs and other expenses attributable to premature motor failure.","It also allows an operator to increase the production by increasing the horsepower capability of existing equipment without raising the temperature.","The cooling effect can be further facilitated if the pump and/or hollow rotor shaft is designed so that the production fluids experience turbulent flow as they pass through the hollow rotor shaft.","The combined cooling effect of the well fluids passing the outside of the motor and through the hollow rotor shaft of the motor provides advantages such as prolonged motor life, increased horsepower density and/or greater electrical efficiency.","It often provides combinations of these benefits.","Surprisingly, these benefits are achieved despite the fact that the production fluids are forced through a reduced diameter area as they pass through the hollow rotor shaft of the motor.","Additional energy is needed to accomplish this, which normally would be expected to increase operating costs and contribute to additional temperature rises.","Unexpectedly, it has been found that this penalty is significantly offset by the benefits of additional heat removal that is achieved by flowing the production fluids in fluid contact with the exterior of the motor and through the motor's hollow rotor shaft.","In a sixth aspect, this invention is a method of pumping fluids with a submergible pumping system including a pump and an electrical motor, wherein the pumping system is submerged in said fluid such that production fluids entering the pumping system pass in fluid contact with exterior portions of the electrical motor and remove heat therefrom before entering the production fluid intakes, wherein the exterior portions of the electrical motor or the interior portions of a surrounding shroud include vortex generators adapted to impart streamwise vorticity to the production fluids as they pass in fluid contact with the exterior portions of the electrical motor.","This aspect of the invention provides an economical means for substantially increasing the efficiency of heat removal from the motor.","This aspect of the invention can be incorporated into the first and second aspects of the invention.","In a seventh aspect, this invention is a mechanism for providing motive force, the mechanism comprising (I) a power unit including: (a) a housing having an exterior wall; (b) a hollow rotor shaft having a longitudinal axis and opposite ends, the hollow rotor shaft being rotatably mounted within the housing for rotation of the hollow rotor shaft relative to the housing, substantially about the longitudinal axis of the hollow rotor shaft; and (c) a drive system mounted within said housing connected to the hollow rotor shaft for causing rotation of the hollow rotor shaft relative to the housing, wherein the drive system includes a plurality of magnets mounted within the housing, located around the hollow rotor shaft, wherein the magnets create magnetic forces for causing the hollow rotor shaft to rotate relative to the housing; and (II) a pumping unit located below the power unit, wherein (i) the pumping unit includes a longitudinal hollow drive shaft that is in fluid communication with the hollow rotor shaft in the power unit, the hollow drive shaft being rotated substantially about its longitudinal axis when the hollow rotor shaft is rotated; (ii) the hollow drive shaft has a shaft inlet proximate to its bottom portion for allowing fluids being pumped to enter the hollow drive shaft; (iii) at least one impeller mounted to the exterior of the hollow drive shaft such that when the hollow drive shaft is rotated, the impeller mounted on the hollow drive shaft pumps fluids downwardly toward the inlet in the hollow drive shaft; and (iv) the pumping unit has fluid intakes proximate to a topmost portion of the pumping unit, through which fluids being pumped enter the pumping unit; the fluid intakes, hollow drive shaft, shaft inlet and impellers being adapted such that when the hollow drive shaft is rotated about its longitudinal axis, fluids are pumped into the pumping unit through said fluid intakes, downwardly within the pumping unit to the shaft inlet of the hollow drive shaft, and then through the hollow drive shaft of the pumping unit and through the hollow rotor shaft of the power unit.","In an eighth aspect, this invention is a method of removing a mixture of gaseous and liquid fluids from a well having at least one point where a mixture of gaseous and liquid fluids enter the well comprising (I) positioning a pumping system in the well above the point or points where the mixture of gaseous and liquid fluids enter the well, wherein: (a) the pumping system includes at least one power unit, at least one pumping unit, and at least one intake through which fluids being pumped enter the pumping system; (b) the power unit includes an electric motor having a hollow rotor shaft in fluid communication said intake, and the pumping system is adapted such that fluids entering the intake are pumped through the hollow rotor shaft of the power unit; (c) the power unit and pumping unit are smaller in diameter than the well; (d) the intake is located below at least a portion of the pumping system and is of smaller diameter than at least that portion of the pumping system above the intake; (e) the intake is adapted such that liquid fluids being pumped by the pumping system are caused to flow downwardly through a section of the intake to enter the pumping system; and (II) operating the pumping system such that (i) the gaseous fluids and the liquid fluids separate at a point proximate to the intake and below that portion of the pumping system above the intake, (ii) the gaseous fluids bypass the pumping system and rise to the surface of the well, and (iii) the liquid fluids being pumped flow upwardly to the intake, then downwardly through a section of the intake and then through the hollow rotor shaft of the motor and subsequently out of the well.","FIG.","1 is a schematic view of two conventional ESP systems.","FIGS.","2 and 2A are cross-sectional views of embodiments of this invention.","FIGS.","3 and 3A represent a schematic view and detail of an embodiment of a motor having vortex generators for use in preferred aspects of the invention.","FIGS.","4-6 are cross-sectional views of embodiments of various aspects of the invention.","In this invention, a submergible pumping system is used to pump a fluid.","The system includes a pump and an electrical motor.","The electrical motor has a hollow rotor shaft and said pumping system is adapted to pump said fluid through said hollow rotor shaft.","External cooling is also provided by the production fluids.","In some embodiments, the pumping system is submerged in the fluid such that production fluids entering the pumping system pass exterior portions of the electrical motor and remove heat therefrom before entering the production fluid intakes, and said production fluids then pass through the hollow rotor shaft of the electrical motor as the fluid is pumped, and remove additional heat from the motor.","The conditions of the fluid flow are preferably such that the fluid flow is turbulent, has streamwise vortices, or both as the fluid passes the exterior portions of the electrical motor, and is turbulent as it passes through the hollow rotor shaft.","In other embodiments, the production fluids are split as they are pumped, with some being pumped through a hollow rotor shaft and the remainder being pumped past the exterior surface of the motor.","Typically, the pumping system is installed in a well, or else has a mechanical means for inducing the fluid to pass in fluid contact with exterior portions of the electrical motor.","An example of such a mechanical means is a shroud which partially covers the pumping system, and has an opening to the fluid being pumped at the opposite end of the pumping system from where the production fluid intakes are located.","This shroud permits the fluids to pass in fluid contact with the exterior portions of the electric motor and enter the production fluid intakes.","The spacing between the shroud and the exterior portions of the electrical motor is preferably such that the fluids exhibit turbulent flow as they pass in fluid contact with the exterior of the motor and do not impair installation in the well or create excessive head loss.","Alternatively, or in addition, vortex generators impart streamwise vortices to the production fluids as they flow through the spacing between the shroud and the exterior portions of the electrical motor.","In preferred embodiments of the invention, the pumping system is installed in a well.","The well may be vertical, horizontal, or a so-called “divert” type.","The well has a casing that has perforations through which production fluids enter the well.","The pumping system includes an electrical motor having a hollow rotor shaft.","The pumping system has production fluid intakes through which the production fluids enter.","These are located below said electrical motor (in the case of a horizontal well, opposite from the wellhead).","The pumping system is adapted to pump said production fluids through the hollow rotor shaft of the electrical motor and then on to the wellhead.","By “production fluids”, it is meant any fluids which are desired to be withdrawn from the well.","Examples of such fluids include water, oil, natural gas, and the like, with liquids being of particular interest and oil being of special interest.","By “perforations”, it is merely meant openings in a well casing through which the production fluids enter the well.","No particular configuration or method of forming these openings is critical to the invention.","The pumping system is located below at least some of the perforations in the casing.","Thus, production fluids entering those perforations that are above the pumping system flow in fluid contact past the exterior portions of the electrical motor and into the production fluid intakes, by force of gravity and/or the action of the pumping system.","In general, the pumping system should be below enough of the perforations that sufficient production fluids pass the exterior of the electrical motor to provide a cooling effect.","Preferably, at least half of the production fluids will pass by the exterior of the motor before entering the production fluid intakes.","More preferably, the pumping system is below substantially all of the perforations, and substantially the entire volume of production fluids passes in fluid contact with the exterior of the motor.","This tends to maximize the desired cooling effect.","Having the pumping system below substantially all of the perforations also facilitates separation of gaseous and liquid production fluids before the liquids enter the pumping system.","This will increase the production rate when the well produces a mixture of gasses and liquids.","Having the gasses circumvent the pump avoids various associated problems such as gas-lock or pumping inefficiencies that are attributable to entrained gasses.","As gasses have lower heat capacities than liquids, having the gasses circumvent the motor in this manner further improves heat transfer and, thus, motor cooling.","Additionally, because the pumping unit does not require oversized equipment or a gas separator as are typically used in conventional systems installed above the perforations, no significant additional power is consumed.","The pumping system includes an electrical motor and a submergible pump that is driven by the electrical motor.","The motor and pump may have a unitary structure (i.e., share a common housing), but it is often preferred that they are separate units which are connected together directly or indirectly as they are installed in the well.","Two embodiments of the process of the invention are illustrated in FIG.","2.In FIG.","2, well casing 210 extends along the periphery of well 201, which as shown has been bored through a producing subterranean rock stratum 440 and into a lower, non-producing subterranean rock stratum 441.Producing stratum 440 contains production fluids that are to be pumped to the well head.","Casing 210 includes perforations 211 through which the production fluids are admitted into the well for pumping to the surface.","Located within casing 210 is a pumping system that includes motor 220 and pump 230.Both motor 220 and pump 230 are located below perforations 211.Motor 220 is affixed to production pipe 250, which is shown in section.","If desired, production pipe 250 can take the form of a coiled tube.","Motor 220 is shown in section to reveal hollow rotor shaft 221, which rotates when motor 220 is operated to provide motive force to operate pump 230.The production fluids pass through the hollow rotor shaft on their way to the wellhead through production pipe 250.Hollow rotor shaft 221 is mechanically connected, directly or indirectly, to pump 230 in a manner such that pump 230 is operated when hollow rotor shaft 221 is rotated.","As shown, the drive shaft of pump 230 is directly connected to hollow rotor shaft 221, without intermediate apparatus.","However, it may be desirable to include various types of intermediate apparatus such as a sealing section between motor 220 and pump 230.The manner through which motor 220 is affixed to production pipe 250 and pump 230 is not critical; a variety of fasteners, interlocking devices and the like can be used.","Power is provided to the motor from the wellhead through a cable or similar device, which is not shown.","Intake 260 is attached to pump 230.When the pumping system is activated, hollow rotor shaft 221 rotates substantially along its longitudinal axis, driving the action of pump 230.Production fluids enter the pumping system through intake 260 and enter pump 230.Hollow rotor shaft 221 is in liquid communication with production pipe 250 and pump 230, so that fluids pumped upwardly by pump 230 pass through hollow rotor shaft 221 and then enter production pipe 250 through which they are delivered to the wellhead.","Because motor 220 is below perforations 211, production fluids that enter well 201 flow in fluid contact past the exterior of motor 220 before entering intake 260.This can be due to simple gravity, the pumping action of pump 230, or some combination of these.","Arrows 290 indicate the direction of flow of the production fluids.","As the production fluids must flow between well casing 210 and motor 220 as they travel toward the intake, the motor is somewhat smaller than the diameter of well casing 210, creating an annulus through which the production fluids can move.","As the production fluids move in fluid contact past the exterior portion of motor 220, they remove waste heat and thus provide cooling.","As the production fluids then are pumped through hollow rotor shaft 221 of motor 220, they remove additional waste heat and thus provide additional cooling.","The embodiment of FIG.","2 is a so-called “inverted” pumping system in which the motor is above the pump.","Although this is preferred to have the motor above the pump, it is not critical to the invention, and FIG.","2A illustrates an embodiment in which the motor is below the pump.","In FIG.","2A, well 201A has casing 210A.","The pumping system includes production pipe 250A, pump 230A, motor 220A and intake 260A.","Motor 220A includes hollow rotor shaft 221A through which production fluids pass.","As before, hollow rotor shaft 221A is in mechanical communication with pump 230A so that as motor 220A operates, hollow rotor shaft 221A causes pump 230A to operate and pump the production fluids through hollow rotor shaft 221A and then into production pipe 250A and up to the wellhead.","In the embodiment shown in FIG.","2A, production fluids enter the well through perforations 211A, travel past the exterior of motor 220A, enter intake 260A, travel upwardly through hollow rotor shaft 221A and into pump 230A and then through production pipe 250A to the wellhead.","Motor 220A is cooled twice by the production fluids; once as they pass in fluid contact with the exterior of motor 220A and again as they pass through hollow rotor shaft 221A in motor 220A).","In FIG.","4, a pumping system including electrical motor 402 and pump 411 is located in a well having casing 401 and perforations 415.Fluid intakes 420 are located below electrical motor 402 below packer 414.In the embodiment shown, fluid intakes 420 lead directly into pump 411, which is located below electrical motor 402.However, it is possible to employ multiple pumps and multiple motors in the submergible pumping system, provided that the intakes are located below at least one electrical motor with a hollow rotor shaft.","In the preferred embodiment shown, hollow rotor shaft 403 of electrical motor 402 rotates about its longitudinal axis when electrical motor 402 is operated.","This is conveniently achieved by affixing stationary magnets 404 to hollow rotor shaft 403 and supplying stators 405 to exterior housing 410 of electrical motor 402.Hollow rotor shaft 403 is coupled directly or indirectly to drive shaft 412 of pump 411, so that drive shaft 412 rotates when hollow rotor shaft 403 rotates, thereby supplying mechanical energy to pump 411.Impellers 413 are affixed to drive shaft 412 which provides motive force to impeller 413 when shaft 412 is rotated.","The embodiment shown in FIG.","4 includes optional features thrust bearing and seal 416 and packer 414.Thrust bearing and seal 416 connects electrical motor 402 with pump 411 and provides a seal against leakage of production fluids.","Packer 414 assists in supporting the weight of the pumping system and prevents pressurized fluids from the pump from flowing downwardly to the pump intake.","These components are well known in the art.","Intakes 420 are in fluid communication with hollow rotor shaft 403 of electrical motor 402.In the embodiment shown, fluids entering intakes 420 flow through pump 411 in the general direction indicated by arrows 408.A first portion of the fluids flow in the general direction indicated by arrows 406, entering hollow rotor shaft 403 through openings 421 and flowing upwardly through hollow rotor shaft 403 and up to the wellhead.","A second portion of the fluids flow in the general direction indicated by arrows 407, passing outwardly through pump housing 409 via openings 417, and then upwardly in a space between well casing 401 and exterior housing 410 of electrical motor 402.The embodiment shown in FIG.","4 permits the electrical motor to be cooled by the production fluids both internally (i.e., as the fluids pass through the hollow rotor shaft) and externally (ie., as the fluids pass between the exterior of the electrical motor and the well casing), without the need to use a shroud, fluid recirculation or locate the pumping system below the perforations in the well.","This avoids having to drill the well down below the perforations, i.e., no “rathole” is required to achieve the benefits of this aspect of the invention.","This embodiment also permits one to avoid exposing the pumping system to sandy conditions as often exist below the perforations.","In FIG.","5, a pumping system includes electrical motor 502 and pump 511.These are located in a well having casing 601 with perforations 515.As shown, perforations 615 are located above pump intakes 517, as is preferred, but the perforations may be located at the level of or below pump intakes 517.Electrical motor 502 contains hollow rotor shaft 503.In the preferred embodiment shown, motor 502 includes a drive system including magnets 504 located about hollow rotor shaft 503 and stators 505 which are located on the interior of housing 510.When operated, hollow rotor shaft 503 rotates about its longitudinal axis.","Pump 511 includes hollow drive shaft 512 that is in fluid communication with hollow rotor shaft 503 of motor 502.When hollow rotor shaft 503 rotates about its longitudinal axis as electrical motor 502 operates, hollow drive shaft 512 of pump 511 likewise rotates about its own longitudinal axis.","Hollow drive shaft 512 includes exterior (to the shaft) impellers 513 that pump the production fluids when the pumping system is operated.","Hollow drive shaft 512 includes shaft inlet 622, through which fluids enter as the pumping system is operated.","Pump 511 includes housing 509 having fluid intakes 517.Fluid intakes 517 are located above shaft inlet 522 and below perforations 515.In operation, production fluids enter the well through perforations 515, flow downwardly past the exterior of electrical motor housing 510 and into intakes 517.The fluids are then pumped downwardly in pump 511 by impellers 513, where they enter shaft inlet 522 and are pumped upwardly through hollow drive shaft 512 of pump 511 and then through hollow rotor shaft 503 of motor 502, all in the direction indicated by arrows 506.The embodiment shown in FIG.","5 includes preferred thrust bearing and seal 516.In this invention, the downward flow of the fluids through pump 511 before entering shaft inlet 522 reduces the downward force produced on pump 511.This in turn relieves the pump thrust carried by thrust bearing and seal 516, thereby prolonging its useful life and reducing the need for service and repair.","In addition, this aspect of the invention permits the electrical motor to be cooled both on its exterior and its interior by the production fluids.","In FIG.","6, a pumping system including pump 611 and electrical motor 602 is located in a well having casing 601 with perforations 615, where production fluids enter the well.","Perforations 615 are located below the pumping system.","The production fluids are a mixture of at least one liquid and at least one gas.","In this embodiment, intake 620 is located below the rest of the pumping system, although it is possible that one or more pumps or electrical motors can be located below the intakes.","Intake 620 has diameter D, which is smaller than the diameter D′ of electrical motor 602 (and, as shown, pump 611) which is immediately above intake 620.Electrical motor 609 includes hollow rotor shaft 603.In the preferred embodiment shown, motor 602 includes a drive system including magnets 604 located about hollow rotor shaft 603 and stators 605 which are located on the interior of housing 610.When operated, hollow rotor shaft 603 rotates about its longitudinal axis.","Hollow rotor shaft 603 is in fluid communication with intake 620.In the embodiment shown, motor 602 is directly above intake 620, and hollow rotor shaft 603 communicates directly with intake 620.However, one or more pumps can be located between motor 602 and intake 620, provided that hollow rotor shaft 603 is in fluid communication with intake 620 indirectly through the intervening pump.","In operation, a mixture of gaseous and liquid fluids enters the well through perforations 615.These move upwardly in the direction indicated by arrows 606 into vortex zones indicated in FIG.","6 by reference numerals 622.Vortex zones 622 are created in part by virtue of the diameter of intake 620 being smaller than that of electrical motor 602.In vortex zones 622, liquids and gasses separate efficiently.","Gasses move upwardly in the direction indicated by arrows 608 between casing 602 and the exterior housings 610 and 609 of electrical motor 602 and pump 611.Liquids enter intake 620, flow downwardly through a section of intake 620, then enter hollow rotor shaft 603, and then are pumped upwardly to the wellhead.","Arrows 607 in FIG.","6 indicate the direction of the liquids.","This last aspect of the invention is readily combined with, for example, the fifth aspect of the invention, so as to obtain the combined benefits of both.","In any of the embodiments of the invention, the pumping system may contain additional elements as may be necessary or desirable in any particular application.","For example, seal chamber sections are often provided in pumping systems for deep hole wells.","Such seal sections may also carry pump thrust and can perform additional functions as well, as described by Brookbank in “Inverted Pump Systems Design and Applications”, ESP Workshop, Houston, Apr.","26-28, 2000.As described by Brookbank, the functions performed by the seal chamber section can be divided among several pieces of apparatus if desired.","In this invention, a seal chamber section or other device for carrying pump thrust is preferably part of the pumping system.","In so-called “inverted” embodiments, it is preferred that some means of carrying pump thrust is included in the pump or motor design or as a separate piece of apparatus above or below the pump.","If desired, seal sections may be located above the pumping system, or between the motor and the pump.","Any apparatus situated between the motor and pump must be adapted so that motive force is transmitted through the apparatus from the motor to the pump, and so that production fluids travel through the hollow rotor shaft in the motor, as described above.","Similarly, apparatus such as sand skirts, packers, various types of connectors and the like can be incorporated into the pumping system or used in conjunction with the pumping system.","The pumping system may contain anti-cavitation devices like a primer pump to prevent cavitation of the fluid in the hollow rotor shaft or pump.","These may be especially useful in configurations where the pump is above the motor.","In producing deep bore wells, it is common practice to use a pumping system that is made up of separate components of a relatively short length.","This approach can be adopted in the process of this invention as well.","The motor, pump, intakes, seal sections and other apparatus may be constructed as two or more separate sections that are connected together to form the overall pumping system.","A wide variety of electrical motor designs can be used in the pumping system of the invention, provided that the motor contains a hollow rotor shaft through which the production fluids can flow to provide cooling and/or vortex generators in the annulus between the motor and the well casing or shroud.","Induction motors and brushless DC motors are useful, among others.","Suitable motors of that type are described in U.S. Pat.","Nos.","5,951,262 and 6,000,915, both to Hartman, both incorporated herein by reference in their entirety.","No special motor design is required, except that (1) it is adapted for submergible applications, (2) it contains the hollow rotor shaft as described herein and (3) it is of a size and shape to fit within the well casing and allow production fluids to pass in fluid contact with the exterior of the motor.","Conventional electric motors as are commonly used for downhole pumping applications can be used, if they are adapted to provide them with a hollow rotor shaft through which the production fluids can be pumped.","In its simplest case, the motor can be any submergible electric motor having a longitudinal, rotating rotor shaft, in which the rotor shaft has a longitudinal bore.","Conventional electric motors having a longitudinal rotor shaft in some cases can be retrofitted for use in this invention simply by boring out the rotor shaft to form the hollow rotor shaft.","The motor may be a single piece or a tandem configuration.","Multiple motors can be used and, if desired, pumps can be placed both above and below the motor.","Seal sections and other components may be installed between the motor and pump.","As the motor will be submerged in the production fluids, it is preferably adapted to operate in those conditions.","The motor preferably will contain a motor fluid that provides lubrication but more importantly retards the entrainment of production fluids in the motor.","The motor fluid may also contain various well-treating materials such as scale inhibitors, emulsifiers, anti-emulsion agents, surfactants, water, and the like.","Various types of seals can be incorporated into the motor to retard the leakage of production fluids into it, and as mentioned above, seal chamber sections can be used to accommodate thermal expansion of the motor fluid and help equalize pressure between the inside and outside of the motor.","In a preferred type of motor, the motor fluid is at a positive pressure relative to the hollow rotor shaft of the motor and has leaking seals, so that motor fluid slowly leaks from the motor into the production fluid.","A source of fresh motor fluid is provided, either from a reservoir in the pumping system or through a tube or capillary system from the wellhead.","By maintaining a positive motor fluid pressure, displacement of the motor fluid by the production fluids can be reduced or eliminated, and motor life can be prolonged.","A pressure independent modulating flow control valve, such as a SkoFlo™ or SubSea™ valve is suitable for maintaining a suitable positive pressure and flow of fresh lubrication fluid into the motor.","A particularly preferred motor has vortex generators on its exterior surface, or else is enclosed within a shroud that has vortex generators on its interior surface.","The vortex generators operate to generate streamwise vortices in the production fluids as they pass the exterior of the motor.","The vortex generators are preferably static devices having geometry and dimensions such that when the production fluids flow through and past the generators, a swirl in the flow is imparted.","These streamwise vortices greatly improve heat transfer from the exterior of the motor to the production fluids, even further increasing the benefits of this invention.","An example of a motor containing vortex generators is schematically illustrated in FIG.","3.Well 300 has casing 310 and perforations 311.The internal diameter of well 300 is D. Motor 320 and pump 330, each having a diameter d, are disposed in the well.","Motor 320 is affixed to production pipe 350, which extends to the wellhead.","Hollow rotor shaft 321 connects motor 320 and pump 330, in the same manner as described with respect to FIG.","2.Pump 330 includes intake 360, where production fluids enter the pumping system.","In the embodiment shown, production fluids enter the well at perforations 311, flow past the exterior of motor 320, into intake 360, through pump 330, through hollow rotor shaft 321 in motor 320 and up through production pipe 350 to the well head, as indicated by arrows 390.In the embodiment shown in FIG.","3, the exterior surface of motor 320 has vortex generators.","An alternative arrangement would be to surround motor 320 in a shroud that has vortex generators on its interior surface.","In the embodiment in FIGS.","3 and 3A, the vortex generators take the form of a plurality of fins 366.Fins 366 are slightly offset to the direction of flow of the production fluids, and adjacent fins 366 are offset in opposite directions.","Thus, each pair of fins 366 define a gap which narrows in the direction of flow.","The dimensions of fins 366 and the offset angle are sufficient to create streamwise vortices in the production fluids as they flow past the fins.","Suitable offset angles are typically +/−10-30, preferably 10-20 degrees from the direction of flow of the production fluids.","Suitable dimensions for fins 366 are illustrated in FIG.","3A.","In FIG.","3A, the gap between motor 320 and well casing 311 is defined by (D-d)/2, where D and d are as described above.","A suitable fin 366 will extend outwardly from the exterior of motor 320 (or inwardly from the exterior surface of a surrounding shroud) approximately 1/3-¾ of the width of the gap between the motor and well casing or, when a shroud is used, between the motor and interior surface of the shroud; in FIG.","3A an especially preferred dimension of ½ the gap width is illustrated.","In FIG.","3A, numeral 325 designates either the exterior surface of the motor or the internal surface of a surrounding shroud.","As shown in FIG.","3A, fin 366 is preferably beveled with increasing width in the direction of flow, reaching its maximum width at a point approximately 1/3-¾ down its length.","However, if desired, leading edge 399 can take other shapes, including those shown in outline in FIG.","3A.","Overall length is preferably about 1 to about 4, more preferably about 1.5 to about 3 times the width of the gap.","In FIG.","3A, the length is shown as equal to the gap width.","In the embodiment shown in FIG.","3, two rows of fins 366 are used.","A single row can be used, or greater than two rows can be used.","When multiple rows are used, a preferred spacing between the rows is about 10-30 times the gap distance.","However, the spacing may be adjusted to trade off pressure loss with heat transfer.","Other suitable vortex generator designs can be used, such as are described, for example, by Paulie and Eaton, Report #MD51, August 1988, “The Fluid Dynamics and Heat Transfer Effect of Streamwise Vortices Embedded in a Turbulent Boundary Layer”.","The pump itself has no special design requirements, other than it is adapted to pump production fluids through the hollow rotor shaft of the motor.","Generally, the particulars of the pump design will be selected to fit the particular application.","The pump is in liquid communication with the hollow rotor shaft of the motor.","This is accomplished by building the pump and motor as a single unit or incorporating the pump into the motor, as described in U.S. Pat.","Nos.","5,951,262 and 6,000,915, by designing the pump to mate with the hollow rotor shaft of the motor, or in some other manner.","Pumps of the type conventionally used in ESPs are entirely suitable, and can easily be adapted for use in this invention through the design of the connection between the pump outlet (or inlet) and the motor.","Progressive cavity pumps are also preferred types.","The pump may be one piece or in tandem sections.","Multiple pumps may be used.","If desired, separate pumps can be provided above and below the motor.","The method of this invention is useful in a variety of wells, including water, oil and natural gas wells.","The pumping method is particularly advantageous in wells where, using a conventional ESP, any flow of production fluids through the annulus between the motor housing and the well casing would be expected to be laminar.","Wells of this type include those having well conditions such as high viscosity production fluids, the formation of emulsions, low flow rates, multiphase flows, deviated and horizontal wells and large motor and/or well diameters, especially in the oil industry.","The well operator can take advantage of the enhanced heat transfer and improved cooling of the motor in several ways.","At equivalent power usage, the motor will be more efficiently cooled, and the operating temperature will be lower.","The operator may choose to take advantage of this lower operating temperature to prolong the motor life.","The lower temperature also tends to reduce electrical resistance, thus allowing equivalent work to be done with less power consumption.","Conversely, the operator may elect to increase the power to the motor so that it runs at higher temperatures similar to those that would be experienced in prior art processes.","In this case, the operator chooses to forego longer motor life in return for higher production rates that are achieved because of the additional power that is used.","In order to increase cooling efficiency as the production fluids flow through hollow rotor shaft of the motor, it is preferred to operate under conditions that produce turbulent flow within the hollow rotor shaft.","More preferably, there is turbulent flow both inside the hollow motor shaft, and turbulent flow or streamwise vortices in the annulus between the motor housing and the well casing where the production fluid passes in contact with the exterior of the motor.","Turbulent flow can be expressed in terms of Reynolds number (a dimensionless parameter), which is a function of the average fluid velocity, kinematic viscosity of the fluid and diameter of the hollow rotor shaft.","A Reynolds number of about 2300 or higher is typically indicative of turbulent flow.","Preferably, flow conditions of the production fluids through the hollow rotor shaft of the motor is such that the Reynolds number is at least about 3000.A Reynolds number in excess of 5000 to 10000 is more preferred.","Another parameter, Nusselt number, is a dimensionless measure of heat transfer.","The Nusselt number is a function of the Reynolds number, the Prantl number, and the absolute viscosity of the bulk fluid and fluid at the wall.","With turbulent flow, a high Nusselt number (exceeding 10) represents a high heat transfer rate for a given temperature difference.","The more turbulent the flow the higher the Reynolds and the Nusselt numbers.","With laminar flow, low Nusselt numbers (below 5) are indicative of poor convective heat transfer.","Enhanced heat transfer from the motor to the production fluid can be expected when the Nusselt number is at least 10, preferably at least 50, and the method of the invention is preferably operated under conditions that achieve such Nusselt numbers.","Operating conditions preferably are also chosen so as to provide a Brinkmann number (another dimensionless parameter) of less than 2, preferably less than 0.5.The Brinkmann number indicates the direction of heat transfer within a viscous fluid.","It is a function of the average velocity of the fluid, its absolute viscosity, the thermal conductivity of the fluid and the temperature difference between the fluid and the inside wall of the hollow rotor shaft.","When conditions are such that the Brinkmann number is less than 2, heat will travel from the hot motor to the cooler production fluid and fluid friction will not add additional heat to the motor.","Viscous dissipation effects are negligible, when the Brinkmann number is less than 0.5.Since the hollow rotor shaft is the drive shaft of the motor, operating conditions can be further described with reference to a Rossby number (still another dimensionless parameter).","The Rossby number provides an indication as to whether spinning flow will or will not dominate axial flow in the hollow rotor shaft.","Conditions generating a Rossby number of at least about 0.5, preferably at least about 1.0 are preferred.","The Rossby number is a function of average fluid velocity, fluid angular velocity and the inside diameter of the hollow rotor shaft.","Yet another advantage of this invention is that it permits gaseous production fluids to separate from liquids before entering the pumping system.","Gasses entering the well through perforations above the pumping system will tend to travel directly upward to the wellhead without passing through the pumping system, due to the natural buoyancy of the gasses.","Liquids will flow downwardly and enter the pumping system, without significant entrained gas, for delivery to the wellhead.","Thus, a ready separation of production gasses and production liquids is made.","Because the pumping system will process less gas, pumping problems and inefficiencies associated with pumping mixtures of gasses and liquids are largely mitigated.","Moreover, by eliminating the need for oversized equipment or a gas separator in a typical system installed above the perforations, no additional failure modes or power losses are introduced."]],"string":"[\n [\n \"Method for pumping fluids\",\n \"The production rate, useful life and operating efficiency of electric submergible pumping systems (ESP) is improved by operating the system so that the motor (220, 320) is cooled by two passes of production.\",\n \"Production fluids remove waste heat from the motor (220, 320) by passing the fluid in contact with the exterior of the motor (220, 320).\",\n \"The improved cooling permits the motor to be operated at a lower temperature improving life and efficiency, and/or to be operated at higher power at a similar temperature.\",\n \"Additionally, oversized and excess equipment is not required, further improving performance and economics for the user.\",\n \"In another aspect, the invention is a method of pumping fluids using a motor having vortex generators (366) on its exterior surfaces or on the interior surfaces of a surrounding shroud.\"\n ],\n [\n \"1.A method comprising pumping a fluid with a submergible pumping system including a pump and an electrical motor, wherein the electrical motor has a hollow rotor shaft and said pumping system is adapted to pump at least a portion of said fluid through said hollow rotor shaft, and wherein during operation at least a portion of said fluid passes in fluid contact with exterior portions of the electrical motor and removes heat therefrom.\",\n \"2.The method of claim 1, wherein the pumping system is submerged in said fluid such that production fluids entering the pumping system pass in fluid contact with exterior portions of the electrical motor and remove heat therefrom before entering the pumping system, and said liquid production fluids then pass through the hollow rotor shaft of the electrical motor as the fluid is pumped, and remove additional heat from the motor.\",\n \"3.A method for producing fluids from a well having a casing that contains perforations through which production fluids enter the well, comprising (a) positioning an electrical submergible pumping system including an electric motor within said casing such that the electric motor is at or below at least some of said perforations, and (b) pumping production fluids from the well with said pumping system, wherein (1) the pumping system includes a pump, an electrical motor having a hollow rotor shaft and production fluid intakes located below said electrical motor, said pumping system being adapted to pump said production fluids through said hollow rotor shaft of the electrical motor, (2) production fluids entering the well through at least some of the perforations in the casing pass in fluid contact with exterior portions of the electrical motor and remove heat therefrom before entering the production fluid intakes, and (3) production fluids pass through the hollow rotor shaft of the electrical motor as the fluid travels to the wellhead, and remove additional heat from the motor.\",\n \"4.The method of claim 3 wherein the production fluids pass through the hollow rotor shaft of the electrical motor under conditions of turbulent flow.\",\n \"5.The method of claim 4 wherein the electrical motor is located between the wellhead and the pump.\",\n \"6.The method of claim 5 wherein production fluids entering the well through the perforations in the casing travel downward in fluid contact with exterior portions of the electrical motor to an intake, and the liquid production fluids are subsequently pumped upwardly through the hollow rotor shaft of the electrical motor and then to the wellhead.\",\n \"7.The method of claim 6, wherein the flow of production fluid through the hollow rotor shaft is characterized by a Reynolds number of at least 2300.8.The method of claim 7, wherein the motor contains a motor fluid at a positive pressure to the exterior of the motor, and has seals which allow motor fluid to leak into the production fluid.\",\n \"9.The method of claim 7, wherein conditions are selected such that heat transfer from at least one of the hollow rotor shaft and the exterior surfaces of the motor to the production fluid is characterized by a Nusselt number of at least 10.10.The method of claim 7 wherein conditions are selected so as to generate a Brinkmann number of less than 2.11.The method of claim 7 wherein conditions are selected so as to generate a Rossby number of more than 0.5.12.The method of claim 4 wherein the pump is located between the wellhead and the electric motor.\",\n \"13.The method of claim 4 wherein the electrical submergible pumping system contains two pumps, one of which is located above the electric motor and one of which is located below the electric motor.\",\n \"14.The method of claim 1 wherein (a) the electrical motor has a hollow rotor shaft and the pumping system has a fluid intake below the electrical motor and in liquid communication with said hollow rotor shaft; (b) the pumping system has at least one outlet located at or below the electrical motor, said outlet being in liquid communication with the fluid intake, (c) the pumping system is submerged in a well having a well casing; (d) the cross-section that the electrical motor is such that the electrical motor fits within the well casing and a space is defined between the well casing and the electrical motor; and (e) said pumping system is adapted so that during operation a portion of the fluid entering the fluid intake is pumped through the hollow rotor shaft of the electrical motor, and a portion of the fluid entering the fluid intake is pumped through the outlet and upwardly in fluid contact with the outside of the electrical motor.\",\n \"15.A method of pumping fluids, comprising (I) positioning a pumping system in a well with the pump being located between the electrical motor and the wellhead; wherein (a) electrical motor has a hollow rotor shaft which is in liquid communication with the pump, (e) the cross-section of the electrical motor is such that the electrical motor fits within the well casing and a space is defined between the well casing and the electrical motor; (f) the pumping system has a first fluid intake in liquid communication with said hollow rotor shaft; (g) the pumping system has a second fluid intake above the motor in liquid communication with the space defined between the well casing and the electrical motor, with the at least one outlet located at or below the electrical motor, said outlet being in liquid communication with the fluid intake; and (II) operating said pumping system under conditions such that during operation a portion of the fluids enter said first fluid intake and is pumped through the hollow rotor shaft of the electrical motor, and a second portion of the fluids are pumped through the space defined between the well casing and the electrical motor and in fluid contact with the exterior of the electrical motor to enter the second fluid intake, and said first and second portions of the fluids are then pumped to the wellhead.\",\n \"16.A mechanism for providing motive force, the mechanism comprising (I) a power unit including: (a) a housing having an exterior wall; (b) a hollow rotor shaft having a longitudinal axis and opposite ends, the hollow rotor shaft being rotatably mounted within the housing for rotation of the hollow rotor shaft relative to the housing, substantially about the longitudinal axis of the hollow rotor shaft; and (c) a drive system mounted within said housing connected to the hollow rotor shaft for causing rotation of the hollow rotor shaft relative to the housing, wherein the drive system includes a plurality of magnets mounted within the housing, located around the hollow rotor shaft, wherein the magnets create magnetic forces for causing the hollow rotor shaft to rotate relative to the housing; and (II) a pumping unit located below the power unit, wherein (i) the pumping unit includes a longitudinal hollow drive shaft that is in fluid communication with the hollow rotor shaft in the power unit, the hollow drive shaft being rotated substantially about its longitudinal axis when the hollow rotor shaft of the power unit is rotated; (ii) the hollow drive shaft of the pumping unit has a shaft inlet proximate to its bottom portion for allowing fluids being pumped to enter the hollow drive shaft; (iii) at least one impeller mounted to the exterior of the hollow drive shaft of the pumping unit such that when the hollow drive shaft is rotated, the impeller pumps fluids downwardly toward the inlet in the hollow drive shaft; and (iv) the pumping unit has fluid intakes proximate to a topmost portion thereof through which fluids being pumped enter the pumping unit; the fluid intakes, hollow drive shaft, shaft inlet and impellers being adapted such that when the hollow drive shaft is rotated about its longitudinal axis, fluids are pumped into the pumping unit through said fluid intakes, downwardly within the pumping unit to the shaft inlet, and then through the hollow drive shaft of the pumping unit and through the hollow rotor shaft of the power unit.\",\n \"17.A method of removing a mixture of gaseous and liquid fluids from a well having at least one point where a mixture of gaseous and liquid fluids enter the well, comprising (I) positioning a pumping system in the well above the point or points where the mixture of gaseous and liquid fluids enter the well, wherein: (a) the pumping system includes at least one power unit, at least one pumping unit, and at least one intake through which fluids being pumped enter the pumping system; (b) the power unit includes a hollow rotor shaft in fluid communication said intake, and the pumping system is adapted such that fluids entering the intake are pumped through the hollow rotor shaft of the motor; (c) the power unit and pumping unit are smaller in diameter than the well; (d) the intake is located below at least a portion of the pumping system and is of smaller diameter than at least that portion of the pumping system above the intake; (e) the intake is adapted such that liquid fluids being pumped by the pumping system are caused to flow downwardly through a section of the intake to enter the pumping system; and (II) operating the pumping system such that (i) the gaseous fluids and the liquid fluids separate at a point proximate to the intake and below that portion of the pumping system above the intake, (ii) the gaseous fluids bypass the pumping system and rise to the surface of the well, and (iii) the liquid fluids being pumped flow upwardly to the intake, then downwardly through a section of the intake and then through the hollow rotor shaft of the motor and out of the well.\",\n \"18.A method of pumping fluids with a submergible pumping system including a pump and an electrical motor, wherein the pumping system is submerged in production fluids such that said fluids flow across vortex generators adapted to impart streamwise vorticity to the production fluids as they pass in fluid contact with the exterior portions of the electrical motor.\",\n \"19.The method of claim 18 wherein the vortex generators are attached to the exterior portions of the electric motor.\",\n \"20.The method of claim 18 wherein the vortex generators are attached to the interior surface of a motor shroud.\"\n ],\n [\n \"The present invention relates to methods for pumping fluids, primarily from wells.\",\n \"Water, oil and natural gas are commonly produced in wells.\",\n \"The wells are formed by drilling a hole into a rock formation to the depth where the fluid reservoir lies.\",\n \"A well casing is inserted into the hole following the drilling.\",\n \"If natural gas is present in the well, it is readily recovered from the well due to its natural buoyancy, while in most cases water and oil must be pumped to the surface.\",\n \"Various types of pumping systems are used to produce fluids from wells.\",\n \"Among these are sucker-rod/beam systems, plunger lift systems, continuous or intermittent gas lift systems, hydraulic reciprocating pumps, progressive cavity pumps and electrical submergible pumps (ESPs).\",\n \"An ESP includes an electric motor that drives a submergible pump that forces fluids from the reservoir to the surface.\",\n \"ESPs are often remotely operated in wells commonly at great depths, in harsh environmental conditions.\",\n \"A challenge in using ESPs is effective heat removal.\",\n \"Resistance in the electric motor windings generates a significant amount of waste heat in operation, as do mechanical friction and fluid friction.\",\n \"If this waste heat is not sufficiently removed, the motor temperature can rise significantly.\",\n \"Increasing the motor temperature leads to a number of problems.\",\n \"Motor life becomes considerably shorter as temperatures increase.\",\n \"Motor winding insulation, bearings, seals, and lubrication are all adversely affected by high temperature.\",\n \"As a result, ESPs commonly are removed from wells more often than desired in order to replace or repair the electric motor.\",\n \"In the oil market in particular, this results in high maintenance and repair costs as well as significant losses of revenue due to lost production.\",\n \"Alternatively, to keep motor temperatures within reasonable limits, sometimes the production is reduced to a rate lower than desired, equipment is oversized or excess equipment is used, compromising the efficiency and profitability of the well.\",\n \"Electrical efficiency also tends to decrease as the temperature rises and additional horsepower is necessary when excess equipment, like gas separators or additional seals, are used.\",\n \"This results in increased energy costs, as more electrical power is needed to perform a given amount of work.\",\n \"Sometimes this problem is exacerbated because of particular well conditions or operator choices.\",\n \"Well conditions such as high gas content, high viscosity production fluids, the formation of emulsions, low flow rates, multiphase flows, steam flood production, deviated and horizontal wells, increasing well depth and other factors can contribute to high motor temperatures.\",\n \"Some of these conditions make it difficult for a well operator to properly size and operate an ESP.\",\n \"The production fluid is often used to remove some of the waste heat and provide some temperature control.\",\n \"In conventional ESP installations, production fluids are drawn or pumped past the motor and remove some heat.\",\n \"This effect is illustrated in FIGS.\",\n \"1 and 1A, which represent conventional ESP and so-called “inverted ESP” pumping systems.\",\n \"In FIG.\",\n \"1, well 1 has casing 2 that has perforations 7 through which production fluids enter the well.\",\n \"The ESP system includes pump 3, motor 4 and seal section 5.Production fluids enter the well at a point below or near the bottom of motor 4, pass by motor 4 in the direction indicated by arrows 8, and then enter pump 3 at pump intake 6.The production fluids are forced upwardly by pump 3, exiting at the top of pump 3 and traveling to the surface in the direction indicated by arrow 9.Cable 10 provides electrical power to motor 4 from the surface.\",\n \"As shown, a packer 16 may be used in the assembly.\",\n \"In the “inverted ESP” system shown in FIG.\",\n \"1A, the relative positions of the pump and motor are reversed.\",\n \"Well 11 has casing 12 and perforations 17 as before.\",\n \"Production fluids enter pump intake 22 in the direction indicated by arrow 18.The production fluids are pumped upwardly through pump 13, exiting from outlet 21 and then flowing upwardly past seal section 15 and motor 14 in the direction indicated by arrow 19.As before, cable 20 provides electrical power from the surface.\",\n \"A packer 16 is again illustrated in this assemblage.\",\n \"In the systems shown in FIGS.\",\n \"1 and 1A, the production fluids remove some heat as they flow past the motor.\",\n \"However, the amount of heat that is removed is often insufficient to optimize motor performance and life.\",\n \"As a result, well operators have attempted in various ways to improve motor performance and life.\",\n \"One way of accomplishing this is to use special high temperature windings in the motor so that it can better tolerate higher temperatures.\",\n \"Unfortunately, this does nothing to reduce heat in the bearings, seals, and lubrication, and significantly increases the cost of the motor.\",\n \"Additionally, running at higher temperatures makes the equipment more prone to scale and corrosion and lowers electrical efficiency.\",\n \"Another technique is to add a shroud around the motor to increase the fluid velocity.\",\n \"Except in installations where fluid does not flow across the motor housing, shrouds will not significantly change the character of the flow (laminar or turbulent), are expensive and can be difficult or impossible to install in deviated and slim-hole wells.\",\n \"Another attempt to improve production using ESPs is described in “Operating Electric Submersible Pumps Below Perforations, J. of Petrol.\",\n \"Techn., p. 742 (July 1997) and by Sison in “Use of A Motor Cooling By-Pass System in An Electric Submersible Pump to Increase the Economic Life of Gas Wells”, Feb. 28, 2000.In these methods, an ESP system is installed in the well below the perforations.\",\n \"Production fluids enter the well and the pump intake without passing in fluid contact with the motor.\",\n \"Without convective cooling, a portion of the production fluids must be drawn off from the pump and recirculated down to the base of the motor and released to remove heat.\",\n \"This typically requires that a second, recirculation pump be used together with special recirculation tubing, and thus is complex, expensive and less efficient than desired.\",\n \"It is also difficult to effectively recirculate enough fluid to provide adequate heat removal.\",\n \"Another approach is the so-called Framco ESP system, which also involves circulating a cooling fluid through the motor from the wellhead.\",\n \"This is described in “The Framco ESP System: A New Approach to Downhole Electric Submersible Pumping”, presented by Jon A. Svaeren and Frank Mohn at the ESP Workshop, Houston, Tex., Apr.\",\n \"30-May 2, 1990.Again, the complexity and expense of such a system makes it undesirable.\",\n \"Another approach is described in U.S. Pat.\",\n \"Nos.\",\n \"5,951,262 and 6,000,915 to Hartman.\",\n \"The Hartman patents describe a pump motor having a hollow rotor shaft that can drive a separate pumping unit.\",\n \"The pumping unit is in fluid communication with the hollow rotor shaft, so that production fluids are pumped through the hollow rotor shaft of the motor, thereby providing cooling to the interior of the motor and allowing it to operate more efficiently.\",\n \"Thus, an improved ESP system would be highly desirable, particularly one which enables more effective and efficient heat removal from the electrical motor particularly if this can be achieved without the expense of additional components or unnecessary features.\",\n \"A further concern in many wells is the separation of liquids and gasses.\",\n \"This problem is often seen in wells that produce both natural gas and oil.\",\n \"Pumping a gas/liquid mixture creates inefficiencies that can be avoided if the gasses and liquids can be separated easily before the liquids enter the pump intakes.\",\n \"It would be desirable to provide a method by which gaseous and liquid fluids can be easily and inexpensively separated before the liquids enter a pumping system.\",\n \"In a first aspect, this invention is method of pumping a fluid with a submergible pumping system including a pump and an electrical motor, wherein the electrical motor has a hollow rotor shaft and said pumping system is adapted to pump at least a portion of said fluid through said hollow rotor shaft, and wherein at least a portion of said fluid passes in fluid contact with exterior portions of the electrical motor and remove heat from the electrical motor.\",\n \"In a second aspect, this invention is a method of pumping fluids with a submergible pumping system including a pump and an electrical motor, wherein (a) the electrical motor has a hollow rotor shaft and said pumping system is adapted to pump said fluid through said hollow rotor shaft, and (b) the pumping system is submerged in said fluid such that production fluids entering the pumping system pass in fluid contact with exterior portions of the electrical motor and remove heat therefrom before entering the production fluid intakes, and said production fluids then pass through the hollow rotor shaft of the electrical motor as the fluid is pumped, and remove additional heat from the motor.\",\n \"In a third aspect, this invention is a method for pumping fluids from a well having a casing that contains perforations through which production fluids enter the well, comprising (a) positioning an electrical submergible pumping system including an electric motor within said casing such that the electric motor is at or below at least some of said perforations, and (b) pumping production fluids from the well with said pumping system, wherein (i) the pumping system includes a pump, an electrical motor having a hollow rotor shaft and production fluid intakes located below said electrical motor, said pumping system being adapted to pump said production fluids through said hollow rotor shaft of the electrical motor, (ii) liquid production fluids entering the well through at least some of the perforations in the well casing come into fluid contact with exterior portions of the electrical motor and remove heat therefrom before entering the production fluid intakes, and (iii) liquid production fluids pass through the hollow rotor shaft of the electrical motor as the fluid travels toward the wellhead, and remove additional heat from the motor.\",\n \"In a fourth aspect, this invention is a method comprising pumping a fluid with a submergible pumping system including a pump and an electrical motor, wherein (a) the electrical motor has a hollow rotor shaft and the pumping system has a fluid intake below the electrical motor in liquid communication with said hollow rotor shaft; (b) the pumping system has at least one outlet located at or below the electrical motor, said outlet being in liquid communication with the fluid intake; (c) the pumping system is submerged in a well having a well casing; (d) the cross-section of the electrical motor is such that the electrical motor fits within the well casing and a space is defined between the well casing and exterior of the electrical motor; and said pumping system is adapted so that during operation a portion of the fluid entering the fluid intake is pumped through the hollow rotor shaft, and a portion of the fluid entering the fluid intake is pumped through the outlet and upwardly in fluid contact with the of outside of the electrical motor in the space defined between the well casing and the exterior of the electrical motor.\",\n \"In a fifth aspect, this invention is a method of pumping a fluid from a well having a wellhead and a well casing, using a submergible pumping system including a pump and an electrical motor, comprising (I) positioning a pumping system in a well with the pump being located between the electrical motor and the wellhead; wherein (a) electrical motor has a hollow rotor shaft which is in liquid communication with the pump, (b) the cross-section of the electrical motor is such that the electrical motor fits within the well casing and a space is defined between the well casing and the electrical motor; (c) the pumping system has a first fluid intake in liquid communication with said hollow rotor shaft; (d) the pumping system has a second fluid intake above the motor in liquid communication with the space defined between the well casing and the electrical motor, with the at least one outlet located at or below the electrical motor, said outlet being in liquid communication with the fluid intake; and (II) operating said pumping system under conditions such that during operation a portion of the fluids enter said first fluid intake and is pumped through the hollow rotor shaft of the electrical motor, and a second portion of the fluids are pumped through the space defined between the well casing and the electrical motor and in fluid contact with the exterior of the electrical motor to enter the second fluid intake, and said first and second portions of the fluids are then pumped to the wellhead.\",\n \"The methods of these aspects of the invention provide several significant benefits.\",\n \"Production fluids in laminar or turbulent flow that come into fluid contact with the exterior of the electrical motor provide heat removal.\",\n \"In gassy wells, the increased flow rate made possible by operating the ESP below the perforations may foster turbulent flow in the annulus between the motor housing and the well casing, contributing to a significant increase in heat transfer.\",\n \"An additional cooling effect is seen when the production fluids pass through the hollow rotor shaft; thus, the production fluids are used to remove heat twice in this invention, once as they pass outside of the motor and a second time as they pass through hollow rotor shaft.\",\n \"The combined cooling effects help maintain the motor within optimal or desired temperature limits.\",\n \"This prolongs motor life and reduces maintenance costs and other expenses attributable to premature motor failure.\",\n \"It also allows an operator to increase the production by increasing the horsepower capability of existing equipment without raising the temperature.\",\n \"The cooling effect can be further facilitated if the pump and/or hollow rotor shaft is designed so that the production fluids experience turbulent flow as they pass through the hollow rotor shaft.\",\n \"The combined cooling effect of the well fluids passing the outside of the motor and through the hollow rotor shaft of the motor provides advantages such as prolonged motor life, increased horsepower density and/or greater electrical efficiency.\",\n \"It often provides combinations of these benefits.\",\n \"Surprisingly, these benefits are achieved despite the fact that the production fluids are forced through a reduced diameter area as they pass through the hollow rotor shaft of the motor.\",\n \"Additional energy is needed to accomplish this, which normally would be expected to increase operating costs and contribute to additional temperature rises.\",\n \"Unexpectedly, it has been found that this penalty is significantly offset by the benefits of additional heat removal that is achieved by flowing the production fluids in fluid contact with the exterior of the motor and through the motor's hollow rotor shaft.\",\n \"In a sixth aspect, this invention is a method of pumping fluids with a submergible pumping system including a pump and an electrical motor, wherein the pumping system is submerged in said fluid such that production fluids entering the pumping system pass in fluid contact with exterior portions of the electrical motor and remove heat therefrom before entering the production fluid intakes, wherein the exterior portions of the electrical motor or the interior portions of a surrounding shroud include vortex generators adapted to impart streamwise vorticity to the production fluids as they pass in fluid contact with the exterior portions of the electrical motor.\",\n \"This aspect of the invention provides an economical means for substantially increasing the efficiency of heat removal from the motor.\",\n \"This aspect of the invention can be incorporated into the first and second aspects of the invention.\",\n \"In a seventh aspect, this invention is a mechanism for providing motive force, the mechanism comprising (I) a power unit including: (a) a housing having an exterior wall; (b) a hollow rotor shaft having a longitudinal axis and opposite ends, the hollow rotor shaft being rotatably mounted within the housing for rotation of the hollow rotor shaft relative to the housing, substantially about the longitudinal axis of the hollow rotor shaft; and (c) a drive system mounted within said housing connected to the hollow rotor shaft for causing rotation of the hollow rotor shaft relative to the housing, wherein the drive system includes a plurality of magnets mounted within the housing, located around the hollow rotor shaft, wherein the magnets create magnetic forces for causing the hollow rotor shaft to rotate relative to the housing; and (II) a pumping unit located below the power unit, wherein (i) the pumping unit includes a longitudinal hollow drive shaft that is in fluid communication with the hollow rotor shaft in the power unit, the hollow drive shaft being rotated substantially about its longitudinal axis when the hollow rotor shaft is rotated; (ii) the hollow drive shaft has a shaft inlet proximate to its bottom portion for allowing fluids being pumped to enter the hollow drive shaft; (iii) at least one impeller mounted to the exterior of the hollow drive shaft such that when the hollow drive shaft is rotated, the impeller mounted on the hollow drive shaft pumps fluids downwardly toward the inlet in the hollow drive shaft; and (iv) the pumping unit has fluid intakes proximate to a topmost portion of the pumping unit, through which fluids being pumped enter the pumping unit; the fluid intakes, hollow drive shaft, shaft inlet and impellers being adapted such that when the hollow drive shaft is rotated about its longitudinal axis, fluids are pumped into the pumping unit through said fluid intakes, downwardly within the pumping unit to the shaft inlet of the hollow drive shaft, and then through the hollow drive shaft of the pumping unit and through the hollow rotor shaft of the power unit.\",\n \"In an eighth aspect, this invention is a method of removing a mixture of gaseous and liquid fluids from a well having at least one point where a mixture of gaseous and liquid fluids enter the well comprising (I) positioning a pumping system in the well above the point or points where the mixture of gaseous and liquid fluids enter the well, wherein: (a) the pumping system includes at least one power unit, at least one pumping unit, and at least one intake through which fluids being pumped enter the pumping system; (b) the power unit includes an electric motor having a hollow rotor shaft in fluid communication said intake, and the pumping system is adapted such that fluids entering the intake are pumped through the hollow rotor shaft of the power unit; (c) the power unit and pumping unit are smaller in diameter than the well; (d) the intake is located below at least a portion of the pumping system and is of smaller diameter than at least that portion of the pumping system above the intake; (e) the intake is adapted such that liquid fluids being pumped by the pumping system are caused to flow downwardly through a section of the intake to enter the pumping system; and (II) operating the pumping system such that (i) the gaseous fluids and the liquid fluids separate at a point proximate to the intake and below that portion of the pumping system above the intake, (ii) the gaseous fluids bypass the pumping system and rise to the surface of the well, and (iii) the liquid fluids being pumped flow upwardly to the intake, then downwardly through a section of the intake and then through the hollow rotor shaft of the motor and subsequently out of the well.\",\n \"FIG.\",\n \"1 is a schematic view of two conventional ESP systems.\",\n \"FIGS.\",\n \"2 and 2A are cross-sectional views of embodiments of this invention.\",\n \"FIGS.\",\n \"3 and 3A represent a schematic view and detail of an embodiment of a motor having vortex generators for use in preferred aspects of the invention.\",\n \"FIGS.\",\n \"4-6 are cross-sectional views of embodiments of various aspects of the invention.\",\n \"In this invention, a submergible pumping system is used to pump a fluid.\",\n \"The system includes a pump and an electrical motor.\",\n \"The electrical motor has a hollow rotor shaft and said pumping system is adapted to pump said fluid through said hollow rotor shaft.\",\n \"External cooling is also provided by the production fluids.\",\n \"In some embodiments, the pumping system is submerged in the fluid such that production fluids entering the pumping system pass exterior portions of the electrical motor and remove heat therefrom before entering the production fluid intakes, and said production fluids then pass through the hollow rotor shaft of the electrical motor as the fluid is pumped, and remove additional heat from the motor.\",\n \"The conditions of the fluid flow are preferably such that the fluid flow is turbulent, has streamwise vortices, or both as the fluid passes the exterior portions of the electrical motor, and is turbulent as it passes through the hollow rotor shaft.\",\n \"In other embodiments, the production fluids are split as they are pumped, with some being pumped through a hollow rotor shaft and the remainder being pumped past the exterior surface of the motor.\",\n \"Typically, the pumping system is installed in a well, or else has a mechanical means for inducing the fluid to pass in fluid contact with exterior portions of the electrical motor.\",\n \"An example of such a mechanical means is a shroud which partially covers the pumping system, and has an opening to the fluid being pumped at the opposite end of the pumping system from where the production fluid intakes are located.\",\n \"This shroud permits the fluids to pass in fluid contact with the exterior portions of the electric motor and enter the production fluid intakes.\",\n \"The spacing between the shroud and the exterior portions of the electrical motor is preferably such that the fluids exhibit turbulent flow as they pass in fluid contact with the exterior of the motor and do not impair installation in the well or create excessive head loss.\",\n \"Alternatively, or in addition, vortex generators impart streamwise vortices to the production fluids as they flow through the spacing between the shroud and the exterior portions of the electrical motor.\",\n \"In preferred embodiments of the invention, the pumping system is installed in a well.\",\n \"The well may be vertical, horizontal, or a so-called “divert” type.\",\n \"The well has a casing that has perforations through which production fluids enter the well.\",\n \"The pumping system includes an electrical motor having a hollow rotor shaft.\",\n \"The pumping system has production fluid intakes through which the production fluids enter.\",\n \"These are located below said electrical motor (in the case of a horizontal well, opposite from the wellhead).\",\n \"The pumping system is adapted to pump said production fluids through the hollow rotor shaft of the electrical motor and then on to the wellhead.\",\n \"By “production fluids”, it is meant any fluids which are desired to be withdrawn from the well.\",\n \"Examples of such fluids include water, oil, natural gas, and the like, with liquids being of particular interest and oil being of special interest.\",\n \"By “perforations”, it is merely meant openings in a well casing through which the production fluids enter the well.\",\n \"No particular configuration or method of forming these openings is critical to the invention.\",\n \"The pumping system is located below at least some of the perforations in the casing.\",\n \"Thus, production fluids entering those perforations that are above the pumping system flow in fluid contact past the exterior portions of the electrical motor and into the production fluid intakes, by force of gravity and/or the action of the pumping system.\",\n \"In general, the pumping system should be below enough of the perforations that sufficient production fluids pass the exterior of the electrical motor to provide a cooling effect.\",\n \"Preferably, at least half of the production fluids will pass by the exterior of the motor before entering the production fluid intakes.\",\n \"More preferably, the pumping system is below substantially all of the perforations, and substantially the entire volume of production fluids passes in fluid contact with the exterior of the motor.\",\n \"This tends to maximize the desired cooling effect.\",\n \"Having the pumping system below substantially all of the perforations also facilitates separation of gaseous and liquid production fluids before the liquids enter the pumping system.\",\n \"This will increase the production rate when the well produces a mixture of gasses and liquids.\",\n \"Having the gasses circumvent the pump avoids various associated problems such as gas-lock or pumping inefficiencies that are attributable to entrained gasses.\",\n \"As gasses have lower heat capacities than liquids, having the gasses circumvent the motor in this manner further improves heat transfer and, thus, motor cooling.\",\n \"Additionally, because the pumping unit does not require oversized equipment or a gas separator as are typically used in conventional systems installed above the perforations, no significant additional power is consumed.\",\n \"The pumping system includes an electrical motor and a submergible pump that is driven by the electrical motor.\",\n \"The motor and pump may have a unitary structure (i.e., share a common housing), but it is often preferred that they are separate units which are connected together directly or indirectly as they are installed in the well.\",\n \"Two embodiments of the process of the invention are illustrated in FIG.\",\n \"2.In FIG.\",\n \"2, well casing 210 extends along the periphery of well 201, which as shown has been bored through a producing subterranean rock stratum 440 and into a lower, non-producing subterranean rock stratum 441.Producing stratum 440 contains production fluids that are to be pumped to the well head.\",\n \"Casing 210 includes perforations 211 through which the production fluids are admitted into the well for pumping to the surface.\",\n \"Located within casing 210 is a pumping system that includes motor 220 and pump 230.Both motor 220 and pump 230 are located below perforations 211.Motor 220 is affixed to production pipe 250, which is shown in section.\",\n \"If desired, production pipe 250 can take the form of a coiled tube.\",\n \"Motor 220 is shown in section to reveal hollow rotor shaft 221, which rotates when motor 220 is operated to provide motive force to operate pump 230.The production fluids pass through the hollow rotor shaft on their way to the wellhead through production pipe 250.Hollow rotor shaft 221 is mechanically connected, directly or indirectly, to pump 230 in a manner such that pump 230 is operated when hollow rotor shaft 221 is rotated.\",\n \"As shown, the drive shaft of pump 230 is directly connected to hollow rotor shaft 221, without intermediate apparatus.\",\n \"However, it may be desirable to include various types of intermediate apparatus such as a sealing section between motor 220 and pump 230.The manner through which motor 220 is affixed to production pipe 250 and pump 230 is not critical; a variety of fasteners, interlocking devices and the like can be used.\",\n \"Power is provided to the motor from the wellhead through a cable or similar device, which is not shown.\",\n \"Intake 260 is attached to pump 230.When the pumping system is activated, hollow rotor shaft 221 rotates substantially along its longitudinal axis, driving the action of pump 230.Production fluids enter the pumping system through intake 260 and enter pump 230.Hollow rotor shaft 221 is in liquid communication with production pipe 250 and pump 230, so that fluids pumped upwardly by pump 230 pass through hollow rotor shaft 221 and then enter production pipe 250 through which they are delivered to the wellhead.\",\n \"Because motor 220 is below perforations 211, production fluids that enter well 201 flow in fluid contact past the exterior of motor 220 before entering intake 260.This can be due to simple gravity, the pumping action of pump 230, or some combination of these.\",\n \"Arrows 290 indicate the direction of flow of the production fluids.\",\n \"As the production fluids must flow between well casing 210 and motor 220 as they travel toward the intake, the motor is somewhat smaller than the diameter of well casing 210, creating an annulus through which the production fluids can move.\",\n \"As the production fluids move in fluid contact past the exterior portion of motor 220, they remove waste heat and thus provide cooling.\",\n \"As the production fluids then are pumped through hollow rotor shaft 221 of motor 220, they remove additional waste heat and thus provide additional cooling.\",\n \"The embodiment of FIG.\",\n \"2 is a so-called “inverted” pumping system in which the motor is above the pump.\",\n \"Although this is preferred to have the motor above the pump, it is not critical to the invention, and FIG.\",\n \"2A illustrates an embodiment in which the motor is below the pump.\",\n \"In FIG.\",\n \"2A, well 201A has casing 210A.\",\n \"The pumping system includes production pipe 250A, pump 230A, motor 220A and intake 260A.\",\n \"Motor 220A includes hollow rotor shaft 221A through which production fluids pass.\",\n \"As before, hollow rotor shaft 221A is in mechanical communication with pump 230A so that as motor 220A operates, hollow rotor shaft 221A causes pump 230A to operate and pump the production fluids through hollow rotor shaft 221A and then into production pipe 250A and up to the wellhead.\",\n \"In the embodiment shown in FIG.\",\n \"2A, production fluids enter the well through perforations 211A, travel past the exterior of motor 220A, enter intake 260A, travel upwardly through hollow rotor shaft 221A and into pump 230A and then through production pipe 250A to the wellhead.\",\n \"Motor 220A is cooled twice by the production fluids; once as they pass in fluid contact with the exterior of motor 220A and again as they pass through hollow rotor shaft 221A in motor 220A).\",\n \"In FIG.\",\n \"4, a pumping system including electrical motor 402 and pump 411 is located in a well having casing 401 and perforations 415.Fluid intakes 420 are located below electrical motor 402 below packer 414.In the embodiment shown, fluid intakes 420 lead directly into pump 411, which is located below electrical motor 402.However, it is possible to employ multiple pumps and multiple motors in the submergible pumping system, provided that the intakes are located below at least one electrical motor with a hollow rotor shaft.\",\n \"In the preferred embodiment shown, hollow rotor shaft 403 of electrical motor 402 rotates about its longitudinal axis when electrical motor 402 is operated.\",\n \"This is conveniently achieved by affixing stationary magnets 404 to hollow rotor shaft 403 and supplying stators 405 to exterior housing 410 of electrical motor 402.Hollow rotor shaft 403 is coupled directly or indirectly to drive shaft 412 of pump 411, so that drive shaft 412 rotates when hollow rotor shaft 403 rotates, thereby supplying mechanical energy to pump 411.Impellers 413 are affixed to drive shaft 412 which provides motive force to impeller 413 when shaft 412 is rotated.\",\n \"The embodiment shown in FIG.\",\n \"4 includes optional features thrust bearing and seal 416 and packer 414.Thrust bearing and seal 416 connects electrical motor 402 with pump 411 and provides a seal against leakage of production fluids.\",\n \"Packer 414 assists in supporting the weight of the pumping system and prevents pressurized fluids from the pump from flowing downwardly to the pump intake.\",\n \"These components are well known in the art.\",\n \"Intakes 420 are in fluid communication with hollow rotor shaft 403 of electrical motor 402.In the embodiment shown, fluids entering intakes 420 flow through pump 411 in the general direction indicated by arrows 408.A first portion of the fluids flow in the general direction indicated by arrows 406, entering hollow rotor shaft 403 through openings 421 and flowing upwardly through hollow rotor shaft 403 and up to the wellhead.\",\n \"A second portion of the fluids flow in the general direction indicated by arrows 407, passing outwardly through pump housing 409 via openings 417, and then upwardly in a space between well casing 401 and exterior housing 410 of electrical motor 402.The embodiment shown in FIG.\",\n \"4 permits the electrical motor to be cooled by the production fluids both internally (i.e., as the fluids pass through the hollow rotor shaft) and externally (ie., as the fluids pass between the exterior of the electrical motor and the well casing), without the need to use a shroud, fluid recirculation or locate the pumping system below the perforations in the well.\",\n \"This avoids having to drill the well down below the perforations, i.e., no “rathole” is required to achieve the benefits of this aspect of the invention.\",\n \"This embodiment also permits one to avoid exposing the pumping system to sandy conditions as often exist below the perforations.\",\n \"In FIG.\",\n \"5, a pumping system includes electrical motor 502 and pump 511.These are located in a well having casing 601 with perforations 515.As shown, perforations 615 are located above pump intakes 517, as is preferred, but the perforations may be located at the level of or below pump intakes 517.Electrical motor 502 contains hollow rotor shaft 503.In the preferred embodiment shown, motor 502 includes a drive system including magnets 504 located about hollow rotor shaft 503 and stators 505 which are located on the interior of housing 510.When operated, hollow rotor shaft 503 rotates about its longitudinal axis.\",\n \"Pump 511 includes hollow drive shaft 512 that is in fluid communication with hollow rotor shaft 503 of motor 502.When hollow rotor shaft 503 rotates about its longitudinal axis as electrical motor 502 operates, hollow drive shaft 512 of pump 511 likewise rotates about its own longitudinal axis.\",\n \"Hollow drive shaft 512 includes exterior (to the shaft) impellers 513 that pump the production fluids when the pumping system is operated.\",\n \"Hollow drive shaft 512 includes shaft inlet 622, through which fluids enter as the pumping system is operated.\",\n \"Pump 511 includes housing 509 having fluid intakes 517.Fluid intakes 517 are located above shaft inlet 522 and below perforations 515.In operation, production fluids enter the well through perforations 515, flow downwardly past the exterior of electrical motor housing 510 and into intakes 517.The fluids are then pumped downwardly in pump 511 by impellers 513, where they enter shaft inlet 522 and are pumped upwardly through hollow drive shaft 512 of pump 511 and then through hollow rotor shaft 503 of motor 502, all in the direction indicated by arrows 506.The embodiment shown in FIG.\",\n \"5 includes preferred thrust bearing and seal 516.In this invention, the downward flow of the fluids through pump 511 before entering shaft inlet 522 reduces the downward force produced on pump 511.This in turn relieves the pump thrust carried by thrust bearing and seal 516, thereby prolonging its useful life and reducing the need for service and repair.\",\n \"In addition, this aspect of the invention permits the electrical motor to be cooled both on its exterior and its interior by the production fluids.\",\n \"In FIG.\",\n \"6, a pumping system including pump 611 and electrical motor 602 is located in a well having casing 601 with perforations 615, where production fluids enter the well.\",\n \"Perforations 615 are located below the pumping system.\",\n \"The production fluids are a mixture of at least one liquid and at least one gas.\",\n \"In this embodiment, intake 620 is located below the rest of the pumping system, although it is possible that one or more pumps or electrical motors can be located below the intakes.\",\n \"Intake 620 has diameter D, which is smaller than the diameter D′ of electrical motor 602 (and, as shown, pump 611) which is immediately above intake 620.Electrical motor 609 includes hollow rotor shaft 603.In the preferred embodiment shown, motor 602 includes a drive system including magnets 604 located about hollow rotor shaft 603 and stators 605 which are located on the interior of housing 610.When operated, hollow rotor shaft 603 rotates about its longitudinal axis.\",\n \"Hollow rotor shaft 603 is in fluid communication with intake 620.In the embodiment shown, motor 602 is directly above intake 620, and hollow rotor shaft 603 communicates directly with intake 620.However, one or more pumps can be located between motor 602 and intake 620, provided that hollow rotor shaft 603 is in fluid communication with intake 620 indirectly through the intervening pump.\",\n \"In operation, a mixture of gaseous and liquid fluids enters the well through perforations 615.These move upwardly in the direction indicated by arrows 606 into vortex zones indicated in FIG.\",\n \"6 by reference numerals 622.Vortex zones 622 are created in part by virtue of the diameter of intake 620 being smaller than that of electrical motor 602.In vortex zones 622, liquids and gasses separate efficiently.\",\n \"Gasses move upwardly in the direction indicated by arrows 608 between casing 602 and the exterior housings 610 and 609 of electrical motor 602 and pump 611.Liquids enter intake 620, flow downwardly through a section of intake 620, then enter hollow rotor shaft 603, and then are pumped upwardly to the wellhead.\",\n \"Arrows 607 in FIG.\",\n \"6 indicate the direction of the liquids.\",\n \"This last aspect of the invention is readily combined with, for example, the fifth aspect of the invention, so as to obtain the combined benefits of both.\",\n \"In any of the embodiments of the invention, the pumping system may contain additional elements as may be necessary or desirable in any particular application.\",\n \"For example, seal chamber sections are often provided in pumping systems for deep hole wells.\",\n \"Such seal sections may also carry pump thrust and can perform additional functions as well, as described by Brookbank in “Inverted Pump Systems Design and Applications”, ESP Workshop, Houston, Apr.\",\n \"26-28, 2000.As described by Brookbank, the functions performed by the seal chamber section can be divided among several pieces of apparatus if desired.\",\n \"In this invention, a seal chamber section or other device for carrying pump thrust is preferably part of the pumping system.\",\n \"In so-called “inverted” embodiments, it is preferred that some means of carrying pump thrust is included in the pump or motor design or as a separate piece of apparatus above or below the pump.\",\n \"If desired, seal sections may be located above the pumping system, or between the motor and the pump.\",\n \"Any apparatus situated between the motor and pump must be adapted so that motive force is transmitted through the apparatus from the motor to the pump, and so that production fluids travel through the hollow rotor shaft in the motor, as described above.\",\n \"Similarly, apparatus such as sand skirts, packers, various types of connectors and the like can be incorporated into the pumping system or used in conjunction with the pumping system.\",\n \"The pumping system may contain anti-cavitation devices like a primer pump to prevent cavitation of the fluid in the hollow rotor shaft or pump.\",\n \"These may be especially useful in configurations where the pump is above the motor.\",\n \"In producing deep bore wells, it is common practice to use a pumping system that is made up of separate components of a relatively short length.\",\n \"This approach can be adopted in the process of this invention as well.\",\n \"The motor, pump, intakes, seal sections and other apparatus may be constructed as two or more separate sections that are connected together to form the overall pumping system.\",\n \"A wide variety of electrical motor designs can be used in the pumping system of the invention, provided that the motor contains a hollow rotor shaft through which the production fluids can flow to provide cooling and/or vortex generators in the annulus between the motor and the well casing or shroud.\",\n \"Induction motors and brushless DC motors are useful, among others.\",\n \"Suitable motors of that type are described in U.S. Pat.\",\n \"Nos.\",\n \"5,951,262 and 6,000,915, both to Hartman, both incorporated herein by reference in their entirety.\",\n \"No special motor design is required, except that (1) it is adapted for submergible applications, (2) it contains the hollow rotor shaft as described herein and (3) it is of a size and shape to fit within the well casing and allow production fluids to pass in fluid contact with the exterior of the motor.\",\n \"Conventional electric motors as are commonly used for downhole pumping applications can be used, if they are adapted to provide them with a hollow rotor shaft through which the production fluids can be pumped.\",\n \"In its simplest case, the motor can be any submergible electric motor having a longitudinal, rotating rotor shaft, in which the rotor shaft has a longitudinal bore.\",\n \"Conventional electric motors having a longitudinal rotor shaft in some cases can be retrofitted for use in this invention simply by boring out the rotor shaft to form the hollow rotor shaft.\",\n \"The motor may be a single piece or a tandem configuration.\",\n \"Multiple motors can be used and, if desired, pumps can be placed both above and below the motor.\",\n \"Seal sections and other components may be installed between the motor and pump.\",\n \"As the motor will be submerged in the production fluids, it is preferably adapted to operate in those conditions.\",\n \"The motor preferably will contain a motor fluid that provides lubrication but more importantly retards the entrainment of production fluids in the motor.\",\n \"The motor fluid may also contain various well-treating materials such as scale inhibitors, emulsifiers, anti-emulsion agents, surfactants, water, and the like.\",\n \"Various types of seals can be incorporated into the motor to retard the leakage of production fluids into it, and as mentioned above, seal chamber sections can be used to accommodate thermal expansion of the motor fluid and help equalize pressure between the inside and outside of the motor.\",\n \"In a preferred type of motor, the motor fluid is at a positive pressure relative to the hollow rotor shaft of the motor and has leaking seals, so that motor fluid slowly leaks from the motor into the production fluid.\",\n \"A source of fresh motor fluid is provided, either from a reservoir in the pumping system or through a tube or capillary system from the wellhead.\",\n \"By maintaining a positive motor fluid pressure, displacement of the motor fluid by the production fluids can be reduced or eliminated, and motor life can be prolonged.\",\n \"A pressure independent modulating flow control valve, such as a SkoFlo™ or SubSea™ valve is suitable for maintaining a suitable positive pressure and flow of fresh lubrication fluid into the motor.\",\n \"A particularly preferred motor has vortex generators on its exterior surface, or else is enclosed within a shroud that has vortex generators on its interior surface.\",\n \"The vortex generators operate to generate streamwise vortices in the production fluids as they pass the exterior of the motor.\",\n \"The vortex generators are preferably static devices having geometry and dimensions such that when the production fluids flow through and past the generators, a swirl in the flow is imparted.\",\n \"These streamwise vortices greatly improve heat transfer from the exterior of the motor to the production fluids, even further increasing the benefits of this invention.\",\n \"An example of a motor containing vortex generators is schematically illustrated in FIG.\",\n \"3.Well 300 has casing 310 and perforations 311.The internal diameter of well 300 is D. Motor 320 and pump 330, each having a diameter d, are disposed in the well.\",\n \"Motor 320 is affixed to production pipe 350, which extends to the wellhead.\",\n \"Hollow rotor shaft 321 connects motor 320 and pump 330, in the same manner as described with respect to FIG.\",\n \"2.Pump 330 includes intake 360, where production fluids enter the pumping system.\",\n \"In the embodiment shown, production fluids enter the well at perforations 311, flow past the exterior of motor 320, into intake 360, through pump 330, through hollow rotor shaft 321 in motor 320 and up through production pipe 350 to the well head, as indicated by arrows 390.In the embodiment shown in FIG.\",\n \"3, the exterior surface of motor 320 has vortex generators.\",\n \"An alternative arrangement would be to surround motor 320 in a shroud that has vortex generators on its interior surface.\",\n \"In the embodiment in FIGS.\",\n \"3 and 3A, the vortex generators take the form of a plurality of fins 366.Fins 366 are slightly offset to the direction of flow of the production fluids, and adjacent fins 366 are offset in opposite directions.\",\n \"Thus, each pair of fins 366 define a gap which narrows in the direction of flow.\",\n \"The dimensions of fins 366 and the offset angle are sufficient to create streamwise vortices in the production fluids as they flow past the fins.\",\n \"Suitable offset angles are typically +/−10-30, preferably 10-20 degrees from the direction of flow of the production fluids.\",\n \"Suitable dimensions for fins 366 are illustrated in FIG.\",\n \"3A.\",\n \"In FIG.\",\n \"3A, the gap between motor 320 and well casing 311 is defined by (D-d)/2, where D and d are as described above.\",\n \"A suitable fin 366 will extend outwardly from the exterior of motor 320 (or inwardly from the exterior surface of a surrounding shroud) approximately 1/3-¾ of the width of the gap between the motor and well casing or, when a shroud is used, between the motor and interior surface of the shroud; in FIG.\",\n \"3A an especially preferred dimension of ½ the gap width is illustrated.\",\n \"In FIG.\",\n \"3A, numeral 325 designates either the exterior surface of the motor or the internal surface of a surrounding shroud.\",\n \"As shown in FIG.\",\n \"3A, fin 366 is preferably beveled with increasing width in the direction of flow, reaching its maximum width at a point approximately 1/3-¾ down its length.\",\n \"However, if desired, leading edge 399 can take other shapes, including those shown in outline in FIG.\",\n \"3A.\",\n \"Overall length is preferably about 1 to about 4, more preferably about 1.5 to about 3 times the width of the gap.\",\n \"In FIG.\",\n \"3A, the length is shown as equal to the gap width.\",\n \"In the embodiment shown in FIG.\",\n \"3, two rows of fins 366 are used.\",\n \"A single row can be used, or greater than two rows can be used.\",\n \"When multiple rows are used, a preferred spacing between the rows is about 10-30 times the gap distance.\",\n \"However, the spacing may be adjusted to trade off pressure loss with heat transfer.\",\n \"Other suitable vortex generator designs can be used, such as are described, for example, by Paulie and Eaton, Report #MD51, August 1988, “The Fluid Dynamics and Heat Transfer Effect of Streamwise Vortices Embedded in a Turbulent Boundary Layer”.\",\n \"The pump itself has no special design requirements, other than it is adapted to pump production fluids through the hollow rotor shaft of the motor.\",\n \"Generally, the particulars of the pump design will be selected to fit the particular application.\",\n \"The pump is in liquid communication with the hollow rotor shaft of the motor.\",\n \"This is accomplished by building the pump and motor as a single unit or incorporating the pump into the motor, as described in U.S. Pat.\",\n \"Nos.\",\n \"5,951,262 and 6,000,915, by designing the pump to mate with the hollow rotor shaft of the motor, or in some other manner.\",\n \"Pumps of the type conventionally used in ESPs are entirely suitable, and can easily be adapted for use in this invention through the design of the connection between the pump outlet (or inlet) and the motor.\",\n \"Progressive cavity pumps are also preferred types.\",\n \"The pump may be one piece or in tandem sections.\",\n \"Multiple pumps may be used.\",\n \"If desired, separate pumps can be provided above and below the motor.\",\n \"The method of this invention is useful in a variety of wells, including water, oil and natural gas wells.\",\n \"The pumping method is particularly advantageous in wells where, using a conventional ESP, any flow of production fluids through the annulus between the motor housing and the well casing would be expected to be laminar.\",\n \"Wells of this type include those having well conditions such as high viscosity production fluids, the formation of emulsions, low flow rates, multiphase flows, deviated and horizontal wells and large motor and/or well diameters, especially in the oil industry.\",\n \"The well operator can take advantage of the enhanced heat transfer and improved cooling of the motor in several ways.\",\n \"At equivalent power usage, the motor will be more efficiently cooled, and the operating temperature will be lower.\",\n \"The operator may choose to take advantage of this lower operating temperature to prolong the motor life.\",\n \"The lower temperature also tends to reduce electrical resistance, thus allowing equivalent work to be done with less power consumption.\",\n \"Conversely, the operator may elect to increase the power to the motor so that it runs at higher temperatures similar to those that would be experienced in prior art processes.\",\n \"In this case, the operator chooses to forego longer motor life in return for higher production rates that are achieved because of the additional power that is used.\",\n \"In order to increase cooling efficiency as the production fluids flow through hollow rotor shaft of the motor, it is preferred to operate under conditions that produce turbulent flow within the hollow rotor shaft.\",\n \"More preferably, there is turbulent flow both inside the hollow motor shaft, and turbulent flow or streamwise vortices in the annulus between the motor housing and the well casing where the production fluid passes in contact with the exterior of the motor.\",\n \"Turbulent flow can be expressed in terms of Reynolds number (a dimensionless parameter), which is a function of the average fluid velocity, kinematic viscosity of the fluid and diameter of the hollow rotor shaft.\",\n \"A Reynolds number of about 2300 or higher is typically indicative of turbulent flow.\",\n \"Preferably, flow conditions of the production fluids through the hollow rotor shaft of the motor is such that the Reynolds number is at least about 3000.A Reynolds number in excess of 5000 to 10000 is more preferred.\",\n \"Another parameter, Nusselt number, is a dimensionless measure of heat transfer.\",\n \"The Nusselt number is a function of the Reynolds number, the Prantl number, and the absolute viscosity of the bulk fluid and fluid at the wall.\",\n \"With turbulent flow, a high Nusselt number (exceeding 10) represents a high heat transfer rate for a given temperature difference.\",\n \"The more turbulent the flow the higher the Reynolds and the Nusselt numbers.\",\n \"With laminar flow, low Nusselt numbers (below 5) are indicative of poor convective heat transfer.\",\n \"Enhanced heat transfer from the motor to the production fluid can be expected when the Nusselt number is at least 10, preferably at least 50, and the method of the invention is preferably operated under conditions that achieve such Nusselt numbers.\",\n \"Operating conditions preferably are also chosen so as to provide a Brinkmann number (another dimensionless parameter) of less than 2, preferably less than 0.5.The Brinkmann number indicates the direction of heat transfer within a viscous fluid.\",\n \"It is a function of the average velocity of the fluid, its absolute viscosity, the thermal conductivity of the fluid and the temperature difference between the fluid and the inside wall of the hollow rotor shaft.\",\n \"When conditions are such that the Brinkmann number is less than 2, heat will travel from the hot motor to the cooler production fluid and fluid friction will not add additional heat to the motor.\",\n \"Viscous dissipation effects are negligible, when the Brinkmann number is less than 0.5.Since the hollow rotor shaft is the drive shaft of the motor, operating conditions can be further described with reference to a Rossby number (still another dimensionless parameter).\",\n \"The Rossby number provides an indication as to whether spinning flow will or will not dominate axial flow in the hollow rotor shaft.\",\n \"Conditions generating a Rossby number of at least about 0.5, preferably at least about 1.0 are preferred.\",\n \"The Rossby number is a function of average fluid velocity, fluid angular velocity and the inside diameter of the hollow rotor shaft.\",\n \"Yet another advantage of this invention is that it permits gaseous production fluids to separate from liquids before entering the pumping system.\",\n \"Gasses entering the well through perforations above the pumping system will tend to travel directly upward to the wellhead without passing through the pumping system, due to the natural buoyancy of the gasses.\",\n \"Liquids will flow downwardly and enter the pumping system, without significant entrained gas, for delivery to the wellhead.\",\n \"Thus, a ready separation of production gasses and production liquids is made.\",\n \"Because the pumping system will process less gas, pumping problems and inefficiencies associated with pumping mixtures of gasses and liquids are largely mitigated.\",\n \"Moreover, by eliminating the need for oversized equipment or a gas separator in a typical system installed above the perforations, no additional failure modes or power losses are introduced.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471462"}}},{"rowIdx":597,"cells":{"text":{"kind":"list like","value":[["Receptacle for a programmable, electronic processing device","A mount for a programmable electronic processing device is disclosed.","It is divided into at least a first functional unit (7) and a second functional unit (8) which represent separate, particularly monolithic integrated, components.","The first functional unit (7) defines all input and output interfaces of the processing device with respect to their electrical properties.","All essential connections of the second functional unit (8) are accessible from outside only via the first functional unit (7).","To this end, the first functional unit (7) includes matching circuits which serve to electrically adapt the connections of the second functional unit (8) to the external conditions."],["1.A mount containing a programmable electronic processing device, particularly a microprocessor, which is divided into at least a first functional unit (7) and a second functional unit (8) which represent separate, particularly monolithic integrated, components, characterized in that that the first functional unit (7) defines all input and output interfaces of the processing device with respect to their electrical properties, all essential connections of the second functional unit (8) are accessible from outside only via the first functional unit (7), with direct accesses being normally, and first functional unit (7) comprises matching circuits which serve to electrically adapt the connections of the second functional unit (8) to the external conditions.","2.A mount as claimed in claim 1, characterized in that at first the mount is bare or contains only a portion of the programmable electronic processing device, and that the mount so designed that the remaining portion or the remaining portions of the processing device can be incorporated later.","3.A mount as claimed in claim 2, characterized in that the portion contained in the mount is regarded as forming part of the first functional unit (7).","4.A mount as claimed in claim 1, characterized in that in at least one of the matching circuits, a change of a voltage level takes place.","5.A mount as claimed in claim 4, characterized in that at least one supply terminal of the second functional unit (8) is fed via the first functional unit (7), in which case the matching circuit includes a voltage regulator.","6.A mount as claimed in claim 1, characterized in that in at least one of the matching circuits, a change of a current level takes place.","7.A mount as claimed in claim 1, characterized in that in at least one of the matching circuits, a conversion from a serial data stream to a parallel data stream or from a parallel data stream to a serial data stream takes place.","8.A mount as claimed in claim 1, characterized in that in at least one of the matching circuits, incoming or outgoing data are temporarily stored.","9.A mount as claimed in claim 1, characterized in that at least one of the matching circuits is programmable in terms of its electrical properties.","10.A mount as claimed in claim 9, characterized in that for at least one of the matching circuits, a measurement mode is programmable.","11.A mount as claimed in claim 10, characterized in that in the measurement mode for at least one of the matching circuits, a direct signal path from an external connection of the mount to a connection of the second functional unit (8) can be switched through by means of the program, with the indirect signal path via the matching circuit being off."],["This invention relates to a mount in the sense of a package or an enclosure or a carrier device for a programmable electronic processing device.","As a rule, such processing devices constitute a processor which comprises at least one monolithic integrated circuit.","Both analog and digital signals or data are processed, depending on the application.","The relatively small electronic modules allow these processors to be housed along with additional circuits, such as memories, in a relatively small package.","Because of the small package dimensions, one also speaks of “microprocessors”.","If at least two individual structures are contained in a mount, this is also referred to as a “hybrid circuit”, as distinguished from those cases where only a single monolithic integrated circuit is present.","The use of microprocessors is increasing in all areas of technology, since with their aid, control processes can be optimized.","The additional cost and complexity is relatively low and allows decentralized solutions.","A wide range of applications is in mechanical engineering, particularly in the automotive field, which is increasingly resorting to such microprocessors in order to optimize the behavior of the vehicle at various points.","In many cases where such processors can be used, however, a basic problem arises from the fact that the cycle times differ very widely for the manufacturer and the user of the processors.","When such processors are adopted in a technical system, as uniform a product as possible should be available for the manufacture and for replacement throughout the development, planning, fabrication, and operating times of the associated system.","In the event of a technology change, aside from the changes in electrical connection parameters, the changed sensitivities to overvoltages, polarity reversals, and electromagnetic interference have to be taken into account.","There are also changes in the interfering effect on other circuits, for instance by steeper clock-pulse and data edges which travel as electromagnetic interference signals over relatively long lines.","The development in semiconductor technology is generally ahead of the time frame specified by the user, because it must follow current technology changes so as to be able to implement increasingly complex circuits.","The continued use of obsolete technologies involves the use of parallel production lines, which are usually uneconomical, because full capacity utilization is not ensured.","It is therefore an object of the invention characterized in claim 1 to provide a sgitable solution to the above problems which enables the semiconductor manufacturer, on the one hand, to use state-of-the-art semiconductor processes and, on the other hand, to offer the user a guarantee that a product is constant for a long time.","This object is attained by the teaching set forth in the characterizing part of claims 1.The mount according to the invention and the processing device are not be regarded as independent parts, but the mount is assigned a first functional unit of the processing device which essentially defines the external electrical conditions of the processing device.","The processing proper takes place in a second functional unit, whose essential connections are normally accessible from outside only via the external connections of the first functional unit.","The term “essential connections” as used herein includes those connections which, as seen from outside, are critical in terms of their electrical parameters because their tolerances are tight or because their active or passive disturbance behavior has to be taken into account.","Uncritical connections, such as set or reset inputs, may be included but need not necessarily be routed via the first functional unit, even though this is safer in the long run in view of the foreseeable development of the technology used.","The arrangement comprising a first and a second functional unit in a mount is similar to a hybrid circuit but serves an entirely different purpose.","The first functional unit, which may also consist of more than one monolithic integrated circuit, serves essentially only to define the external conditions and make available suitable matching circuits for the second functional unit.","In this manner, the processing device proper, which is contained in the second functional unit and may also consist of more than one monolithic integrated circuit, is “masked” as viewed from outside.","In addition to the pure matching circuits, the first functional unit may contain subcircuits performing more general functions, such as oscillators, supply voltage regulator circuits, buffer circuits, protective circuits, etc.","The supply voltage regulator circuits permit autonomous operation of the microprocessor in a supply system with an unregulated or excessive supply voltage.","This is particularly important for the operation of the second functional unit, whose supply voltage must, if necessary, be adapted to the technology used, while, on the other hand, the original supply voltage must be retained for other electronic circuits or measuring devices.","Since, as a rule, the processing device also contains memory devices, the write and erase voltages of the latter are also advantageously generated or adapted in the first functional unit.","The division into a first and a second functional unit allows a further technological development of the processing device proper without this having to be taken into account from outsidel provided that the technology is retained for the first functional unit.","Further developments of the technology are generally associated with higher processing rates and lower voltage levels.","Over the relatively long leads, the processing rates increase the interference potential to be taken into account.","On the other hand, the higher processing rates permit sequential processing for many processes for which parallel processing was previously necessary because of the lower processing speed.","Parallel processing generally necessitates parallel data inputs and outputs, whereby the number of contact pads, and thus the amount of chip area required, is increased.","Thus, it is necessary despite the increasing functionality of the processors not to let the number of contact pads increase in proportion thereto, but to limit it.","Accordingly, serial data input and output is desirable in the second functional unit.","This, however, requires suitable conversion devices in the first functional unit.","If the matching circuits are not fixed, but programmable, the flexibility will, of course, be even greater, since adaptation will then be possible via the program.","If the input and output levels of the processor change because a new technology is used, the new voltage levels of the matching circuits will be adapted via the program.","The invention and advantageous developments will now be described in more detail with reference to the accompanying drawings, in which: FIG.","1 is a schematic plan view of a typical mount for a monolithic integrated circuit according to the prior art; FIG.","2 is a schematic plan view of a typical mount for a hybrid circuit according to the prior art; FIG.","3 is a schematic plan view of a mount according to the invention for a lateral arrangement of a first and a second functional unit; FIG.","4 is a schematic plan view of a mount according to the invention for a stacked arrangement of the first and second functional units; FIG.","5 is a schematic plan view of a mount according to the invention for a combination of a stacked arrangement and a lateral arrangement; FIG.","6 is a schematic plan view of a mount according to the invention in which all contacts of the first and second functional units are first connected to a passive carrier; and FIG.","7 is a schematic cross-sectional view of a mount roughly according to FIG.","6, with the external terminals defined by an array of solder pads.","Referring to Fig.","i, there is shown a schematic plan view of the typical arrangement of a monolithic integrated circuit 1 in a mount according to the prior art.","The mount is generally a molded plastic package, whose outline is represented by a line 2.The carrier of the monolithic integrated circuit 1 is, as a rule, a flat metal stamping 3, the so-called frame, which, on the one hand, provides the through connections for the contact pads of the monolithic integrated circuit through the plastic package and, on the other hand, contains a platform 4 for receiving the monolithic integrated circuit i.","As a rule, this platform is connected to an external ground pin.","The contact pads of monolithic integrated circuit 1 are connected to the associated inner leads of frame 3 by bond wires.","All external leads to monolithic integrated circuit 1 run via frame 3.FIG.","2 shows a schematic plan view of a prior-art hybrid circuit.","The hybrid circuit consists of two monolithic integrated circuits 5, 6.If the carrier is again a frame 3 with a platform 4, the two monolithic integrated circuits are arranged on this platform side by side.","Contact is made to the monolithic integrated circuits 5, 6 via wire bonds which run either to the associated leads of frame 3 or directly from circuit to circuit.","In some cases, use is made of a stacked structure, in which the smaller circuit is cemented onto the larger one.","The smaller circuit is generally a memory circuit or a circuit with specific components, such as capacitors, coils, filters, etc.","Hybrid circuits are usually employed where subcircuits with different basic functions interact, for example an analog circuit and a digital circuit, possibly in conjunction with different memory types.","Thus, the optimum technology is available for each circuit.","In this manner it is also possible to combine products of different manufacturers.","Mounts for hybrid circuits may also be entire printed circuit boards, to which connections are also generally made by wire bonding.","The individual circuits and the wire bonds on the board are then protected by a plastic enclosure.","FIG.","3 is a schematic plan view of a mount according to the invention with a lateral arrangement of a first and a second functional unit 7, 8.The lateral arrangement of the two monolithic integrated functional units 7, 8 is relatively similar to the side-by-side arrangement of the two monolithic integrated circuits 5, 6 of FIG.","2.The difference lies in the fact that in FIG.","3, all external contacts are only connected to the first functional unit 7 and that the second functional unit 8 only has connections to the first functional unit 7.The common ground connection via platform 4 represents an exception.","FIG.","3 already shows in the schematic representation that if a frame 3 is used, this mount is limited to relatively few external leads, because the wire bonds allow only a limited bonding geometry, and compensation by the design of the internal frame leads is only partially possible.","A certain remedy is provided by the stacked arrangement shown in FIG.","4.In the schematic stacked arrangement of FIG.","4, comprising two or more subcircuits on a frame platform 4, a central arrangement roughly as in FIG.","1 is preserved, so that all inner contacts of frame 3 can be easily reached from the edge of the first functional unit 7 via bond wires.","Because of the stacked arrangement, the first functional unit 7 is allotted a carrier function for the second functional unit 8.This requires, of course, that the chip area of the first functional unit 7 is substantially greater than that of the second functional unit 8.The package outline or the border of the plastic enclosure is represented by the line 2.FIG.","5 shows schematically an embodiment of a combination of a stacked arrangement and a lateral arrangement on a frame platform 4.The stacked arrangement approximately corresponds to FIG.","4, and the lateral arrangement to FIG.","3.The first functional unit 7 supports the second functional unit 8 and is connected laterally to a further, second functional or auxiliary unit 9 disposed on the common frame platform.","All connections to the outside world go only via the first functional unit 7.The unit 9 may be a memory module, for example.","Whether its connections to the second functional unit 8 are made directly by bond wires or indirectly via the first functional unit 7 will depend primarily on the spatial conditions.","The package outline or the border of the plastic enclosure is represented by the line 2.FIG.","6 is a schematic plan view of an embodiment of the mount in which the problem of inadequate accessibility of the external connections 30 from the first functional unit 7 is solved by the lateral arrangement of the first and second functional units 7, 8.An insulating carrier i0, consisting, for example, of a ceramic body with an interconnection plane Ii deposited on its surface, supports the first and second functional units 7, 8.The interconnection plane II may consist of several, mutually isolated layers of wiring to form all interconnections 12, 13, 14, 15, 16, 17 and associated contact areas 20.By means of wires bonds 40, connections are made from the first and second functional units 7, 8 to the contact areas 20 of the interconnections.","The interconnections are isolated from each other and from the surface, and may therefore cross arbitrarily or pass below the first or second functional unit 7, 8; compare, for example, the crossover of interconnections 15 and 16, and the interconnection 17, which runs below the second functional unit 8.In this manner, all external terminals 30 of the mount can be easily reached from the first functional unit 7.Carrier 10 is also connected to the external terminals 30, or the latter were embedded in the carrier already during its manufacture.","The whole structure is surrounded by a plastic enclosure, whose outline is represented by the dash-dotted line 2.FIG.","7 shows a cross-sectional diagram of a further mount comprising a carrier 10 and a lateral arrangement of the first and second functional units 7, 8.The arrangement is roughly similar to that of FIG.","6.It differs from FIG.","6 in that the external terminals are defined by an array of solder pads 35.This arrangement will be used where the number of external terminals is so high that with a linear arrangement of terminals along the edges, the spacing becomes too small.","Each of the solder pads 35 is connected to a via hole 36, which provides the electrical connection to interconnection plane ii.","In the side view of FIG.","7, the wire bonds 40 from the first and second functional units 7, 8 to the interconnection plane Ii are clearly visible.","With the division into a first and a second functional unit, the invention is, of course, not limited to the use of monolithic integrated circuits.","If appropriate, other manufacturing processes may be used for the individual subcircuits.","What is important is that from outside, an electrically constant performance of the processing device is ensured, which is achieved by masking the processing device proper by means of a first functional unit having constant electrical parameters."]],"string":"[\n [\n \"Receptacle for a programmable, electronic processing device\",\n \"A mount for a programmable electronic processing device is disclosed.\",\n \"It is divided into at least a first functional unit (7) and a second functional unit (8) which represent separate, particularly monolithic integrated, components.\",\n \"The first functional unit (7) defines all input and output interfaces of the processing device with respect to their electrical properties.\",\n \"All essential connections of the second functional unit (8) are accessible from outside only via the first functional unit (7).\",\n \"To this end, the first functional unit (7) includes matching circuits which serve to electrically adapt the connections of the second functional unit (8) to the external conditions.\"\n ],\n [\n \"1.A mount containing a programmable electronic processing device, particularly a microprocessor, which is divided into at least a first functional unit (7) and a second functional unit (8) which represent separate, particularly monolithic integrated, components, characterized in that that the first functional unit (7) defines all input and output interfaces of the processing device with respect to their electrical properties, all essential connections of the second functional unit (8) are accessible from outside only via the first functional unit (7), with direct accesses being normally, and first functional unit (7) comprises matching circuits which serve to electrically adapt the connections of the second functional unit (8) to the external conditions.\",\n \"2.A mount as claimed in claim 1, characterized in that at first the mount is bare or contains only a portion of the programmable electronic processing device, and that the mount so designed that the remaining portion or the remaining portions of the processing device can be incorporated later.\",\n \"3.A mount as claimed in claim 2, characterized in that the portion contained in the mount is regarded as forming part of the first functional unit (7).\",\n \"4.A mount as claimed in claim 1, characterized in that in at least one of the matching circuits, a change of a voltage level takes place.\",\n \"5.A mount as claimed in claim 4, characterized in that at least one supply terminal of the second functional unit (8) is fed via the first functional unit (7), in which case the matching circuit includes a voltage regulator.\",\n \"6.A mount as claimed in claim 1, characterized in that in at least one of the matching circuits, a change of a current level takes place.\",\n \"7.A mount as claimed in claim 1, characterized in that in at least one of the matching circuits, a conversion from a serial data stream to a parallel data stream or from a parallel data stream to a serial data stream takes place.\",\n \"8.A mount as claimed in claim 1, characterized in that in at least one of the matching circuits, incoming or outgoing data are temporarily stored.\",\n \"9.A mount as claimed in claim 1, characterized in that at least one of the matching circuits is programmable in terms of its electrical properties.\",\n \"10.A mount as claimed in claim 9, characterized in that for at least one of the matching circuits, a measurement mode is programmable.\",\n \"11.A mount as claimed in claim 10, characterized in that in the measurement mode for at least one of the matching circuits, a direct signal path from an external connection of the mount to a connection of the second functional unit (8) can be switched through by means of the program, with the indirect signal path via the matching circuit being off.\"\n ],\n [\n \"This invention relates to a mount in the sense of a package or an enclosure or a carrier device for a programmable electronic processing device.\",\n \"As a rule, such processing devices constitute a processor which comprises at least one monolithic integrated circuit.\",\n \"Both analog and digital signals or data are processed, depending on the application.\",\n \"The relatively small electronic modules allow these processors to be housed along with additional circuits, such as memories, in a relatively small package.\",\n \"Because of the small package dimensions, one also speaks of “microprocessors”.\",\n \"If at least two individual structures are contained in a mount, this is also referred to as a “hybrid circuit”, as distinguished from those cases where only a single monolithic integrated circuit is present.\",\n \"The use of microprocessors is increasing in all areas of technology, since with their aid, control processes can be optimized.\",\n \"The additional cost and complexity is relatively low and allows decentralized solutions.\",\n \"A wide range of applications is in mechanical engineering, particularly in the automotive field, which is increasingly resorting to such microprocessors in order to optimize the behavior of the vehicle at various points.\",\n \"In many cases where such processors can be used, however, a basic problem arises from the fact that the cycle times differ very widely for the manufacturer and the user of the processors.\",\n \"When such processors are adopted in a technical system, as uniform a product as possible should be available for the manufacture and for replacement throughout the development, planning, fabrication, and operating times of the associated system.\",\n \"In the event of a technology change, aside from the changes in electrical connection parameters, the changed sensitivities to overvoltages, polarity reversals, and electromagnetic interference have to be taken into account.\",\n \"There are also changes in the interfering effect on other circuits, for instance by steeper clock-pulse and data edges which travel as electromagnetic interference signals over relatively long lines.\",\n \"The development in semiconductor technology is generally ahead of the time frame specified by the user, because it must follow current technology changes so as to be able to implement increasingly complex circuits.\",\n \"The continued use of obsolete technologies involves the use of parallel production lines, which are usually uneconomical, because full capacity utilization is not ensured.\",\n \"It is therefore an object of the invention characterized in claim 1 to provide a sgitable solution to the above problems which enables the semiconductor manufacturer, on the one hand, to use state-of-the-art semiconductor processes and, on the other hand, to offer the user a guarantee that a product is constant for a long time.\",\n \"This object is attained by the teaching set forth in the characterizing part of claims 1.The mount according to the invention and the processing device are not be regarded as independent parts, but the mount is assigned a first functional unit of the processing device which essentially defines the external electrical conditions of the processing device.\",\n \"The processing proper takes place in a second functional unit, whose essential connections are normally accessible from outside only via the external connections of the first functional unit.\",\n \"The term “essential connections” as used herein includes those connections which, as seen from outside, are critical in terms of their electrical parameters because their tolerances are tight or because their active or passive disturbance behavior has to be taken into account.\",\n \"Uncritical connections, such as set or reset inputs, may be included but need not necessarily be routed via the first functional unit, even though this is safer in the long run in view of the foreseeable development of the technology used.\",\n \"The arrangement comprising a first and a second functional unit in a mount is similar to a hybrid circuit but serves an entirely different purpose.\",\n \"The first functional unit, which may also consist of more than one monolithic integrated circuit, serves essentially only to define the external conditions and make available suitable matching circuits for the second functional unit.\",\n \"In this manner, the processing device proper, which is contained in the second functional unit and may also consist of more than one monolithic integrated circuit, is “masked” as viewed from outside.\",\n \"In addition to the pure matching circuits, the first functional unit may contain subcircuits performing more general functions, such as oscillators, supply voltage regulator circuits, buffer circuits, protective circuits, etc.\",\n \"The supply voltage regulator circuits permit autonomous operation of the microprocessor in a supply system with an unregulated or excessive supply voltage.\",\n \"This is particularly important for the operation of the second functional unit, whose supply voltage must, if necessary, be adapted to the technology used, while, on the other hand, the original supply voltage must be retained for other electronic circuits or measuring devices.\",\n \"Since, as a rule, the processing device also contains memory devices, the write and erase voltages of the latter are also advantageously generated or adapted in the first functional unit.\",\n \"The division into a first and a second functional unit allows a further technological development of the processing device proper without this having to be taken into account from outsidel provided that the technology is retained for the first functional unit.\",\n \"Further developments of the technology are generally associated with higher processing rates and lower voltage levels.\",\n \"Over the relatively long leads, the processing rates increase the interference potential to be taken into account.\",\n \"On the other hand, the higher processing rates permit sequential processing for many processes for which parallel processing was previously necessary because of the lower processing speed.\",\n \"Parallel processing generally necessitates parallel data inputs and outputs, whereby the number of contact pads, and thus the amount of chip area required, is increased.\",\n \"Thus, it is necessary despite the increasing functionality of the processors not to let the number of contact pads increase in proportion thereto, but to limit it.\",\n \"Accordingly, serial data input and output is desirable in the second functional unit.\",\n \"This, however, requires suitable conversion devices in the first functional unit.\",\n \"If the matching circuits are not fixed, but programmable, the flexibility will, of course, be even greater, since adaptation will then be possible via the program.\",\n \"If the input and output levels of the processor change because a new technology is used, the new voltage levels of the matching circuits will be adapted via the program.\",\n \"The invention and advantageous developments will now be described in more detail with reference to the accompanying drawings, in which: FIG.\",\n \"1 is a schematic plan view of a typical mount for a monolithic integrated circuit according to the prior art; FIG.\",\n \"2 is a schematic plan view of a typical mount for a hybrid circuit according to the prior art; FIG.\",\n \"3 is a schematic plan view of a mount according to the invention for a lateral arrangement of a first and a second functional unit; FIG.\",\n \"4 is a schematic plan view of a mount according to the invention for a stacked arrangement of the first and second functional units; FIG.\",\n \"5 is a schematic plan view of a mount according to the invention for a combination of a stacked arrangement and a lateral arrangement; FIG.\",\n \"6 is a schematic plan view of a mount according to the invention in which all contacts of the first and second functional units are first connected to a passive carrier; and FIG.\",\n \"7 is a schematic cross-sectional view of a mount roughly according to FIG.\",\n \"6, with the external terminals defined by an array of solder pads.\",\n \"Referring to Fig.\",\n \"i, there is shown a schematic plan view of the typical arrangement of a monolithic integrated circuit 1 in a mount according to the prior art.\",\n \"The mount is generally a molded plastic package, whose outline is represented by a line 2.The carrier of the monolithic integrated circuit 1 is, as a rule, a flat metal stamping 3, the so-called frame, which, on the one hand, provides the through connections for the contact pads of the monolithic integrated circuit through the plastic package and, on the other hand, contains a platform 4 for receiving the monolithic integrated circuit i.\",\n \"As a rule, this platform is connected to an external ground pin.\",\n \"The contact pads of monolithic integrated circuit 1 are connected to the associated inner leads of frame 3 by bond wires.\",\n \"All external leads to monolithic integrated circuit 1 run via frame 3.FIG.\",\n \"2 shows a schematic plan view of a prior-art hybrid circuit.\",\n \"The hybrid circuit consists of two monolithic integrated circuits 5, 6.If the carrier is again a frame 3 with a platform 4, the two monolithic integrated circuits are arranged on this platform side by side.\",\n \"Contact is made to the monolithic integrated circuits 5, 6 via wire bonds which run either to the associated leads of frame 3 or directly from circuit to circuit.\",\n \"In some cases, use is made of a stacked structure, in which the smaller circuit is cemented onto the larger one.\",\n \"The smaller circuit is generally a memory circuit or a circuit with specific components, such as capacitors, coils, filters, etc.\",\n \"Hybrid circuits are usually employed where subcircuits with different basic functions interact, for example an analog circuit and a digital circuit, possibly in conjunction with different memory types.\",\n \"Thus, the optimum technology is available for each circuit.\",\n \"In this manner it is also possible to combine products of different manufacturers.\",\n \"Mounts for hybrid circuits may also be entire printed circuit boards, to which connections are also generally made by wire bonding.\",\n \"The individual circuits and the wire bonds on the board are then protected by a plastic enclosure.\",\n \"FIG.\",\n \"3 is a schematic plan view of a mount according to the invention with a lateral arrangement of a first and a second functional unit 7, 8.The lateral arrangement of the two monolithic integrated functional units 7, 8 is relatively similar to the side-by-side arrangement of the two monolithic integrated circuits 5, 6 of FIG.\",\n \"2.The difference lies in the fact that in FIG.\",\n \"3, all external contacts are only connected to the first functional unit 7 and that the second functional unit 8 only has connections to the first functional unit 7.The common ground connection via platform 4 represents an exception.\",\n \"FIG.\",\n \"3 already shows in the schematic representation that if a frame 3 is used, this mount is limited to relatively few external leads, because the wire bonds allow only a limited bonding geometry, and compensation by the design of the internal frame leads is only partially possible.\",\n \"A certain remedy is provided by the stacked arrangement shown in FIG.\",\n \"4.In the schematic stacked arrangement of FIG.\",\n \"4, comprising two or more subcircuits on a frame platform 4, a central arrangement roughly as in FIG.\",\n \"1 is preserved, so that all inner contacts of frame 3 can be easily reached from the edge of the first functional unit 7 via bond wires.\",\n \"Because of the stacked arrangement, the first functional unit 7 is allotted a carrier function for the second functional unit 8.This requires, of course, that the chip area of the first functional unit 7 is substantially greater than that of the second functional unit 8.The package outline or the border of the plastic enclosure is represented by the line 2.FIG.\",\n \"5 shows schematically an embodiment of a combination of a stacked arrangement and a lateral arrangement on a frame platform 4.The stacked arrangement approximately corresponds to FIG.\",\n \"4, and the lateral arrangement to FIG.\",\n \"3.The first functional unit 7 supports the second functional unit 8 and is connected laterally to a further, second functional or auxiliary unit 9 disposed on the common frame platform.\",\n \"All connections to the outside world go only via the first functional unit 7.The unit 9 may be a memory module, for example.\",\n \"Whether its connections to the second functional unit 8 are made directly by bond wires or indirectly via the first functional unit 7 will depend primarily on the spatial conditions.\",\n \"The package outline or the border of the plastic enclosure is represented by the line 2.FIG.\",\n \"6 is a schematic plan view of an embodiment of the mount in which the problem of inadequate accessibility of the external connections 30 from the first functional unit 7 is solved by the lateral arrangement of the first and second functional units 7, 8.An insulating carrier i0, consisting, for example, of a ceramic body with an interconnection plane Ii deposited on its surface, supports the first and second functional units 7, 8.The interconnection plane II may consist of several, mutually isolated layers of wiring to form all interconnections 12, 13, 14, 15, 16, 17 and associated contact areas 20.By means of wires bonds 40, connections are made from the first and second functional units 7, 8 to the contact areas 20 of the interconnections.\",\n \"The interconnections are isolated from each other and from the surface, and may therefore cross arbitrarily or pass below the first or second functional unit 7, 8; compare, for example, the crossover of interconnections 15 and 16, and the interconnection 17, which runs below the second functional unit 8.In this manner, all external terminals 30 of the mount can be easily reached from the first functional unit 7.Carrier 10 is also connected to the external terminals 30, or the latter were embedded in the carrier already during its manufacture.\",\n \"The whole structure is surrounded by a plastic enclosure, whose outline is represented by the dash-dotted line 2.FIG.\",\n \"7 shows a cross-sectional diagram of a further mount comprising a carrier 10 and a lateral arrangement of the first and second functional units 7, 8.The arrangement is roughly similar to that of FIG.\",\n \"6.It differs from FIG.\",\n \"6 in that the external terminals are defined by an array of solder pads 35.This arrangement will be used where the number of external terminals is so high that with a linear arrangement of terminals along the edges, the spacing becomes too small.\",\n \"Each of the solder pads 35 is connected to a via hole 36, which provides the electrical connection to interconnection plane ii.\",\n \"In the side view of FIG.\",\n \"7, the wire bonds 40 from the first and second functional units 7, 8 to the interconnection plane Ii are clearly visible.\",\n \"With the division into a first and a second functional unit, the invention is, of course, not limited to the use of monolithic integrated circuits.\",\n \"If appropriate, other manufacturing processes may be used for the individual subcircuits.\",\n \"What is important is that from outside, an electrically constant performance of the processing device is ensured, which is achieved by masking the processing device proper by means of a first functional unit having constant electrical parameters.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471467"}}},{"rowIdx":598,"cells":{"text":{"kind":"list like","value":[["Noise cancellation","A noise cancellation circuit has a first and second input for a first signal having a signal element and a noise element, and a second signal comprising at least a smaller amplitude of the said signal element, a first inverter arrangement for producing an inverted signal output that is an inverted form of one of the first and second signals, a first adder for adding the other signal and the inverted signal to produce an intermediate signal, an intermediate inverter arrangement for inverting the intermediate signal to produce an inverted intermediate signal, and a second adder for adding the other signal, the inverted signal and the inverted intermediate signal to produce an output."],["1.A noise cancellation circuit comprising: a first input for a first signal having a signal element and a noise element; a second input for a second signal comprising at least a smaller amplitude of the said signal element; a first inverter arrangement for producing an inverted signal output that is an inverted form of one of the first and second signals; a first adder for adding the other signal and the inverted signal to produce an intermediate signal; an intermediate inverter arrangement for inverting the intermediate signal to produce an inverted intermediate signal; and a second adder for adding the other signal, the inverted signal and the inverted intermediate signal to produce an output.","2.A circuit as claimed in claim 1 in which the first inverter arrangement comprises a first inverter having an output of a first inverted signal which is operably connected with the first adder, and a second inverter having an output of a second inverted signal which is operably connected with the second adder.","3.A circuit as claimed in claim 1, including means for balancing the amplitude of the noise in the first signal, the inverted signal and the inverted intermediate signal, such that it is substantially cancelled in the output.","4.A circuit as claimed in claim 3 in which the means for balancing include at least one variable gain amplifier.","5.A circuit as claimed in claim 4 in which the first inverter includes the variable gain amplifier.","6.A circuit as claimed in claim 4 in which the intermediate inverter includes the variable gain amplifier.","7.A circuit as claimed in claim 4, in which the output of the second adder is connected to the variable gain amplifier.","8.A circuit as claimed in claim 1 in which the intermediate inverter attenuates the intermediate signal.","9.A circuit as claimed in claim 1, including a first transducer operably connected with the first input, and a second transducer operably connected with the second input, the second transducer being constructed and/or arranged to receive at least an attenuated amplitude of the signal element relative to the signal element received by the first transducer.","10.A circuit as claimed in claim 9 in which at least one of the first and second transducers is] constructed and arranged to inhibit the reception of the signal element by the second transducer.","11.A circuit as claimed in claim 9 in which the transducers are microphones.","12.A circuit as claimed in claim 9 in which the transducers are microphones and in which the second microphone is arranged with a baffle to reception of the signal element.","13.A circuit as claimed in claim 12 in which the microphones are directional, the first microphone being arranged in front of the second microphone along the major direction of reception of signal thereby.","14.A circuit as claimed in claim 13 in which the receiving faces of the microphones are spaced by a distance in the range 0.2 mm to 2.5 mm, preferably 0.625 mm.","15.A circuit as claimed in claim 1 in which the first inverter arrangement is arranged to invert the second signal from the second input to produce the inverted signal.","16.A circuit as claimed in claim 1 in which the first and second signals are low pass filtered to attenuate higher frequency noise.","17.A method of noise reduction comprising: comparing a first signal having a signal element and a noise element and a second signal having at least a smaller amplitude of the said signal element to produce an intermediate signal; and subtracting the intermediate signal from a comparison of the first signal and the second signal to produce an output in which the noise is reduced.","18.A microphone arrangement for a noise cancellation circuit, comprising a first microphone arranged to receive a signal element and a noise element, a second microphone arranged with a baffle such that it receives at least a smaller amplitude of signal element relative to the first microphone.","19.A microphone arrangement as claimed in claim 18 in which the second microphone is arranged in relation to the first microphone such that the first microphone acts as a baffle to receipt of the signal element by the second microphone.","20.A circuit as claimed in claim 9 in which the transducers are microphones and in which the second microphone is arranged with a baffle to reception of the signal element and in which the second microphone is arranged at a greater distance from the signal source than the first microphone.","21.A circuit as claimed in claim 9 in which the transducers are microphones and in which the second microphone is arranged at a greater distance from the signal source than the first microphone."],["The present invention relates to noise cancellation.","Noise cancellation in the audio or other frequency ranges is commonly based on the theory of using a first input of the desired signal plus noise, and a second input of noise alone.","One input is phase inverted with respect to the other, and the two are then added so that the noise (common to both inputs) is cancelled, leaving the desired signal.","The techniques used in practice are more sophisticated than this because the basic theory does not take into account other considerations.","The input transducers (for example microphones in the audio frequency range) from which the input signals are derived also are non-ideal and, thus, impose distortions on the two inputs, but to different extents.","Furthermore, the theory demands that the desired signal be absent from the second input or at least attenuated in it.","Previous techniques for improving on the basic approach to noise cancellation include filtering the output signal.","However, filtering neglects to take into account the distortion imposed on the noise signal by the process of transducing it into a manageable electrical quantity, and subsequent signal processing.","Another technique is to digitise the analogue signals and apply digital signal processing to address the residual noise.","Other noise reduction methods that have been proposed involve the use of phased arrays of signal pick-ups (for example microphones).","These are inflexible and expensive.","In spite of the fact that the basic technique of noise cancellation has been known for many decades, the incomplete noise cancellation due to distortions imposed on the two signals at the first and second inputs has only ever been addressed by such additional techniques of filtering, etc.","which are unrelated to any comparison of the signals themselves.","The limit on the extent to which noise cancellation can be effective resides, at least in part, in the distortion of the transduced signals.","For example, microphones used in pairs or other groupings are not identical and do not produce exactly the same signals for a given input.","A matched pair of microphones can be used to minimise this problem, but they will never be identical and, in any event, will cost more.","Another problem is the spacing of the transducers in relation to the source.","Microphones will be located at different positions and, therefore, will be exposed to slightly different noise stimulation.","According to the present invention there is provided a noise cancellation circuit comprising: a first input for a first signal having a signal element and a noise element; a second input for a second signal comprising at least a smaller amplitude of the said signal element; a first inverter arrangement for producing an inverted signal output that is an inverted form of one of the first and second signals; a first adder for adding the other signal and the inverted signal to produce an intermediate signal; an intermediate inverter arrangement for inverting the intermediate signal to produce an inverted intermediate signal; and a second adder for adding the other signal, the inverted signal and the inverted intermediate signal to produce an output.","Also, according to the present invention there is provided a method of noise cancellation comprising comparing a first signal having a signal element and a noise element, with a second signal having at least a smaller amplitude of the said signal element to produce an intermediate signal, and subtracting the intermediate signal from a comparison of the first signal and the second signal to produce an output.","The present invention provides a particularly beneficial effect that is not intuitive.","This is because the circuit of the invention compares the noise in signals received, as transduced at the second input, with itself further to reduce the noise which is reduced in any event by comparing the noise at the first and second inputs separately.","The effect is to cancel noise by comparing similar responses and, thereby, avoid the effects of distortions due to which the cancellation previously effected according to known principles was less effective.","By comparing the first input, the second input and the intermediate signal, the distorting effect at the circuit input is also taken into account.","The noise cancelling effect can be optimised for a given application according to the relative attenuations/amplifications of the signals at the second adder.","Preferably, transducers are connected to the first and second inputs by which signals are received and to which the noise cancelling process is to be applied.","In a particular form of the invention the first inverter arrangement is arranged to invert the second signal from the second input to produce the inverted signal.","The invention is particularly applicable to the audio frequency range, but is not limited to it.","The invention applies to any frequency range and applications where the effects of distortion caused by the input should be taken into account.","Preferably, the second input can be derived by using transducers in which the transducer connected with the first input is constructed and/or arranged to reduce the reception of the signal element by the second transducer.","For example, when the transducers are microphones, one microphone connected with the second input can be arranged to be baffled in its reception of the signal element, by the presence of the microphone connected with the first input in the direction of reception of the signal elements or by an additional baffle.","In the case of microphones, it is found that the receiving faces are preferably spaced by a distance in the range of 0.2 mm to 2.5 mm, preferably 0.625 mm.","Alternatively, the baffle can be introduced to attenuate the signal reaching the noise microphone.","The attenuation of the signal element received by the second (noise) microphone is due to the baffling effect of the microphone in front, and also the distance of the second microphone from the signal source.","Thus, it is preferable that the microphones are directional.","The subtraction and inversion of signals is preferably carried out using operational amplifiers in the analogue domain.","However, other analogue circuit techniques could be used to equal effect, such as transistor amplifiers.","In a preferred embodiment of the invention the first and second signals are each low pass filtered.","This signal conditioning is used to smooth out the characteristic spikes of high frequency noise in the signal.","As a result, the signals to be cancelled present a broader (less acute) target of a lower power spectral density.","The cancellation technique is more effective when applied to the low pass filtered signals because the circuit is less sensitive to phase distortion and timeshifts of either or both of the input signals.","The invention is equally applicable to the digital domain in which some unwanted distortion noise is added at the analogue-to-digital conversion stage when analogue signals are converted into digital data.","The same issues of signal processing distortion can be addressed by comparing the originally digitised signal plus ambient noise, an inverted form of the originally digitised signal with a small amplitude signal element and an inverted form of the intermediate digital signal.","The invention can be put into practice in various ways, some of which will now by described by way of example with reference to the accompanying drawings, in which: FIG.","1 is a circuit diagram of one embodiment of the present invention; FIG.","2 is an illustration of the orientation of audio microphones for use in the present invention; FIG.","3 is a circuit diagram of an alternative form of the circuit of FIG.","1; FIGS.","4 to 7 are graphic illustrations of signals in the circuit of FIG.","1; FIG.","8 is a circuit according to an alternative embodiment: FIG.","9 is a generalised block diagram according to the invention; and FIG.","10 is a circuit according to a further alternative embodiment.","Referring to FIG.","1, a noise cancellation circuit for audio frequencies comprises a first input 10 for an electret voice microphone 12.The microphone is a transducer, converting acoustic signals into analogue electrical signals.","The acoustic signals are accompanied by ambient noise within the dynamic range of the microphone.","It is the noise that must be cancelled as much as possible to derive a more faithful signal at the output of the circuit.","A second input 14 has an electret noise microphone 16 connected to it.","Both first and second inputs 10 and 14 are connected between a negative voltage rail (−) and respective 5 kohm pull-up resistors 18/20 connected to a positive voltage rail (+) in each case.","Other forms of microphone could be used, such as dynamic (electromagnetic), crystal or carbon, that do not require any power connections.","Directional microphones are desirable in order to provide at least some selectivity in picking up the signal.","The signal level for each input 10/14 is buffered by a unity gain non-inverting operational amplifier 22/24.The outputs of the buffer amplifiers 22/24 for the voice/noise microphones 12/14 are labelled as points C and D, respectively, in FIG.","1.The non-inverted signal at point D from the noise microphone 16 is connected to the inverting inputs of a pair of inverting operational amplifiers 26/28 which are also connected to a mid-supply voltage reference level 30 by their respective non-inverting inputs, to centre their output with respect to the full supply voltage swing, The operational amplifier 26 is arranged as an inverting attenuator with a gain of 0.85, providing a signal at point E of the inverted attenuated form of the signal at point D. The operational amplifier 28 is arranged as an inverting attenuator with a gain of 0.72 at point E′ of the signal at point D. Other settings (for example unity gain) for the attenuation of the signals will depend on, for example, the microphones and other circuit components used and the supply potential.","The gains of the op-amps 26 and 28 are preferably the same or close enough to provide signals of similar amplitude in order not to impose undue burdens on the rest of the circuit or undermine the noise cancelling functionality.","The signals at points C and E are combined at point F through resistors R11 and R12 so that the voice signal at C is, in effect, added to the inverted attenuated form of the noise signal at E. The effect of the addition of these two signals at point F should, in theory, realise the cancellation of the common, but antiphase, signals in each (i.e.","the noise) subject to what attenuation of the signal at D is caused by the attenuator 26.However, it is well known that this is not the case in practice.","This is because the signals received at the microphones 12 and 16 are subject to different distortions due, for example, to the non-linearities in the system, and thermal and temporal component drift.","Thus, it is not the case that the noise in one line can simply be a faithful but inverted form of the other.","While, in the past, the thinking has been to approach the residual noise problem by filtering and other more sophisticated techniques, the invention makes use of the result of this comparison to reduce the noise by reapplying it to the circuit.","The reduced noise at point F is buffered by a further unity gain non-inverting amplifier 32 and amplified by a compensating inverting amplifier 34 with a gain of 1.95.The output of the amplifier 32 is an intermediate signal consisting of an inverted form of the signal at point C which is indicated in FIG.","1 as point G. The signal at G is attenuated relative to the signal at the point E. At point H in the circuit, the signal from the second inverting attenuator 28 at point E′ is added to the signal at point C and the intermediate signal at point G through resistors R9, R13 and R14.The combined signal is buffered by a unity gain non-inverting amplifier 36, filtered by an a.c. couple 38 to remove any dc component, and connected to an output 40.FIG.","2 illustrates the arrangement of the microphones 12 and 16 also according to the present invention.","While each microphone can pick up sound from more than one direction, it has a predominant direction of reception and is, to that extent, directional.","It will be seen in FIG.","2 that the noise microphone 16 is arranged with its receiving face about 0.625 mm (¼″) behind the receiving face of the voice microphone.","In this way, the voice microphone is fully exposed to the desired input signal (i.e.","speech), but also presents a baffle to the reception of the same desired signal by the noise microphone so that the desired signal is attenuated at the noise microphone.","In this way, the noise microphone receives a relatively greater proportion of noise signal input than the voice microphone.","The arrangement of the voice and noise microphones may differ according to application, type of microphones used, distance from the source of the desired signal, etc., and can be derived empirically according to circumstances.","FIG.","3 shows a modified form of the circuit in FIG.","1.An amplifier 42 with adjustable gain is used to perform the functions of the amplifiers 26 and 28.Thus, the signals at points E and E′, which are essentially the same are now rendered simply at the point E in FIG.","3, and connected to the resistors R12 and R14 in parallel.","It is found that it is easier to balance the signal by using only a single amplifier at this point.","FIG.","4 illustrates the two transduced signals, as read at points C and D of the circuit of FIG.","1.They each comprise a wanted voice signal (in this case a basic sinusoid for the sake of illustration) and a distorting noise component superimposed upon the wanted signal.","They are similar as both are exposed to the same noise sources, but the voice signal at point D is slightly attenuated by about 0.15 relative to that at point C due, at least in part, to the baffling effect of the voice microphone 12 in front of the noise microphone 16, and/or their relative distances from the source of the sound, as shown in FIG.","2.FIG.","5 shows a comparison of the waveforms at points D and E, which latter waveform is the attenuated inverted form of the waveform at point D. It is also equivalent to the waveform at point E′ as well.","The waveform at point E can be considered as a negative ‘dirty’ form of that at D because a small random variation has been introduced into the signal, associated with small physical and electrical differences between the two microphones 12/16 due, for example, to manufacturing tolerances in the circuit components.","FIG.","6 illustrates the signals at point C, F and G. The signal at point F is the reduced amplitude, reduced noise signal due to the addition of the antiphase signals at points C and E, but it still contains significant noise products due to the dissimilarity in the transduced noise signal components caused by the differently distorting effects of the two microphones.","The signal at point G is the inverted and attenuated form of the signal at point F which is itself used in the circuit.","The signal at point C is shown for comparison with the signal at point F to illustrate that there is noise reduction albeit with an attenuated voice signal as well.","This is an illustration of the point in the circuit at which the prior art would apply filtering and other techniques to address the remaining noise.","FIG.","7 shows the signal at point C compared with the signal at point F (as in FIG.","6) and also as compared with the signal at point H after the signals at point C and E′ have been added to the inverted form of the signal at point F (i.e.","the signal at point G).","As a result of applying the invention, the signal at point H is seen to contain far less noise than at point F for a similar output voice signal amplitude.","The invention addresses the additional distortion in the signal due to the transducers.","The invention provides a technique that does this by adding the signal at G, which is the noise-reduced inverted and attenuated signal at F from the basic difference between the output of the noise microphone at point D. To arrive at balanced settings for the gains of the various op-amps in the circuit by which noise at the point H is cancelled to an improved extent, the resistor R8 on the op-amp 34 is made adjustable.","By placing a microphone arrangement as shown in FIG.","2 so that its predominant reception direction is at right angles to a source of noise (so that both microphones receive equal noise input) the value of R8 is adjusted until the noise output at the op-amp 36 is minimised.","It is also possible to adjust the values of the gains of the other op-amps in the circuit, such as those of the op-amps 26 and 28 for example, to the same end.","However, R8 is the convenient resistance to choose as it limits the number of adjustments that have to be made.","The invention has been described in terms of audio frequencies.","However, the invention is equally applicable to other frequency ranges and applications in which the distorting effect of transducing one signal into another form imposes different distortions on the signals to be compared for the purposes of noise reduction.","The invention provides improvements in signal to noise that are of benefit both objectively and subjectively.","Objectively it is found that the signal to noise improvements have particular advantages in speech decoding schemes such as voice recognition software.","Subjectively, the clarity of the reproduced sound is particularly useful in telephony and radio and other analogue/digital speech communication systems.","It will be appreciated from the description that the preferred embodiment uses very readily available components such as operational amplifiers, basic resistors and capacitors and transducers, and can be implemented on an integrated circuit very easily.","The invention is particularly suited to incorporation into equipment at the manufacturing stage or as additional equipment for existing products, such as in wired and wireless telephony.","FIG.","8 illustrates an alternative embodiment of the invention in which like reference numerals have been used for like parts.","In this circuit input op-amps 50 and 52 for the signal and noise channels, respectively, have gains.","The output also has an op-amp amplifier 54 with non-unity gain.","It is necessary to boost the output for certain applications.","It is found that it is beneficial to do this at least partly by amplifying the inputs, and subjecting any amplification of the noise to the same noise cancelling by the circuit, and to limit the amplification at the output.","To summarise the operations of circuit, the desired signal plus noise at point C is combined both with the inverted form of the more noisy signal at point E′ and the inverted and attenuated form of the noise-cancelled signal at point G produced by comparing the signals at point C and D. This is illustrated in FIG.","9 in block diagram form.","Because of distortions, the noise is not sufficiently cancelled for many applications at point G as there is not complete identity between the signals at points C and D. The addition of this signal in adjusted form with the inverted noise signal and the voice signal substantially reduces still further the noise at the output 40 according to the relative choice of amplification/attenuation factors of the signals at the various points.","FIG.","10 illustrates a further embodiment of the invention.","In the drawing, parts corresponding to those in FIGS.","1 and 8 have been given like reference numerals.","In this embodiment, the signal at the point D is applied to the input of a second order Chebyshev low pass filter 60 with a 4 kHz cut-off frequency.","The output of the filter 60 is inverted by a unity gain inverter 62 to provide the equivalent of point E referred to previously.","Similarly, the signal at point C is low pass filter by a second order Chebyshev filter 64 and inverted by a unity gain inverter 66.The output of the inverter 62 is applied to the input of an adjustable amplifier 68, having a variable feedback resistor 70, providing an output at point E″ that is equivalent to point E in FIG.","3.According to the adjustment of the feedback resistors 70, the amplifier may be adjusted to act as an attenuator.","The signals from the respective microphones 12/16 are now in the form of smooth (high frequency attenuated), inverted and attenuated (in the case of the noise microphone signal) outputs.","These are added at the point F′ (equivalent to the point F in FIGS.","1, 2 and 8) after resistors R11 and R12 to provide a high impedance input to the buffer amplifier 32.They are also added at the point H′ after resistors R13 and R14 to provide a high impedance input to a further adjustable amplifier 72 having a variable feedback resistor 74.The added signals at point H′ are connected to the output of the inverter 76 at point G′ (equivalent to point G in FIGS.","1, 2 and 8).","The added signals at point F′ are buffered by the buffer 32 and inverted by an adjustable gain inverter 76 (equivalent to the inverter 34 in FIGS.","1 and 8), having a variable resistor 78.Thus, the added signals at points F′ and H′ are combined at G′, which is attenuated relative to the signal at point E, as before.","According to this embodiment of the invention, the filtering and antiphasing is carried out on each signal substantially identically but electrically separate.","The filtered and inverted signals are provided as high impedance inputs at resistors R11, R12, R13 and R14.One antiphase (inverted) combination of signals takes place at point F′ and the other separately at point H′.","The antiphase combining at point F′ is used to determine a ‘compensation signal’ that is added to the other antiphase signal at point H′.","The result is a optimally antiphase combination of signals at H′.","The signal at H′ is the basic advantageously noise cancelled output.","This is then buffered by buffer 72 and further low pass filtered in a second order Chebyshev filter 80.The output of the filter is further buffered by buffer 82 before being a.c. coupled at 40, as before, to provide the conditioned noise cancelled output.","To set up the circuit of FIG.","10 for a given situation, microphones and output, the following procedure is used.","1.Set the variable resistors 70 and 78 at zero.","2.Set the variable resistor 74 for full amplifier gain.","3.Connect an oscilloscope to the output 40 and arrange the noise and voice microphone so that their predominant direction of reception is orthogonal to the location of the noise.","4.Adjust the resistor 70 for minimum amplitude output on the oscilloscope.","5.Adjust the resistor 78 further to minimise the output on the oscilloscope.","6.Reorient the microphones towards the noise source.","7.Adjust the resistor 78 and 74 to achieve the desired output amplitude.","It will be appreciated by those of ordinary skill in the art that, as well as the digital implementation of the invention, it is also possible for other components to be used to the same effect for different parts of the circuits disclosed.","For example, the filters in the circuits of FIGS.","1, 8 and 10 could be any suitable active or passive arrangement that provides the required cut-off frequency and attenuation rates.","Examples include Butterworth, Elliptical and Bessel filters, and infinite and finite impulse response filters in the digital domain.","The purpose of the filters 26, 28 or 60, 64 for the microphone inputs is to reduce the typically sharp spikes of noise components in a signal caused by high frequency noise in a given spectrum so that they are in a more smooth (high frequency attenuated) form.","It is found that the attenuation of sharp unfiltered spikes of noise is less effective when there is less than perfect phase and amplitude comparison.","This has the effect of reducing the sensitivity of the circuit to phase shifts introduced by circuit components and/or the microphones.","This significantly improves the noise cancellation performance.","Because the invention does not rely on precise matching of components, the increased tolerance derived from low pass filtering the signals is of particularly beneficial effect.","It will be apparent from the foregoing that the present invention can be realised in many different ways.","The invention is not limited to those described herein, but only according to the spirit and scope of the following claims."]],"string":"[\n [\n \"Noise cancellation\",\n \"A noise cancellation circuit has a first and second input for a first signal having a signal element and a noise element, and a second signal comprising at least a smaller amplitude of the said signal element, a first inverter arrangement for producing an inverted signal output that is an inverted form of one of the first and second signals, a first adder for adding the other signal and the inverted signal to produce an intermediate signal, an intermediate inverter arrangement for inverting the intermediate signal to produce an inverted intermediate signal, and a second adder for adding the other signal, the inverted signal and the inverted intermediate signal to produce an output.\"\n ],\n [\n \"1.A noise cancellation circuit comprising: a first input for a first signal having a signal element and a noise element; a second input for a second signal comprising at least a smaller amplitude of the said signal element; a first inverter arrangement for producing an inverted signal output that is an inverted form of one of the first and second signals; a first adder for adding the other signal and the inverted signal to produce an intermediate signal; an intermediate inverter arrangement for inverting the intermediate signal to produce an inverted intermediate signal; and a second adder for adding the other signal, the inverted signal and the inverted intermediate signal to produce an output.\",\n \"2.A circuit as claimed in claim 1 in which the first inverter arrangement comprises a first inverter having an output of a first inverted signal which is operably connected with the first adder, and a second inverter having an output of a second inverted signal which is operably connected with the second adder.\",\n \"3.A circuit as claimed in claim 1, including means for balancing the amplitude of the noise in the first signal, the inverted signal and the inverted intermediate signal, such that it is substantially cancelled in the output.\",\n \"4.A circuit as claimed in claim 3 in which the means for balancing include at least one variable gain amplifier.\",\n \"5.A circuit as claimed in claim 4 in which the first inverter includes the variable gain amplifier.\",\n \"6.A circuit as claimed in claim 4 in which the intermediate inverter includes the variable gain amplifier.\",\n \"7.A circuit as claimed in claim 4, in which the output of the second adder is connected to the variable gain amplifier.\",\n \"8.A circuit as claimed in claim 1 in which the intermediate inverter attenuates the intermediate signal.\",\n \"9.A circuit as claimed in claim 1, including a first transducer operably connected with the first input, and a second transducer operably connected with the second input, the second transducer being constructed and/or arranged to receive at least an attenuated amplitude of the signal element relative to the signal element received by the first transducer.\",\n \"10.A circuit as claimed in claim 9 in which at least one of the first and second transducers is] constructed and arranged to inhibit the reception of the signal element by the second transducer.\",\n \"11.A circuit as claimed in claim 9 in which the transducers are microphones.\",\n \"12.A circuit as claimed in claim 9 in which the transducers are microphones and in which the second microphone is arranged with a baffle to reception of the signal element.\",\n \"13.A circuit as claimed in claim 12 in which the microphones are directional, the first microphone being arranged in front of the second microphone along the major direction of reception of signal thereby.\",\n \"14.A circuit as claimed in claim 13 in which the receiving faces of the microphones are spaced by a distance in the range 0.2 mm to 2.5 mm, preferably 0.625 mm.\",\n \"15.A circuit as claimed in claim 1 in which the first inverter arrangement is arranged to invert the second signal from the second input to produce the inverted signal.\",\n \"16.A circuit as claimed in claim 1 in which the first and second signals are low pass filtered to attenuate higher frequency noise.\",\n \"17.A method of noise reduction comprising: comparing a first signal having a signal element and a noise element and a second signal having at least a smaller amplitude of the said signal element to produce an intermediate signal; and subtracting the intermediate signal from a comparison of the first signal and the second signal to produce an output in which the noise is reduced.\",\n \"18.A microphone arrangement for a noise cancellation circuit, comprising a first microphone arranged to receive a signal element and a noise element, a second microphone arranged with a baffle such that it receives at least a smaller amplitude of signal element relative to the first microphone.\",\n \"19.A microphone arrangement as claimed in claim 18 in which the second microphone is arranged in relation to the first microphone such that the first microphone acts as a baffle to receipt of the signal element by the second microphone.\",\n \"20.A circuit as claimed in claim 9 in which the transducers are microphones and in which the second microphone is arranged with a baffle to reception of the signal element and in which the second microphone is arranged at a greater distance from the signal source than the first microphone.\",\n \"21.A circuit as claimed in claim 9 in which the transducers are microphones and in which the second microphone is arranged at a greater distance from the signal source than the first microphone.\"\n ],\n [\n \"The present invention relates to noise cancellation.\",\n \"Noise cancellation in the audio or other frequency ranges is commonly based on the theory of using a first input of the desired signal plus noise, and a second input of noise alone.\",\n \"One input is phase inverted with respect to the other, and the two are then added so that the noise (common to both inputs) is cancelled, leaving the desired signal.\",\n \"The techniques used in practice are more sophisticated than this because the basic theory does not take into account other considerations.\",\n \"The input transducers (for example microphones in the audio frequency range) from which the input signals are derived also are non-ideal and, thus, impose distortions on the two inputs, but to different extents.\",\n \"Furthermore, the theory demands that the desired signal be absent from the second input or at least attenuated in it.\",\n \"Previous techniques for improving on the basic approach to noise cancellation include filtering the output signal.\",\n \"However, filtering neglects to take into account the distortion imposed on the noise signal by the process of transducing it into a manageable electrical quantity, and subsequent signal processing.\",\n \"Another technique is to digitise the analogue signals and apply digital signal processing to address the residual noise.\",\n \"Other noise reduction methods that have been proposed involve the use of phased arrays of signal pick-ups (for example microphones).\",\n \"These are inflexible and expensive.\",\n \"In spite of the fact that the basic technique of noise cancellation has been known for many decades, the incomplete noise cancellation due to distortions imposed on the two signals at the first and second inputs has only ever been addressed by such additional techniques of filtering, etc.\",\n \"which are unrelated to any comparison of the signals themselves.\",\n \"The limit on the extent to which noise cancellation can be effective resides, at least in part, in the distortion of the transduced signals.\",\n \"For example, microphones used in pairs or other groupings are not identical and do not produce exactly the same signals for a given input.\",\n \"A matched pair of microphones can be used to minimise this problem, but they will never be identical and, in any event, will cost more.\",\n \"Another problem is the spacing of the transducers in relation to the source.\",\n \"Microphones will be located at different positions and, therefore, will be exposed to slightly different noise stimulation.\",\n \"According to the present invention there is provided a noise cancellation circuit comprising: a first input for a first signal having a signal element and a noise element; a second input for a second signal comprising at least a smaller amplitude of the said signal element; a first inverter arrangement for producing an inverted signal output that is an inverted form of one of the first and second signals; a first adder for adding the other signal and the inverted signal to produce an intermediate signal; an intermediate inverter arrangement for inverting the intermediate signal to produce an inverted intermediate signal; and a second adder for adding the other signal, the inverted signal and the inverted intermediate signal to produce an output.\",\n \"Also, according to the present invention there is provided a method of noise cancellation comprising comparing a first signal having a signal element and a noise element, with a second signal having at least a smaller amplitude of the said signal element to produce an intermediate signal, and subtracting the intermediate signal from a comparison of the first signal and the second signal to produce an output.\",\n \"The present invention provides a particularly beneficial effect that is not intuitive.\",\n \"This is because the circuit of the invention compares the noise in signals received, as transduced at the second input, with itself further to reduce the noise which is reduced in any event by comparing the noise at the first and second inputs separately.\",\n \"The effect is to cancel noise by comparing similar responses and, thereby, avoid the effects of distortions due to which the cancellation previously effected according to known principles was less effective.\",\n \"By comparing the first input, the second input and the intermediate signal, the distorting effect at the circuit input is also taken into account.\",\n \"The noise cancelling effect can be optimised for a given application according to the relative attenuations/amplifications of the signals at the second adder.\",\n \"Preferably, transducers are connected to the first and second inputs by which signals are received and to which the noise cancelling process is to be applied.\",\n \"In a particular form of the invention the first inverter arrangement is arranged to invert the second signal from the second input to produce the inverted signal.\",\n \"The invention is particularly applicable to the audio frequency range, but is not limited to it.\",\n \"The invention applies to any frequency range and applications where the effects of distortion caused by the input should be taken into account.\",\n \"Preferably, the second input can be derived by using transducers in which the transducer connected with the first input is constructed and/or arranged to reduce the reception of the signal element by the second transducer.\",\n \"For example, when the transducers are microphones, one microphone connected with the second input can be arranged to be baffled in its reception of the signal element, by the presence of the microphone connected with the first input in the direction of reception of the signal elements or by an additional baffle.\",\n \"In the case of microphones, it is found that the receiving faces are preferably spaced by a distance in the range of 0.2 mm to 2.5 mm, preferably 0.625 mm.\",\n \"Alternatively, the baffle can be introduced to attenuate the signal reaching the noise microphone.\",\n \"The attenuation of the signal element received by the second (noise) microphone is due to the baffling effect of the microphone in front, and also the distance of the second microphone from the signal source.\",\n \"Thus, it is preferable that the microphones are directional.\",\n \"The subtraction and inversion of signals is preferably carried out using operational amplifiers in the analogue domain.\",\n \"However, other analogue circuit techniques could be used to equal effect, such as transistor amplifiers.\",\n \"In a preferred embodiment of the invention the first and second signals are each low pass filtered.\",\n \"This signal conditioning is used to smooth out the characteristic spikes of high frequency noise in the signal.\",\n \"As a result, the signals to be cancelled present a broader (less acute) target of a lower power spectral density.\",\n \"The cancellation technique is more effective when applied to the low pass filtered signals because the circuit is less sensitive to phase distortion and timeshifts of either or both of the input signals.\",\n \"The invention is equally applicable to the digital domain in which some unwanted distortion noise is added at the analogue-to-digital conversion stage when analogue signals are converted into digital data.\",\n \"The same issues of signal processing distortion can be addressed by comparing the originally digitised signal plus ambient noise, an inverted form of the originally digitised signal with a small amplitude signal element and an inverted form of the intermediate digital signal.\",\n \"The invention can be put into practice in various ways, some of which will now by described by way of example with reference to the accompanying drawings, in which: FIG.\",\n \"1 is a circuit diagram of one embodiment of the present invention; FIG.\",\n \"2 is an illustration of the orientation of audio microphones for use in the present invention; FIG.\",\n \"3 is a circuit diagram of an alternative form of the circuit of FIG.\",\n \"1; FIGS.\",\n \"4 to 7 are graphic illustrations of signals in the circuit of FIG.\",\n \"1; FIG.\",\n \"8 is a circuit according to an alternative embodiment: FIG.\",\n \"9 is a generalised block diagram according to the invention; and FIG.\",\n \"10 is a circuit according to a further alternative embodiment.\",\n \"Referring to FIG.\",\n \"1, a noise cancellation circuit for audio frequencies comprises a first input 10 for an electret voice microphone 12.The microphone is a transducer, converting acoustic signals into analogue electrical signals.\",\n \"The acoustic signals are accompanied by ambient noise within the dynamic range of the microphone.\",\n \"It is the noise that must be cancelled as much as possible to derive a more faithful signal at the output of the circuit.\",\n \"A second input 14 has an electret noise microphone 16 connected to it.\",\n \"Both first and second inputs 10 and 14 are connected between a negative voltage rail (−) and respective 5 kohm pull-up resistors 18/20 connected to a positive voltage rail (+) in each case.\",\n \"Other forms of microphone could be used, such as dynamic (electromagnetic), crystal or carbon, that do not require any power connections.\",\n \"Directional microphones are desirable in order to provide at least some selectivity in picking up the signal.\",\n \"The signal level for each input 10/14 is buffered by a unity gain non-inverting operational amplifier 22/24.The outputs of the buffer amplifiers 22/24 for the voice/noise microphones 12/14 are labelled as points C and D, respectively, in FIG.\",\n \"1.The non-inverted signal at point D from the noise microphone 16 is connected to the inverting inputs of a pair of inverting operational amplifiers 26/28 which are also connected to a mid-supply voltage reference level 30 by their respective non-inverting inputs, to centre their output with respect to the full supply voltage swing, The operational amplifier 26 is arranged as an inverting attenuator with a gain of 0.85, providing a signal at point E of the inverted attenuated form of the signal at point D. The operational amplifier 28 is arranged as an inverting attenuator with a gain of 0.72 at point E′ of the signal at point D. Other settings (for example unity gain) for the attenuation of the signals will depend on, for example, the microphones and other circuit components used and the supply potential.\",\n \"The gains of the op-amps 26 and 28 are preferably the same or close enough to provide signals of similar amplitude in order not to impose undue burdens on the rest of the circuit or undermine the noise cancelling functionality.\",\n \"The signals at points C and E are combined at point F through resistors R11 and R12 so that the voice signal at C is, in effect, added to the inverted attenuated form of the noise signal at E. The effect of the addition of these two signals at point F should, in theory, realise the cancellation of the common, but antiphase, signals in each (i.e.\",\n \"the noise) subject to what attenuation of the signal at D is caused by the attenuator 26.However, it is well known that this is not the case in practice.\",\n \"This is because the signals received at the microphones 12 and 16 are subject to different distortions due, for example, to the non-linearities in the system, and thermal and temporal component drift.\",\n \"Thus, it is not the case that the noise in one line can simply be a faithful but inverted form of the other.\",\n \"While, in the past, the thinking has been to approach the residual noise problem by filtering and other more sophisticated techniques, the invention makes use of the result of this comparison to reduce the noise by reapplying it to the circuit.\",\n \"The reduced noise at point F is buffered by a further unity gain non-inverting amplifier 32 and amplified by a compensating inverting amplifier 34 with a gain of 1.95.The output of the amplifier 32 is an intermediate signal consisting of an inverted form of the signal at point C which is indicated in FIG.\",\n \"1 as point G. The signal at G is attenuated relative to the signal at the point E. At point H in the circuit, the signal from the second inverting attenuator 28 at point E′ is added to the signal at point C and the intermediate signal at point G through resistors R9, R13 and R14.The combined signal is buffered by a unity gain non-inverting amplifier 36, filtered by an a.c. couple 38 to remove any dc component, and connected to an output 40.FIG.\",\n \"2 illustrates the arrangement of the microphones 12 and 16 also according to the present invention.\",\n \"While each microphone can pick up sound from more than one direction, it has a predominant direction of reception and is, to that extent, directional.\",\n \"It will be seen in FIG.\",\n \"2 that the noise microphone 16 is arranged with its receiving face about 0.625 mm (¼″) behind the receiving face of the voice microphone.\",\n \"In this way, the voice microphone is fully exposed to the desired input signal (i.e.\",\n \"speech), but also presents a baffle to the reception of the same desired signal by the noise microphone so that the desired signal is attenuated at the noise microphone.\",\n \"In this way, the noise microphone receives a relatively greater proportion of noise signal input than the voice microphone.\",\n \"The arrangement of the voice and noise microphones may differ according to application, type of microphones used, distance from the source of the desired signal, etc., and can be derived empirically according to circumstances.\",\n \"FIG.\",\n \"3 shows a modified form of the circuit in FIG.\",\n \"1.An amplifier 42 with adjustable gain is used to perform the functions of the amplifiers 26 and 28.Thus, the signals at points E and E′, which are essentially the same are now rendered simply at the point E in FIG.\",\n \"3, and connected to the resistors R12 and R14 in parallel.\",\n \"It is found that it is easier to balance the signal by using only a single amplifier at this point.\",\n \"FIG.\",\n \"4 illustrates the two transduced signals, as read at points C and D of the circuit of FIG.\",\n \"1.They each comprise a wanted voice signal (in this case a basic sinusoid for the sake of illustration) and a distorting noise component superimposed upon the wanted signal.\",\n \"They are similar as both are exposed to the same noise sources, but the voice signal at point D is slightly attenuated by about 0.15 relative to that at point C due, at least in part, to the baffling effect of the voice microphone 12 in front of the noise microphone 16, and/or their relative distances from the source of the sound, as shown in FIG.\",\n \"2.FIG.\",\n \"5 shows a comparison of the waveforms at points D and E, which latter waveform is the attenuated inverted form of the waveform at point D. It is also equivalent to the waveform at point E′ as well.\",\n \"The waveform at point E can be considered as a negative ‘dirty’ form of that at D because a small random variation has been introduced into the signal, associated with small physical and electrical differences between the two microphones 12/16 due, for example, to manufacturing tolerances in the circuit components.\",\n \"FIG.\",\n \"6 illustrates the signals at point C, F and G. The signal at point F is the reduced amplitude, reduced noise signal due to the addition of the antiphase signals at points C and E, but it still contains significant noise products due to the dissimilarity in the transduced noise signal components caused by the differently distorting effects of the two microphones.\",\n \"The signal at point G is the inverted and attenuated form of the signal at point F which is itself used in the circuit.\",\n \"The signal at point C is shown for comparison with the signal at point F to illustrate that there is noise reduction albeit with an attenuated voice signal as well.\",\n \"This is an illustration of the point in the circuit at which the prior art would apply filtering and other techniques to address the remaining noise.\",\n \"FIG.\",\n \"7 shows the signal at point C compared with the signal at point F (as in FIG.\",\n \"6) and also as compared with the signal at point H after the signals at point C and E′ have been added to the inverted form of the signal at point F (i.e.\",\n \"the signal at point G).\",\n \"As a result of applying the invention, the signal at point H is seen to contain far less noise than at point F for a similar output voice signal amplitude.\",\n \"The invention addresses the additional distortion in the signal due to the transducers.\",\n \"The invention provides a technique that does this by adding the signal at G, which is the noise-reduced inverted and attenuated signal at F from the basic difference between the output of the noise microphone at point D. To arrive at balanced settings for the gains of the various op-amps in the circuit by which noise at the point H is cancelled to an improved extent, the resistor R8 on the op-amp 34 is made adjustable.\",\n \"By placing a microphone arrangement as shown in FIG.\",\n \"2 so that its predominant reception direction is at right angles to a source of noise (so that both microphones receive equal noise input) the value of R8 is adjusted until the noise output at the op-amp 36 is minimised.\",\n \"It is also possible to adjust the values of the gains of the other op-amps in the circuit, such as those of the op-amps 26 and 28 for example, to the same end.\",\n \"However, R8 is the convenient resistance to choose as it limits the number of adjustments that have to be made.\",\n \"The invention has been described in terms of audio frequencies.\",\n \"However, the invention is equally applicable to other frequency ranges and applications in which the distorting effect of transducing one signal into another form imposes different distortions on the signals to be compared for the purposes of noise reduction.\",\n \"The invention provides improvements in signal to noise that are of benefit both objectively and subjectively.\",\n \"Objectively it is found that the signal to noise improvements have particular advantages in speech decoding schemes such as voice recognition software.\",\n \"Subjectively, the clarity of the reproduced sound is particularly useful in telephony and radio and other analogue/digital speech communication systems.\",\n \"It will be appreciated from the description that the preferred embodiment uses very readily available components such as operational amplifiers, basic resistors and capacitors and transducers, and can be implemented on an integrated circuit very easily.\",\n \"The invention is particularly suited to incorporation into equipment at the manufacturing stage or as additional equipment for existing products, such as in wired and wireless telephony.\",\n \"FIG.\",\n \"8 illustrates an alternative embodiment of the invention in which like reference numerals have been used for like parts.\",\n \"In this circuit input op-amps 50 and 52 for the signal and noise channels, respectively, have gains.\",\n \"The output also has an op-amp amplifier 54 with non-unity gain.\",\n \"It is necessary to boost the output for certain applications.\",\n \"It is found that it is beneficial to do this at least partly by amplifying the inputs, and subjecting any amplification of the noise to the same noise cancelling by the circuit, and to limit the amplification at the output.\",\n \"To summarise the operations of circuit, the desired signal plus noise at point C is combined both with the inverted form of the more noisy signal at point E′ and the inverted and attenuated form of the noise-cancelled signal at point G produced by comparing the signals at point C and D. This is illustrated in FIG.\",\n \"9 in block diagram form.\",\n \"Because of distortions, the noise is not sufficiently cancelled for many applications at point G as there is not complete identity between the signals at points C and D. The addition of this signal in adjusted form with the inverted noise signal and the voice signal substantially reduces still further the noise at the output 40 according to the relative choice of amplification/attenuation factors of the signals at the various points.\",\n \"FIG.\",\n \"10 illustrates a further embodiment of the invention.\",\n \"In the drawing, parts corresponding to those in FIGS.\",\n \"1 and 8 have been given like reference numerals.\",\n \"In this embodiment, the signal at the point D is applied to the input of a second order Chebyshev low pass filter 60 with a 4 kHz cut-off frequency.\",\n \"The output of the filter 60 is inverted by a unity gain inverter 62 to provide the equivalent of point E referred to previously.\",\n \"Similarly, the signal at point C is low pass filter by a second order Chebyshev filter 64 and inverted by a unity gain inverter 66.The output of the inverter 62 is applied to the input of an adjustable amplifier 68, having a variable feedback resistor 70, providing an output at point E″ that is equivalent to point E in FIG.\",\n \"3.According to the adjustment of the feedback resistors 70, the amplifier may be adjusted to act as an attenuator.\",\n \"The signals from the respective microphones 12/16 are now in the form of smooth (high frequency attenuated), inverted and attenuated (in the case of the noise microphone signal) outputs.\",\n \"These are added at the point F′ (equivalent to the point F in FIGS.\",\n \"1, 2 and 8) after resistors R11 and R12 to provide a high impedance input to the buffer amplifier 32.They are also added at the point H′ after resistors R13 and R14 to provide a high impedance input to a further adjustable amplifier 72 having a variable feedback resistor 74.The added signals at point H′ are connected to the output of the inverter 76 at point G′ (equivalent to point G in FIGS.\",\n \"1, 2 and 8).\",\n \"The added signals at point F′ are buffered by the buffer 32 and inverted by an adjustable gain inverter 76 (equivalent to the inverter 34 in FIGS.\",\n \"1 and 8), having a variable resistor 78.Thus, the added signals at points F′ and H′ are combined at G′, which is attenuated relative to the signal at point E, as before.\",\n \"According to this embodiment of the invention, the filtering and antiphasing is carried out on each signal substantially identically but electrically separate.\",\n \"The filtered and inverted signals are provided as high impedance inputs at resistors R11, R12, R13 and R14.One antiphase (inverted) combination of signals takes place at point F′ and the other separately at point H′.\",\n \"The antiphase combining at point F′ is used to determine a ‘compensation signal’ that is added to the other antiphase signal at point H′.\",\n \"The result is a optimally antiphase combination of signals at H′.\",\n \"The signal at H′ is the basic advantageously noise cancelled output.\",\n \"This is then buffered by buffer 72 and further low pass filtered in a second order Chebyshev filter 80.The output of the filter is further buffered by buffer 82 before being a.c. coupled at 40, as before, to provide the conditioned noise cancelled output.\",\n \"To set up the circuit of FIG.\",\n \"10 for a given situation, microphones and output, the following procedure is used.\",\n \"1.Set the variable resistors 70 and 78 at zero.\",\n \"2.Set the variable resistor 74 for full amplifier gain.\",\n \"3.Connect an oscilloscope to the output 40 and arrange the noise and voice microphone so that their predominant direction of reception is orthogonal to the location of the noise.\",\n \"4.Adjust the resistor 70 for minimum amplitude output on the oscilloscope.\",\n \"5.Adjust the resistor 78 further to minimise the output on the oscilloscope.\",\n \"6.Reorient the microphones towards the noise source.\",\n \"7.Adjust the resistor 78 and 74 to achieve the desired output amplitude.\",\n \"It will be appreciated by those of ordinary skill in the art that, as well as the digital implementation of the invention, it is also possible for other components to be used to the same effect for different parts of the circuits disclosed.\",\n \"For example, the filters in the circuits of FIGS.\",\n \"1, 8 and 10 could be any suitable active or passive arrangement that provides the required cut-off frequency and attenuation rates.\",\n \"Examples include Butterworth, Elliptical and Bessel filters, and infinite and finite impulse response filters in the digital domain.\",\n \"The purpose of the filters 26, 28 or 60, 64 for the microphone inputs is to reduce the typically sharp spikes of noise components in a signal caused by high frequency noise in a given spectrum so that they are in a more smooth (high frequency attenuated) form.\",\n \"It is found that the attenuation of sharp unfiltered spikes of noise is less effective when there is less than perfect phase and amplitude comparison.\",\n \"This has the effect of reducing the sensitivity of the circuit to phase shifts introduced by circuit components and/or the microphones.\",\n \"This significantly improves the noise cancellation performance.\",\n \"Because the invention does not rely on precise matching of components, the increased tolerance derived from low pass filtering the signals is of particularly beneficial effect.\",\n \"It will be apparent from the foregoing that the present invention can be realised in many different ways.\",\n \"The invention is not limited to those described herein, but only according to the spirit and scope of the following claims.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10471470"}}},{"rowIdx":599,"cells":{"text":{"kind":"list like","value":[["Method for determining synthetic term senses using reference text","A method for determining term senses by identifying terms having multiple “senses” or meanings.","For a given source term and trigger term, a list of important terms having high relevance to a combination of the source term and trigger term is created.","The list of important terms is determined to be a “sense” for the source term in accordance with the present invention.","A sense is assigned to a given term of a given document by determining similarity of the document to one or more senses and assigning a sense as a function of the degree of similarity.","A sense-indicative index is created to treat multiple occurrences of a term distinctly, as a function of each respective assigned sense.","Accordingly, the index may be used for sense-relevant information retrieval when a sense of a query term is discernible or specified."],["1.A method for determining a sense of a source term of a document, the method comprising the steps of: (a) identifying a trigger term relating to said source term; (b) identifying a plurality of important terms relating to a combination of said source term and said trigger term; and (c) establishing a sense of said source term, said sense comprising said plurality of important terms.","2.The method of claim 1, wherein step (c) comprises the step of: (c1) storing said plurality of important terms in association with said source term.","3.The method of claim 1, wherein step (a) comprises the step of: (a1) searching a group of documents for terms important to said source term.","4.The method of claim 1, wherein step (a) comprises the steps of: (a1) determining a reference frequency for each of a plurality of terms of a reference text, said reference frequency comprising a frequency of occurrence within said reference text; (a2) identifying a sample text comprising documents comprising said source term; (a3) determining a sample frequency for each of a plurality of terms of said sample text, said sample frequency comprising a frequency of occurrence within said sample text; (a4) for each of said plurality of terms of said sample text, comparing a respective sample frequency to a respective reference frequency to determine importance as a function of said respective frequencies by calculating a difference between said respective sample frequency and said respective reference frequency; (a5) assigning an importance score to each of said plurality of terms of said sample text, said importance score being determined as a function of said difference; and (a6) defining a plurality of trigger terms to comprise each of said plurality of terms of said sample text having a respective importance score exceeding a threshold.","5.The method of claim 1, wherein step (b) comprises the step of: (b1) searching a group of documents for terms important to said source term.","6.The method of claim 1, wherein step (b) comprises the steps of: (b1) determining a reference frequency for each of a plurality of terms of a reference text, said reference frequency comprising a frequency of occurrence within said reference text; (b2) identifying a sample text comprising documents comprising said source term; (b3) determining a sample frequency for each of a plurality of terms of said sample text, said sample frequency comprising a frequency of occurrence within said sample text; (b4) for each of said plurality of terms of said sample text, comparing a respective sample frequency to a respective reference frequency to determine importance as a function of said respective frequencies by calculating a difference between said respective sample frequency and said respective reference frequency; (b5) assigning an importance score to each of said plurality of terms of said sample text, said importance score being determined as a function of said difference; and (b6) defining said plurality of important terms to comprise each of said plurality of terms having a respective importance score exceeding a threshold.","7.A method for determining senses of a source term, the method comprising the steps of: (a) identifying a plurality of trigger terms relating to said source term; (b) for one of said plurality of trigger terms, identifying a plurality of important terms relating to a combination of said source term and said one of said plurality of trigger terms, said plurality of important terms comprising a sense of said source term; (c) removing from said plurality of trigger terms all of said plurality of important terms, if any, to define a reduced plurality of trigger terms; and (d) for one of said reduced plurality of trigger terms, identifying a next 11 plurality of important terms relating to a combination of said source term and said one of said reduced plurality of trigger terms, said next plurality of important terms comprising a next sense of said source term.","8.The method of claim 7, wherein step (a) comprises the steps of: (a1) determining a reference frequency for each of a plurality of terms of a reference text, said reference frequency comprising a frequency of occurrence within said reference text; (a2) identifying a sample text comprising documents comprising said source term; (a3) determining a sample frequency for each of a plurality of terms of said sample text, said sample frequency comprising a frequency of occurrence within said sample text; (a4) for each of said plurality of terms of said sample text, comparing a respective sample frequency to a respective reference frequency to determine importance as a function of said respective frequencies by calculating a difference between said respective sample frequency and said respective reference frequency; (a5) assigning an importance score to each of said plurality of terms of said sample text, said importance score being determined as a function of said difference; and (a6) defining said plurality of trigger terms to comprise each of said plurality of terms having a respective importance score exceeding a threshold.","9.A method for assigning a sense to a document's term for facilitating sense-relevant retrieval of said document by an information retrieval system, the method comprising the steps of: identifying a term of said document; identifying a first sense for said term, said first sense comprising terms relating to said term; comparing said first sense to said document to determine similarity; assigning said first sense to said term if said first sense and said document are sufficiently similar.","10.The method of claim 9, further comprising the steps of: identifying a next sense for said term if said first sense and said document are not sufficiently similar, said sense comprising terms relating to said term; comparing said next sense to said document to determine similarity; and assigning said next sense to said term if said next sense and said document are sufficiently similar.","11.The method of claim 9, wherein said comparing step is performed using a cosine measure technique.","12.The method of claim 9, wherein said assigning step comprises storing data associating said term with said first sense.","13.The method of claim 9, wherein said identifying step comprises referencing a memory storing a plurality of senses, each of said plurality of senses comprising a plurality of terms.","14.A method for assigning a sense to a document's term for facilitating sense-relevant retrieval of said document by an information retrieval system, the method comprising the steps of: identifying a term of said document; identifying a plurality of senses for said term, each of said plurality of senses 6 comprising a plurality of terms; comparing each of said plurality of senses to said document to determine similarity, assigning to said term a sense of said plurality of senses determined to have the greatest similarity.","15.The method of claim 14, wherein said comparing step comprises the step of generating a similarity score for each of said plurality of senses, and wherein said assigning step comprises the step of assigning to said term said sense of said plurality of senses determined to have the highest similarity score.","16.A method for preparing a group of documents for sense-relevant retrieval by an information retrieval system, the method comprising the steps of: (a) creating an index for said group of documents, said index associating each of a plurality of term identifiers with a corresponding set of document identifiers, each of said plurality of term identifiers being associated with a term, each of said set of document identifiers being associated with a document; (b) for each of said term identifiers, identifying a sense corresponding to a respective term and a respective document associated with a respective one of said corresponding set of document identifiers, said sense comprising a plurality of important terms; (c) for each of said term identifiers, creating at least one sensed term identifier, each said sensed term identifier corresponding to a respective term identifier and a corresponding sense; and (d) creating a sensed index for said group of documents, said sensed index associating each of said sensed term identifiers with a corresponding set of document identifiers, each of said sensed term identifiers being associated with a respective term and a respective sense.","17.An information processing system for determining a sense of a source term of a document, the system comprising: a central processing unit (CPU) for executing programs; a memory operatively connected to said CPU; a first program stored in said memory and executable by said CPU for identifying a trigger term relating to said source term; and a second program stored in said memory and executable by said CPU for identifying a plurality of important terms relating to a combination of said source term and said trigger term, said sense comprising said plurality of important terms.","18.An information processing system for assigning a sense to a document's term for facilitating sense-relevant retrieval of the document by an information retrieval system, the system comprising: a central processing unit (CPU) for executing programs; a memory operatively connected to said CPU; a first program stored in said memory and executable by said CPU for identifying a term of said document; a second program stored in said memory and executable by said CPU for identifying a first sense for said term, said sense comprising terms relating to said term; a third program stored in said memory and executable by said CPU for comparing 11 said first sense to said document to determine similarity; and a fourth program stored in said memory and executable by said CPU for assigning said first sense to said term if said first sense and said document are sufficiently similar.","19.An information processing system for preparing a group of documents for sense-relevant retrieval by an information retrieval system, the system comprising: a central processing unit (CPU) for executing programs; a memory operatively connected to said CPU; a first program stored in said memory and executable by said CPU for creating an index for said group of documents, said index associating each of a plurality of term identifiers with a corresponding set of document identifiers, each of said plurality of term identifiers being associated with a term, each of said set of document identifiers being associated with a document; a second program stored in said memory and executable by said CPU for identifying, for each of said term identifiers, a sense corresponding to a respective term and a respective document associated with a respective one of said corresponding set of document identifiers, said sense comprising a plurality of important terms; a third program stored in said memory and executable by said CPU for creating, for each of said term identifiers, at least one sensed term identifier, each said sensed term identifier corresponding to a respective term identifier and a corresponding sense; and a fourth program stored in said memory and executable by said CPU for creating a sensed index for said group of documents, said sensed index associating each of said sensed term identifiers with a corresponding set of document identifiers, each of said sensed term identifiers being associated with a respective term and a respective sense.","20.An information processing system for facilitating sense-relevant retrieval of a document by an information retrieval system, the system comprising: a central processing unit (CPU) for executing programs; a memory operatively connected to said CPU; a document stored in said memory, said document comprising a term; and data stored in said memory associating said term with a sense comprising a plurality of terms.","21.The information processing system of claim 20, further comprising: data stored in said memory identifying said plurality of terms.","22.The information processing system of claim 21, further comprising: a first program stored in said memory and executable by said CPU for comparing said plurality of terms to a search query.","23.The information processing system of claim 22, further comprising: a second program stored in said memory and executable by said CPU for identifying said document as a relevant search result for said search query if said first program determines sufficient similarity between said plurality of terms and said search query."],["<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates generally to computerized systems for processing, searching and retrieving information.","In particular, the present invention relates to techniques for determining a “sense” for a term that may be used, for example, to provide more accurate search results by seeking to match not only search terms but also the search term's sense.","2.Description of the Related Art Computerized text-based information retrieval systems are now in widespread use in database, intranet and internet (e.g., World Wide Web) applications.","Such systems typically search a large group of documents according to search query terms provided by a user to identify a subset of documents of the group of documents that are likely of interest to the user based on the search query terms.","Since a very large number of search results is typically obtained, it is burdensome to the user to identify the relatively few documents that are actually of interest to the user.","This problem is exacerbated by the fact that many document terms and search terms have multiple contextual meanings.","As used herein, “term” is used broadly and may include, for example, a word, a stemmed word, a character n-gram, phrase, or any other grouping that may be used to characterize text.","Term meanings are typically not accounted for as part of the automated search process.","For example, when a term has more than one “sense”, documents are often returned as search results when there is a term match, but not necessarily a sense match.","For illustrative purposes, consider a Web-based search engine operated by a user to search the Web for documents relating to the search term “jaguar”.","The term “jaguar” has multiple senses, including an automotive sense, an animal sense, and a sports sense.","As a result, when the search engine retrieves documents that contain the word “jaguar”, some will likely relate to Jaguar cars, others will likely relate to jaguar animals, and yet others will likely relate to a Jaguars football team.","This is undesirable because a user must review an unnecessarily large set of search results including all such senses of the search term, although the user may be interested only in documents in which the term is used in one of the term's senses.","What is needed is a method for determining a term's sense for facilitating automated sense-relevant information retrieval."],["<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is directed toward a method and apparatus for determining synthetic term senses for facilitating automated sense-relevant information processing, such as automated sense-relevant information retrieval.","Conceptually, the present invention can be embodied in two principal aspects, which may be combined.","The first aspect may involve using reference text, e.g.","a group of documents, to determine senses of terms.","In effect, this establishes a “library” of senses for terms in the large body of documents.","This may be performed before any search queries are processed, and/or may be ongoing as new documents are added to the collection.","In contrast to using a dictionary to identify a “sense”, this method is advantageous because it provides “synthetic senses”—some of which likely cannot be found in a dictionary.","Additionally, this method excludes dictionary senses that do not appear in the reference text, and therefore are of little relevance when searching reference text.","The senses are “synthetic” in that they are based on document collection statistics, e.g.","term frequencies, rather than dictionary definitions.","The second aspect involves assigning an appropriate sense to a term in a given document or query.","In effect, this provides an indication of the terms' senses for each document in the large body of documents.","For example, the term's sense for a given document may be used for sense-relevant information retrieval, cross-language translations, etc.","Once term senses have been determined and/or assigned in accordance with the present invention, sense-relevant automated information retrieval may be performed using known information retrieval and indexing techniques by using appropriate term senses in the index and in the query instead of unsensed terms, i.e.","terms to which senses have not been assigned.","In addition to sense-relevant automated information retrieval, the present invention may be used to provide senses for use in the context of a thesaurus, a cross-language dictionary, or a document index."],["CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of prior filed co-pending U.S.","Application Nos.","60/271,962 and 60/271,960, both filed Feb. 28, 2001, the disclosures of which are hereby incorporated herein by reference.","BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates generally to computerized systems for processing, searching and retrieving information.","In particular, the present invention relates to techniques for determining a “sense” for a term that may be used, for example, to provide more accurate search results by seeking to match not only search terms but also the search term's sense.","2.Description of the Related Art Computerized text-based information retrieval systems are now in widespread use in database, intranet and internet (e.g., World Wide Web) applications.","Such systems typically search a large group of documents according to search query terms provided by a user to identify a subset of documents of the group of documents that are likely of interest to the user based on the search query terms.","Since a very large number of search results is typically obtained, it is burdensome to the user to identify the relatively few documents that are actually of interest to the user.","This problem is exacerbated by the fact that many document terms and search terms have multiple contextual meanings.","As used herein, “term” is used broadly and may include, for example, a word, a stemmed word, a character n-gram, phrase, or any other grouping that may be used to characterize text.","Term meanings are typically not accounted for as part of the automated search process.","For example, when a term has more than one “sense”, documents are often returned as search results when there is a term match, but not necessarily a sense match.","For illustrative purposes, consider a Web-based search engine operated by a user to search the Web for documents relating to the search term “jaguar”.","The term “jaguar” has multiple senses, including an automotive sense, an animal sense, and a sports sense.","As a result, when the search engine retrieves documents that contain the word “jaguar”, some will likely relate to Jaguar cars, others will likely relate to jaguar animals, and yet others will likely relate to a Jaguars football team.","This is undesirable because a user must review an unnecessarily large set of search results including all such senses of the search term, although the user may be interested only in documents in which the term is used in one of the term's senses.","What is needed is a method for determining a term's sense for facilitating automated sense-relevant information retrieval.","SUMMARY OF THE INVENTION The present invention is directed toward a method and apparatus for determining synthetic term senses for facilitating automated sense-relevant information processing, such as automated sense-relevant information retrieval.","Conceptually, the present invention can be embodied in two principal aspects, which may be combined.","The first aspect may involve using reference text, e.g.","a group of documents, to determine senses of terms.","In effect, this establishes a “library” of senses for terms in the large body of documents.","This may be performed before any search queries are processed, and/or may be ongoing as new documents are added to the collection.","In contrast to using a dictionary to identify a “sense”, this method is advantageous because it provides “synthetic senses”—some of which likely cannot be found in a dictionary.","Additionally, this method excludes dictionary senses that do not appear in the reference text, and therefore are of little relevance when searching reference text.","The senses are “synthetic” in that they are based on document collection statistics, e.g.","term frequencies, rather than dictionary definitions.","The second aspect involves assigning an appropriate sense to a term in a given document or query.","In effect, this provides an indication of the terms' senses for each document in the large body of documents.","For example, the term's sense for a given document may be used for sense-relevant information retrieval, cross-language translations, etc.","Once term senses have been determined and/or assigned in accordance with the present invention, sense-relevant automated information retrieval may be performed using known information retrieval and indexing techniques by using appropriate term senses in the index and in the query instead of unsensed terms, i.e.","terms to which senses have not been assigned.","In addition to sense-relevant automated information retrieval, the present invention may be used to provide senses for use in the context of a thesaurus, a cross-language dictionary, or a document index.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 is a flow diagram illustrating an exemplary method for determining senses for a source term according to the present invention; FIG.","2 is a flow diagram illustrating an exemplary method for assigning a sense to a term of a document according to the present; FIG.","3 is a flow diagram illustrating an exemplary method for creating a sense-indicative index according to the present invention; FIG.","4 is a flow diagram illustrating an exemplary method for invention refining senses for a source term according to the present invention; and FIG.","5 is a block diagram of an information retrieval system in accordance with the present invention.","DETAILED DESCRIPTION FIG.","1 is a flow diagram 10 illustrating an exemplary method for determining senses for a source term according a first aspect of the present invention.","As used herein, a “source term” is a term for which it is desired to determine senses.","As shown in FIG.","1, the method starts with identification of a source term, as shown at steps 11 and 12.The source term may be identified in any suitable manner.","For example, a source term may be arbitrarily or randomly selected, specified by a user, etc.","Alternatively, for a given group of documents that are desired to be classified and/or searched by sense, the documents may be indexed to identify the most frequently occurring terms and terms having a frequency above a desired threshold may be treated as source terms in order of decreasing frequencies.","Various indexing techniques are well known in the art.","Any suitable alternative may be used to identify a source term, as will be understood by those skilled in the art.","Preferably, a method for selecting source terms is used so that all or nearly all document terms (e.g.","excluding stop terms) are eventually selected as source terms.","A list of trigger terms is then identified, as shown at step 14.As used herein, a “trigger term” is a term that is selected for use to identify senses for a source term.","Accordingly, it is often advantageous that each of the trigger terms has a relatively high relevance to the source term, i.e.","it is related to the source term.","Any suitable method may be used for identification of trigger terms.","Various techniques are known in the art for finding terms relating to a given term, such as those that involve use of the Jaccard coefficient, the cosine coefficient, and Yule's coefficient, as are well known in the art.","For example, trigger terms may be identified as terms of an affinity set as described in U.S.","Provisional Application No.","60/271,962.Generally, the affinity set technique identifies as terms important to a subset of documents, those terms that appear more frequently in the subset than in the entire set of documents.","For example, that technique involves determining a frequency of occurrence within sample text of terms of the sample text.","Each term's frequency for the sample text is then compared with a reference frequency, such as the term's frequency of occurrence within a reference text, e.g.","a large text sample.","Importance to the sample text is determined as a function of a difference between the frequencies.","An importance score is assigned to each of the plurality of terms of the target text and an affinity set is defined to include terms of sufficient importance, e.g.","as reflected by an importance score exceeding a desired threshold.","By using the source term as a search query to identify the sample text, the important terms of the sample text are terms related to the source term, and these important terms may be used to help define different senses of the source term, and so are useful as trigger terms for the present invention.","As an illustration of steps 12 and 14, consider that “red” is selected as a source term, and that “wine, cabernet, cross, merlot, fiscal” are identified as trigger terms that are related to red and that are likely usable to determine senses of “red.” For this example, it is assumed that each trigger term has been assigned an importance score and that the list has been sorted in order of decreasing importance score.","It must then be determined what are the senses of “red”, or more specifically, what words should be grouped with “red” to define a first sense, a second sense, etc.","This is achieved in steps 16-24, as discussed below.","A first term from the list of trigger terms is then selected, as shown at step 16 of FIG.","1.For example, the trigger term having the highest importance score may be selected first, if the trigger terms have been assigned importance scores.","Subsequent trigger terms may then be selected in order of decreasing importance score.","Accordingly, consider that “wine” is selected as the first trigger.","A list of important terms having high relevance to a combination of the source term and the trigger term is then identified, as shown at step 18 of FIG.","1.This may be carried out using any suitable method.","For example, the source term and trigger term may be combined in a search query with a Boolean AND operator, and the search query may be executed to search reference text, the search results being used as the sample text, and the affinity set technique described above may be used to identify an affinity set including important terms of the sample text.","Alternatively, the important terms may be the terms that appear most frequently, the terms appearing with a frequency above a threshold, the top X most frequently appearing terms, etc.","Other suitable methods for determining important terms will be apparent to those skilled in the art and any suitable method may be used.","This list of important terms is referred to herein as a “sense” of the source term.","More specifically, the sense is a list of terms that often co-occur with the source term when the source term is used in an associated sense, with a certain meaning, in a certain context, etc.","To illustrate step 18 of FIG.","1, consider that a query including “red” and “wine” is executed to identify relevant documents.","The relevant documents are then analyzed, e.g., using the affinity set technique described in U.S.","Provisional Application No.","60/271,962, to identify important terms including “cabernet, merlot, blush, grape, vineyard, tasting”.","These important terms define a first sense for the term “red”, namely, a sense associated with wine.","Accordingly, any documents containing these terms, and the term “red”, is probably using “red” in the context of wine.","Additionally, any document containing the term “red”, and being significantly similar to this sense, i.e.","list of important terms, is probably using “red” in the context of wine.","Various methods are known for determining similarity of documents, and any suitable method may be used, such as a cosine measure, a probabilistic measure, etc.","According to the present invention, the list of terms of the sense is treated as a document and a known similarity technique is used to determine similarity to assign a sense, as discussed in greater detail below with reference to FIG.","2.Optionally, as shown in FIG.","1, each new sense determined in step 18 is compared to each previously determined sense for a given source term to determine similarity, as shown at step 20.Various techniques are known for determining such similarity and any suitable technique may be used.","For example, such techniques include cosine measures, probabilistic measures, and other techniques well known in the art.","If a high degree of similarity exists (according to user or system preferences), it may be advantageous to skip to the next trigger term (e.g.","by skipping to step 24, as shown), or to stop the process of FIG.","1 (i.e.","to end by skipping to step 27) and/or to repeat the process of FIG.","1 for a next source term.","Such similarity indicates that the same or very similar senses are being repeatedly identified.","It has been determined that once this begins to happen, it happens very frequently, and relatively few new senses will likely be found, at least during the present iteration of FIG.","1.Optionally, the sense for the source term determined in step 18 is stored as a first sense, e.g.","in a memory of an information processing system, as shown at step 22 of FIG.","1.This sense may be referenced later as necessary, e.g.","when assigning a sense to a term of a document, as discussed below with reference to FIG.","4.Although a first sense of the source term has been identified, it is likely that additional senses exist.","Accordingly, it is useful to repeat the process to identify additional senses for the source term.","To enhance efficiency of the process, and to prevent identification of highly similar senses, the terms from the first sense are first deleted, removed, or otherwise flagged as used or unusable, etc., from the list of trigger terms identified in step 14, as shown at step 24.In this manner, terms associated with the first sense will not be used as trigger terms when trying to find a next (different) sense.","Then, if the end of the list of trigger terms has not yet been reached, a next term is selected from the list of trigger terms and steps 16-26 are repeated, as shown.","The process of FIG.","1 ends when the end of the list of trigger terms is reached, as shown at steps 26 and 27.At this point, multiple senses have likely been determined for the source term.","To illustrate, consider that the terms “cabernet” and “merlot” (the only terms appearing in the sense list of important terms and the trigger term list) are deleted from the trigger term list in step 24.The next trigger term, “cross” is then selected in step 16 and is combined with “red” to form a query used to identify another set of search-relevant documents.","Important terms of these documents are identified as “blood, donations, international” in step 18 and define a second sense for the term red, namely a sense associated with the Red Cross organization.","Accordingly, any documents containing these terms, and the term red, is probably using “red” in the context of the Red Cross.","Preferably, the method of FIG.","1 is repeated for subsequent source terms until multiple senses have been identified for all source terms.","This creates a “library” of senses for use in assigning senses to document terms as discussed below with reference to FIG.","2.FIG.","2 is a flow diagram 30 illustrating an exemplary method for assigning a sense to a term of a document according to a second aspect of the present invention.","This method considers that a “library” of senses has already been established.","As shown in FIG.","2, the method starts with the identifying of a term of a document, as shown at steps 31 and 32.The term may be identified in any suitable manner.","For example, the document may be indexed to find a frequency of occurrence of each term within the document and the terms may be identified in order of decreasing frequency.","Alternatively, the terms may be identified in order of their first appearance in a document.","A set of senses for the term is next identified, as shown at step 34.For example, this step may include referencing a database of senses stored in a memory of an information processing system, e.g.","those stored in step 20 of FIG.","1.In the example of FIG.","2, each sense is compared to the document and assigned a respective similarity score according to its degree of similarity to the document, as shown at step 36.Many suitable techniques are known in the art for determining document similarity and assigning a similarity score.","Any suitable method for determining similarity may be used.","In accordance with the present invention, the sense, i.e.","the list of terms, is treated as a document for the purpose of determining similarity to another document.","In the example of FIG.","2, the term is assigned the sense having the highest respective similarity score, as shown at step 38, and the method ends, as shown at step 39.Suitable alternatives for assigning a sense having sufficient similarity will be apparent to those skilled in the art.","For example, senses may be identified and compared sequentially until a similarity score above a desired threshold is obtained, at which point the corresponding sense may be assigned to the term.","It should be noted that the method of FIG.","2 may be used to assign a single sense to all occurrences of the term in the document, which is generally acceptable.","Alternatively, the document may be divided into smaller units (e.g.","sentences, or the n terms preceding and the n terms following an occurrence of a source term), and senses may be assigned to each such unit separately.","In some embodiments, if a term is not sufficiently similar to any known (previously identified) sense, the term is assigned a generic sense indicating that the sense is presently unknown or cannot yet be classified.","Terms assigned to such a class may be later assigned a sense as additional senses are identified (e.g.","as the steps of FIGS.","1 and 2 are repeated) and/or through sense-refinement, as discussed below with respect to FIG.","4.Preferably, but not exclusively, the method of FIG.","2 is repeated for each term (or selected term) of the document, and for each document of the group of documents.","Accordingly, each document is provided with sense-relevant information that may be used for sense-relevant information retrieval purposes, cross-language translations, etc.","Accordingly, after step 2, terms that have been assigned a sense according to FIG.","2 are distinguishable from one another.","In particular, the terms may be distinguishable by indexing software.","Various indexing software and techniques are well known in the art.","Various approaches can be used to cause standard/known indexing software to distinguish between terms to which senses have been assigned.","In one embodiment, sense-distinctive string equivalents may be inserted into the text.","For example, consider that the term “bank” is determined to have ten different senses, and that “bank” in a certain document has the fourth sense.","Accordingly, the sense-distinctive string “bank#4” may be substituted into the document for each occurrence of “bank” so that when a usual indexing method is performed on the document “bank#4” appears distinct from “bank#3”, “bank#2”, etc.","appearing in other documents.","When a search query is performed for “bank#4”, or a user provides a search term of “bank” and it is specified by the user or determined by a system that the fourth sense is sought, appropriate documents may be identified in a straightforward manner using known information retrieval techniques, as will be apparent to those skilled in the art.","Exemplary, but not limiting, methods for identifying a sense for a search query are provided below.","In an alternate embodiment, sense determinations are made dynamically as a document is indexed.","More specifically, as each term of the document is identified for indexing purposes as known in the art, the term's sense is determined according to the method of FIG.","2.Then, a reference representing that sense of the word may be created or specified for further processing in the same way that the system represents a new, distinct term, e.g.","the string “bank#1” or an integer not already used to represent any other term or sense.","This requires straightforward modification of known indexing techniques to incorporate the method of FIG.","2 where appropriate, as will be apparent to those skilled in the art.","In yet another alternative embodiment, senses are assigned to an internal representation of each document, not to the document itself.","For example, an information retrieval system may internally represent a document as a document identifier, followed by a list of terms or term identifiers (collectively “term identifiers”) contained in the document, and an occurrence count for each of those term identifiers.","A sensed version, i.e.","a version in which senses have been assigned to term identifiers, of such an internal representation may be created by performing the process of FIG.","2 for each term identifier to determine which sense of the term identifier is most similar to the document, and replacing the original term identifier with a new term identifier representing the desired sense, e.g.","to replace “bank” with “bank#3”.","This process may be applied to each document representation.","Other data structures (e.g., an ‘inverted index’) that are part of the index can then be rebuilt by referring to the new document representations including the new term identifiers.","Alternatively, other data structures may be modified to reflect the identified senses.","For example, an inverted index typically includes a pairing of each term identifier with a list of document identifiers indicating the documents that contain that term.","To create a sense-indicative index, each such pairing is replaced by a set of pairings, one for each term sense, each of which associates the term identifier for a certain sense with a list of document identifiers indicating the documents associated with that sense.","The sense-indicative index may then be used for information retrieval purposes in a straightforward manner, provided that a sense of a search term and/or query may be discerned or is provided.","By way of further example, FIG.","3 is a flow diagram 40 illustrating an exemplary method for creating a sense-indicative index according to the present invention that may be used, for example, for sense relevant information retrieval.","As shown in FIG.","3, the method starts with creating an index for a group of documents using any well-known indexing method, as shown at steps 41 and 42.In this example, the index associates term identifiers with document identifiers to indicate that a term corresponding to a given term identifier may be found in documents corresponding to the associated document identifiers.","For each term identifier, a corresponding term is identified, and the term is assigned a sense for each document in which it appears, using the method of FIG.","2 as described above.","This is repeated for all document identifiers (and corresponding documents) for a given term identifier (and corresponding term), as shown at steps 44-52.When all document identifiers have been considered, and sense assigned, all documents corresponding to the document identifiers associated with the given term identifier are grouped by sense to create sense subgroups, as shown at step 54.For example, if term identifier A is associated with document identifiers 1,2 and 3, and the term is assigned sense Y for documents 1 and 3 and sense Z for document 2, documents 1 and 3 are grouped for sense Y and document 2 is grouped for sense Z.","A sensed term identifier is then created for each sense subgroup, e.g.","TermIdentifierY, TermIdentifierZ, as shown at step 56.The sensed term identifier is then stored in association with all document identifiers corresponding to the documents in the sense subgroup, as shown at step 58.For example, TermIdentifierY would be stored in correspondence with the document identifiers for documents 1 and 3.This is repeated for all term identifiers as shown at step 60.From this information, a sensed index is created as shown at step 62 to associate the sensed term identifiers with corresponding document identifiers.","In this manner, a new index has been created that is sense-indicative, and each term identifier from the index is replaced with one or more sense-indicative term identifiers, each of which is associated with a different sense of the associated term.","Each of the sense-indicative identifiers can then be treated as a unique term by an information retrieval system referencing the sense-indicative index to allow for sense relevant information retrieval when a sense for a search term and/or query is known.","Because the sense differentiation is performed at the document and/or index level, known information retrieval techniques may be used with little or no modification, as will be apparent to those skilled in the art.","A sense for a search term and/or query may be determined in various ways.","For example, a search query provided by a user is treated as a document and the method of FIG.","2 is used to identify a sense for each term of the search query.","Alternatively, a user entering a term into a search engine is asked to specify a sense, e.g.","by picking from a pick list displayed to the user and showing terms for each sense, using prior knowledge of the user or a user-profile to determine a likely sense, etc.","Alternatively, the search query may be executed on an unsensed group of documents, i.e.","a group to which senses have not been assigned, to retrieve documents, the senses of terms in those documents may be identified “on the fly”, e.g.","as part of the document retrieval process, or in a pre-search processing step, and a most common sense for each of the search term(s) may be used as the query that is executed against the sensed collection of documents.","Various other techniques will be apparent to those skilled in the art and any suitable technique may be used.","It should be noted that sensing of the documents and/or an index of the documents may be refined by repeating the steps of FIGS.","1 and 2 for a given body of documents.","In effect, the first iteration applies sense to an unsensed collection of documents, while the second iteration applies senses to a sensed collection of documents.","By identifying senses in a collection of documents to which senses have already been assigned, cross-pollution of senses is reduced, and more subtle senses may be detected.","The resulting sense may be applied either to the sensed document collection, or to the original unsensed collection.","FIG.","4 is a flow diagram 70 illustrating an exemplary method for iteratively refining a sense for a source term according to the present invention.","Optionally, this method for refining senses for a source term may be used to implement step 18 of FIG.","1.It may be desirable to use this method because a sense initially identified may include terms that actually represent more than one sense.","This method helps to refine the senses so that multiple “senses” of a term are not conflated in a single sense.","As shown in FIG.","4, the method starts with creation of an initially empty set of subtrigger lists, as shown at steps 71 and 72.An initial subtrigger list is identified that includes subtrigger terms having high relevance to a combination of the source term and the trigger term, as shown at step 74.As used herein, a “subtrigger term” is a term that is selected for use to identify senses for a source term, and preferably is a term related to the source term.","Such terms may be identified using the affinity set technique described in U.S.","Provisional Application No.","60/271,962 and as discussed above.","More specifically, the source term and trigger term are used to identify documents used as the sample text, and important terms of such documents (e.g.","as determined using the affinity set technique) are used as the subtrigger terms.","The initial subtrigger list is then added to the set of subtrigger lists and all subtrigger lists are combined to form a unified subtrigger list including all subtrigger terms from all subtrigger lists, as shown at steps 76 and 78.The terms of the unified subtrigger list are preferably ranked in order of importance, e.g.","in order of decreasing importance score.","For example, the importance score for a term in the unified subtrigger list that appears on one or more subtrigger lists may be determined to be equal to its worst rank on any individual substrigger list, or equal to its worst score on any individual subtrigger list.","A first subtrigger term is then selected from the unified subtrigger list, as shown at step 80.This subtrigger term is a term that has not already been selected as a trigger term (e.g.","in FIG.","1) or as a subtrigger term (e.g.","in FIG.","4, as discussed further below).","A new subtrigger list is then identified that includes terms having high relevance to a combination of the source term and the first subtrigger term, as shown at step 82.For example, such terms can be identified using the affinity set technique, wherein the source term and first subtrigger term are used to identify documents used as the sample text, and wherein the important terms of such documents are used as the new subtrigger terms.","All terms from the new subtrigger list are then deleted from the list of trigger terms identified in step 14 of FIG.","1, as shown at step 84.The new subtrigger list is then added to the set of subtrigger lists as shown at step 86.These steps (78 to 86) may be repeated for as many iterations as desired, as shown at step 88.More iterations generally providing a higher degree of sense refinement but an increased processing costs.","It has been found that between 1 and 3 iterations provide adequate results.","If the desired number of iterations has been completed, the sense refinement process ends with combination of all subtrigger lists to form a list of important terms, as shown at steps 88, 90 and 91.The list of important terms is then stored as a sense for the source term, as shown at step 22 of FIG.","1.By way of example, each important term appearing on multiple term lists may be assigned a respective importance score equal to its worst rank or its worst score on any individual subtrigger list before those lists are combined and ordered according to importance score.","It should be noted that the present invention is useful for various purposes beyond automated information retrieval discussed above for illustrative purposes.","For example, the present invention may be used in the context of a thesaurus, a dictionary for cross-language translation, for document translation, or to compose a search query in a foreign language to search foreign documents as a function of a search query in a primary (native) language.","For example, consider that English is the primary language and translations in French are desired to be searched.","Further consider that an English language large text sample and a French language large text sample that is an aligned, parallel collection of the English language large text sample, meaning that it has a one-to-one correspondence of documents.","For example the Hansards of the Canadian legislature are published in both French and English and may be used as aligned, parallel collections of text.","Such a collection may be used to find an appropriate French translation for an English word by searching the English collection for documents relevant to the word to be translated, selecting the set of French documents that are translations of the retrieved English documents, extracting a set of important terms from the French documents so selected, and identifying the set's term or terms having the greatest importance as possible translation(s).","When this method is applied to an unsensed collection, a word that has many different translations (such as ‘bank,’ which may be translated into French either as ‘banque’ for a financial bank or as ‘rive’ for a river bank) may lead to an inappropriate translation.","If, however, the English documents have senses assigned to their words, then each such sense may be translated to different, sense appropriate, terms.","More specifically, a first French term of the set of important terms may be used as a trigger term and combined with the English search term to find a plurality of important terms relating to their combination, those terms defining a sense.","This can be performed for multiple French terms of the set of important terms to define multiple senses that may define alternative French “translations” for the corresponding English term.","FIG.","5 is a block diagram of an information processing system in accordance with the present invention.","As is well known in the art, the information processing system of FIG.","5 includes a general purpose microprocessor (CPU) 202 and a bus 204 employed to connect and enable communication between the microprocessor 202 and the components of the information processing system 200 in accordance with known techniques.","The information processing system 200 typically includes a user interface adapter 206, which connects the microprocessor 202 via the bus 204 to one or more interface devices, such as a keyboard 208, mouse 210, and/or other interface devices 212, which can be any user interface device, such as a touch sensitive screen, digitized entry pad, etc.","The bus 204 also connects a display device 214, such as an LCD screen or monitor, to the microprocessor 202 via a display adapter 216.The bus 204 also connects the microprocessor 202 to memory 218 and long-term storage 220 (collectively, “memory”) which can include a hard drive, diskette drive, tape drive, etc.","The information processing system 200 may communicate with other computers or networks of computers, for example via a communications channel, network card or modem 222.The information processing system 200 may be associated with such other computers in a local area network (LAN) or a wide area network (WAN), or the information processing system 200 can be a client or server in a client/server arrangement with another computer, etc.","All of these configurations, as well as the appropriate communications hardware and software, are known in the art.","Software programming code for carrying out this inventive method is typically stored in memory.","Accordingly, the information processing system 200 stores in its memory microprocessor executable instructions.","When the information processing system 200 is configured for determining a sense of a source term of a document, the system stores in its memory a first program for identifying a trigger term relating to a source term, and a second program for identifying a plurality of important terms relating to a combination of the source term and the trigger term.","When the information processing system 200 is configured for assigning a sense to a document's term for facilitating sense-relevant retrieval of the document by an information retrieval system, it stores in its memory a first program for identifying a term of the document, a second program stored for identifying a first sense for the term, a third program for comparing the first sense to the document to determine similarity, and a fourth program for assigning the first sense to the term if the first sense and the document are sufficiently similar.","When the information processing system 200 is configured for preparing a group of documents for sense-relevant retrieval by an information retrieval system, it stores in its memory a first program for creating an index for the group of documents.","The index associates each of a plurality of term identifiers with a corresponding set of document identifiers.","Each of the plurality of term identifiers is associated with a term and each of the sets of document identifiers is associated with a document.","It further stores in its memory a second program for identifying, for each of the term identifiers, a sense corresponding to a respective term and a respective document associated with a respective one of the corresponding set of document identifiers.","The sense includes a plurality of important terms.","It also stores a third program for creating, for each of the term identifiers, at least one sensed term identifier, each sensed term identifier corresponding to a respective term identifier and a corresponding sense; and a fourth program stored for creating a sensed index for the group of documents.","The sensed index associates each of the sensed term identifiers with a corresponding set of document identifiers.","Each of the sensed term identifiers is associated with a term and a sense.","Having thus described particular embodiments of the invention, various alterations, modifications, and improvements will readily occur to those skilled in the art.","Such alterations, modifications and improvements as are made obvious by this disclosure are intended to be part of this description though not expressly stated herein, and are intended to be within the spirit and scope of the invention.","Accordingly, the foregoing description is by way of example only, and not limiting.","The invention is limited only as defined in the following claims and equivalents thereto."]],"string":"[\n [\n \"Method for determining synthetic term senses using reference text\",\n \"A method for determining term senses by identifying terms having multiple “senses” or meanings.\",\n \"For a given source term and trigger term, a list of important terms having high relevance to a combination of the source term and trigger term is created.\",\n \"The list of important terms is determined to be a “sense” for the source term in accordance with the present invention.\",\n \"A sense is assigned to a given term of a given document by determining similarity of the document to one or more senses and assigning a sense as a function of the degree of similarity.\",\n \"A sense-indicative index is created to treat multiple occurrences of a term distinctly, as a function of each respective assigned sense.\",\n \"Accordingly, the index may be used for sense-relevant information retrieval when a sense of a query term is discernible or specified.\"\n ],\n [\n \"1.A method for determining a sense of a source term of a document, the method comprising the steps of: (a) identifying a trigger term relating to said source term; (b) identifying a plurality of important terms relating to a combination of said source term and said trigger term; and (c) establishing a sense of said source term, said sense comprising said plurality of important terms.\",\n \"2.The method of claim 1, wherein step (c) comprises the step of: (c1) storing said plurality of important terms in association with said source term.\",\n \"3.The method of claim 1, wherein step (a) comprises the step of: (a1) searching a group of documents for terms important to said source term.\",\n \"4.The method of claim 1, wherein step (a) comprises the steps of: (a1) determining a reference frequency for each of a plurality of terms of a reference text, said reference frequency comprising a frequency of occurrence within said reference text; (a2) identifying a sample text comprising documents comprising said source term; (a3) determining a sample frequency for each of a plurality of terms of said sample text, said sample frequency comprising a frequency of occurrence within said sample text; (a4) for each of said plurality of terms of said sample text, comparing a respective sample frequency to a respective reference frequency to determine importance as a function of said respective frequencies by calculating a difference between said respective sample frequency and said respective reference frequency; (a5) assigning an importance score to each of said plurality of terms of said sample text, said importance score being determined as a function of said difference; and (a6) defining a plurality of trigger terms to comprise each of said plurality of terms of said sample text having a respective importance score exceeding a threshold.\",\n \"5.The method of claim 1, wherein step (b) comprises the step of: (b1) searching a group of documents for terms important to said source term.\",\n \"6.The method of claim 1, wherein step (b) comprises the steps of: (b1) determining a reference frequency for each of a plurality of terms of a reference text, said reference frequency comprising a frequency of occurrence within said reference text; (b2) identifying a sample text comprising documents comprising said source term; (b3) determining a sample frequency for each of a plurality of terms of said sample text, said sample frequency comprising a frequency of occurrence within said sample text; (b4) for each of said plurality of terms of said sample text, comparing a respective sample frequency to a respective reference frequency to determine importance as a function of said respective frequencies by calculating a difference between said respective sample frequency and said respective reference frequency; (b5) assigning an importance score to each of said plurality of terms of said sample text, said importance score being determined as a function of said difference; and (b6) defining said plurality of important terms to comprise each of said plurality of terms having a respective importance score exceeding a threshold.\",\n \"7.A method for determining senses of a source term, the method comprising the steps of: (a) identifying a plurality of trigger terms relating to said source term; (b) for one of said plurality of trigger terms, identifying a plurality of important terms relating to a combination of said source term and said one of said plurality of trigger terms, said plurality of important terms comprising a sense of said source term; (c) removing from said plurality of trigger terms all of said plurality of important terms, if any, to define a reduced plurality of trigger terms; and (d) for one of said reduced plurality of trigger terms, identifying a next 11 plurality of important terms relating to a combination of said source term and said one of said reduced plurality of trigger terms, said next plurality of important terms comprising a next sense of said source term.\",\n \"8.The method of claim 7, wherein step (a) comprises the steps of: (a1) determining a reference frequency for each of a plurality of terms of a reference text, said reference frequency comprising a frequency of occurrence within said reference text; (a2) identifying a sample text comprising documents comprising said source term; (a3) determining a sample frequency for each of a plurality of terms of said sample text, said sample frequency comprising a frequency of occurrence within said sample text; (a4) for each of said plurality of terms of said sample text, comparing a respective sample frequency to a respective reference frequency to determine importance as a function of said respective frequencies by calculating a difference between said respective sample frequency and said respective reference frequency; (a5) assigning an importance score to each of said plurality of terms of said sample text, said importance score being determined as a function of said difference; and (a6) defining said plurality of trigger terms to comprise each of said plurality of terms having a respective importance score exceeding a threshold.\",\n \"9.A method for assigning a sense to a document's term for facilitating sense-relevant retrieval of said document by an information retrieval system, the method comprising the steps of: identifying a term of said document; identifying a first sense for said term, said first sense comprising terms relating to said term; comparing said first sense to said document to determine similarity; assigning said first sense to said term if said first sense and said document are sufficiently similar.\",\n \"10.The method of claim 9, further comprising the steps of: identifying a next sense for said term if said first sense and said document are not sufficiently similar, said sense comprising terms relating to said term; comparing said next sense to said document to determine similarity; and assigning said next sense to said term if said next sense and said document are sufficiently similar.\",\n \"11.The method of claim 9, wherein said comparing step is performed using a cosine measure technique.\",\n \"12.The method of claim 9, wherein said assigning step comprises storing data associating said term with said first sense.\",\n \"13.The method of claim 9, wherein said identifying step comprises referencing a memory storing a plurality of senses, each of said plurality of senses comprising a plurality of terms.\",\n \"14.A method for assigning a sense to a document's term for facilitating sense-relevant retrieval of said document by an information retrieval system, the method comprising the steps of: identifying a term of said document; identifying a plurality of senses for said term, each of said plurality of senses 6 comprising a plurality of terms; comparing each of said plurality of senses to said document to determine similarity, assigning to said term a sense of said plurality of senses determined to have the greatest similarity.\",\n \"15.The method of claim 14, wherein said comparing step comprises the step of generating a similarity score for each of said plurality of senses, and wherein said assigning step comprises the step of assigning to said term said sense of said plurality of senses determined to have the highest similarity score.\",\n \"16.A method for preparing a group of documents for sense-relevant retrieval by an information retrieval system, the method comprising the steps of: (a) creating an index for said group of documents, said index associating each of a plurality of term identifiers with a corresponding set of document identifiers, each of said plurality of term identifiers being associated with a term, each of said set of document identifiers being associated with a document; (b) for each of said term identifiers, identifying a sense corresponding to a respective term and a respective document associated with a respective one of said corresponding set of document identifiers, said sense comprising a plurality of important terms; (c) for each of said term identifiers, creating at least one sensed term identifier, each said sensed term identifier corresponding to a respective term identifier and a corresponding sense; and (d) creating a sensed index for said group of documents, said sensed index associating each of said sensed term identifiers with a corresponding set of document identifiers, each of said sensed term identifiers being associated with a respective term and a respective sense.\",\n \"17.An information processing system for determining a sense of a source term of a document, the system comprising: a central processing unit (CPU) for executing programs; a memory operatively connected to said CPU; a first program stored in said memory and executable by said CPU for identifying a trigger term relating to said source term; and a second program stored in said memory and executable by said CPU for identifying a plurality of important terms relating to a combination of said source term and said trigger term, said sense comprising said plurality of important terms.\",\n \"18.An information processing system for assigning a sense to a document's term for facilitating sense-relevant retrieval of the document by an information retrieval system, the system comprising: a central processing unit (CPU) for executing programs; a memory operatively connected to said CPU; a first program stored in said memory and executable by said CPU for identifying a term of said document; a second program stored in said memory and executable by said CPU for identifying a first sense for said term, said sense comprising terms relating to said term; a third program stored in said memory and executable by said CPU for comparing 11 said first sense to said document to determine similarity; and a fourth program stored in said memory and executable by said CPU for assigning said first sense to said term if said first sense and said document are sufficiently similar.\",\n \"19.An information processing system for preparing a group of documents for sense-relevant retrieval by an information retrieval system, the system comprising: a central processing unit (CPU) for executing programs; a memory operatively connected to said CPU; a first program stored in said memory and executable by said CPU for creating an index for said group of documents, said index associating each of a plurality of term identifiers with a corresponding set of document identifiers, each of said plurality of term identifiers being associated with a term, each of said set of document identifiers being associated with a document; a second program stored in said memory and executable by said CPU for identifying, for each of said term identifiers, a sense corresponding to a respective term and a respective document associated with a respective one of said corresponding set of document identifiers, said sense comprising a plurality of important terms; a third program stored in said memory and executable by said CPU for creating, for each of said term identifiers, at least one sensed term identifier, each said sensed term identifier corresponding to a respective term identifier and a corresponding sense; and a fourth program stored in said memory and executable by said CPU for creating a sensed index for said group of documents, said sensed index associating each of said sensed term identifiers with a corresponding set of document identifiers, each of said sensed term identifiers being associated with a respective term and a respective sense.\",\n \"20.An information processing system for facilitating sense-relevant retrieval of a document by an information retrieval system, the system comprising: a central processing unit (CPU) for executing programs; a memory operatively connected to said CPU; a document stored in said memory, said document comprising a term; and data stored in said memory associating said term with a sense comprising a plurality of terms.\",\n \"21.The information processing system of claim 20, further comprising: data stored in said memory identifying said plurality of terms.\",\n \"22.The information processing system of claim 21, further comprising: a first program stored in said memory and executable by said CPU for comparing said plurality of terms to a search query.\",\n \"23.The information processing system of claim 22, further comprising: a second program stored in said memory and executable by said CPU for identifying said document as a relevant search result for said search query if said first program determines sufficient similarity between said plurality of terms and said search query.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates generally to computerized systems for processing, searching and retrieving information.\",\n \"In particular, the present invention relates to techniques for determining a “sense” for a term that may be used, for example, to provide more accurate search results by seeking to match not only search terms but also the search term's sense.\",\n \"2.Description of the Related Art Computerized text-based information retrieval systems are now in widespread use in database, intranet and internet (e.g., World Wide Web) applications.\",\n \"Such systems typically search a large group of documents according to search query terms provided by a user to identify a subset of documents of the group of documents that are likely of interest to the user based on the search query terms.\",\n \"Since a very large number of search results is typically obtained, it is burdensome to the user to identify the relatively few documents that are actually of interest to the user.\",\n \"This problem is exacerbated by the fact that many document terms and search terms have multiple contextual meanings.\",\n \"As used herein, “term” is used broadly and may include, for example, a word, a stemmed word, a character n-gram, phrase, or any other grouping that may be used to characterize text.\",\n \"Term meanings are typically not accounted for as part of the automated search process.\",\n \"For example, when a term has more than one “sense”, documents are often returned as search results when there is a term match, but not necessarily a sense match.\",\n \"For illustrative purposes, consider a Web-based search engine operated by a user to search the Web for documents relating to the search term “jaguar”.\",\n \"The term “jaguar” has multiple senses, including an automotive sense, an animal sense, and a sports sense.\",\n \"As a result, when the search engine retrieves documents that contain the word “jaguar”, some will likely relate to Jaguar cars, others will likely relate to jaguar animals, and yet others will likely relate to a Jaguars football team.\",\n \"This is undesirable because a user must review an unnecessarily large set of search results including all such senses of the search term, although the user may be interested only in documents in which the term is used in one of the term's senses.\",\n \"What is needed is a method for determining a term's sense for facilitating automated sense-relevant information retrieval.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is directed toward a method and apparatus for determining synthetic term senses for facilitating automated sense-relevant information processing, such as automated sense-relevant information retrieval.\",\n \"Conceptually, the present invention can be embodied in two principal aspects, which may be combined.\",\n \"The first aspect may involve using reference text, e.g.\",\n \"a group of documents, to determine senses of terms.\",\n \"In effect, this establishes a “library” of senses for terms in the large body of documents.\",\n \"This may be performed before any search queries are processed, and/or may be ongoing as new documents are added to the collection.\",\n \"In contrast to using a dictionary to identify a “sense”, this method is advantageous because it provides “synthetic senses”—some of which likely cannot be found in a dictionary.\",\n \"Additionally, this method excludes dictionary senses that do not appear in the reference text, and therefore are of little relevance when searching reference text.\",\n \"The senses are “synthetic” in that they are based on document collection statistics, e.g.\",\n \"term frequencies, rather than dictionary definitions.\",\n \"The second aspect involves assigning an appropriate sense to a term in a given document or query.\",\n \"In effect, this provides an indication of the terms' senses for each document in the large body of documents.\",\n \"For example, the term's sense for a given document may be used for sense-relevant information retrieval, cross-language translations, etc.\",\n \"Once term senses have been determined and/or assigned in accordance with the present invention, sense-relevant automated information retrieval may be performed using known information retrieval and indexing techniques by using appropriate term senses in the index and in the query instead of unsensed terms, i.e.\",\n \"terms to which senses have not been assigned.\",\n \"In addition to sense-relevant automated information retrieval, the present invention may be used to provide senses for use in the context of a thesaurus, a cross-language dictionary, or a document index.\"\n ],\n [\n \"CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of prior filed co-pending U.S.\",\n \"Application Nos.\",\n \"60/271,962 and 60/271,960, both filed Feb. 28, 2001, the disclosures of which are hereby incorporated herein by reference.\",\n \"BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates generally to computerized systems for processing, searching and retrieving information.\",\n \"In particular, the present invention relates to techniques for determining a “sense” for a term that may be used, for example, to provide more accurate search results by seeking to match not only search terms but also the search term's sense.\",\n \"2.Description of the Related Art Computerized text-based information retrieval systems are now in widespread use in database, intranet and internet (e.g., World Wide Web) applications.\",\n \"Such systems typically search a large group of documents according to search query terms provided by a user to identify a subset of documents of the group of documents that are likely of interest to the user based on the search query terms.\",\n \"Since a very large number of search results is typically obtained, it is burdensome to the user to identify the relatively few documents that are actually of interest to the user.\",\n \"This problem is exacerbated by the fact that many document terms and search terms have multiple contextual meanings.\",\n \"As used herein, “term” is used broadly and may include, for example, a word, a stemmed word, a character n-gram, phrase, or any other grouping that may be used to characterize text.\",\n \"Term meanings are typically not accounted for as part of the automated search process.\",\n \"For example, when a term has more than one “sense”, documents are often returned as search results when there is a term match, but not necessarily a sense match.\",\n \"For illustrative purposes, consider a Web-based search engine operated by a user to search the Web for documents relating to the search term “jaguar”.\",\n \"The term “jaguar” has multiple senses, including an automotive sense, an animal sense, and a sports sense.\",\n \"As a result, when the search engine retrieves documents that contain the word “jaguar”, some will likely relate to Jaguar cars, others will likely relate to jaguar animals, and yet others will likely relate to a Jaguars football team.\",\n \"This is undesirable because a user must review an unnecessarily large set of search results including all such senses of the search term, although the user may be interested only in documents in which the term is used in one of the term's senses.\",\n \"What is needed is a method for determining a term's sense for facilitating automated sense-relevant information retrieval.\",\n \"SUMMARY OF THE INVENTION The present invention is directed toward a method and apparatus for determining synthetic term senses for facilitating automated sense-relevant information processing, such as automated sense-relevant information retrieval.\",\n \"Conceptually, the present invention can be embodied in two principal aspects, which may be combined.\",\n \"The first aspect may involve using reference text, e.g.\",\n \"a group of documents, to determine senses of terms.\",\n \"In effect, this establishes a “library” of senses for terms in the large body of documents.\",\n \"This may be performed before any search queries are processed, and/or may be ongoing as new documents are added to the collection.\",\n \"In contrast to using a dictionary to identify a “sense”, this method is advantageous because it provides “synthetic senses”—some of which likely cannot be found in a dictionary.\",\n \"Additionally, this method excludes dictionary senses that do not appear in the reference text, and therefore are of little relevance when searching reference text.\",\n \"The senses are “synthetic” in that they are based on document collection statistics, e.g.\",\n \"term frequencies, rather than dictionary definitions.\",\n \"The second aspect involves assigning an appropriate sense to a term in a given document or query.\",\n \"In effect, this provides an indication of the terms' senses for each document in the large body of documents.\",\n \"For example, the term's sense for a given document may be used for sense-relevant information retrieval, cross-language translations, etc.\",\n \"Once term senses have been determined and/or assigned in accordance with the present invention, sense-relevant automated information retrieval may be performed using known information retrieval and indexing techniques by using appropriate term senses in the index and in the query instead of unsensed terms, i.e.\",\n \"terms to which senses have not been assigned.\",\n \"In addition to sense-relevant automated information retrieval, the present invention may be used to provide senses for use in the context of a thesaurus, a cross-language dictionary, or a document index.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is a flow diagram illustrating an exemplary method for determining senses for a source term according to the present invention; FIG.\",\n \"2 is a flow diagram illustrating an exemplary method for assigning a sense to a term of a document according to the present; FIG.\",\n \"3 is a flow diagram illustrating an exemplary method for creating a sense-indicative index according to the present invention; FIG.\",\n \"4 is a flow diagram illustrating an exemplary method for invention refining senses for a source term according to the present invention; and FIG.\",\n \"5 is a block diagram of an information retrieval system in accordance with the present invention.\",\n \"DETAILED DESCRIPTION FIG.\",\n \"1 is a flow diagram 10 illustrating an exemplary method for determining senses for a source term according a first aspect of the present invention.\",\n \"As used herein, a “source term” is a term for which it is desired to determine senses.\",\n \"As shown in FIG.\",\n \"1, the method starts with identification of a source term, as shown at steps 11 and 12.The source term may be identified in any suitable manner.\",\n \"For example, a source term may be arbitrarily or randomly selected, specified by a user, etc.\",\n \"Alternatively, for a given group of documents that are desired to be classified and/or searched by sense, the documents may be indexed to identify the most frequently occurring terms and terms having a frequency above a desired threshold may be treated as source terms in order of decreasing frequencies.\",\n \"Various indexing techniques are well known in the art.\",\n \"Any suitable alternative may be used to identify a source term, as will be understood by those skilled in the art.\",\n \"Preferably, a method for selecting source terms is used so that all or nearly all document terms (e.g.\",\n \"excluding stop terms) are eventually selected as source terms.\",\n \"A list of trigger terms is then identified, as shown at step 14.As used herein, a “trigger term” is a term that is selected for use to identify senses for a source term.\",\n \"Accordingly, it is often advantageous that each of the trigger terms has a relatively high relevance to the source term, i.e.\",\n \"it is related to the source term.\",\n \"Any suitable method may be used for identification of trigger terms.\",\n \"Various techniques are known in the art for finding terms relating to a given term, such as those that involve use of the Jaccard coefficient, the cosine coefficient, and Yule's coefficient, as are well known in the art.\",\n \"For example, trigger terms may be identified as terms of an affinity set as described in U.S.\",\n \"Provisional Application No.\",\n \"60/271,962.Generally, the affinity set technique identifies as terms important to a subset of documents, those terms that appear more frequently in the subset than in the entire set of documents.\",\n \"For example, that technique involves determining a frequency of occurrence within sample text of terms of the sample text.\",\n \"Each term's frequency for the sample text is then compared with a reference frequency, such as the term's frequency of occurrence within a reference text, e.g.\",\n \"a large text sample.\",\n \"Importance to the sample text is determined as a function of a difference between the frequencies.\",\n \"An importance score is assigned to each of the plurality of terms of the target text and an affinity set is defined to include terms of sufficient importance, e.g.\",\n \"as reflected by an importance score exceeding a desired threshold.\",\n \"By using the source term as a search query to identify the sample text, the important terms of the sample text are terms related to the source term, and these important terms may be used to help define different senses of the source term, and so are useful as trigger terms for the present invention.\",\n \"As an illustration of steps 12 and 14, consider that “red” is selected as a source term, and that “wine, cabernet, cross, merlot, fiscal” are identified as trigger terms that are related to red and that are likely usable to determine senses of “red.” For this example, it is assumed that each trigger term has been assigned an importance score and that the list has been sorted in order of decreasing importance score.\",\n \"It must then be determined what are the senses of “red”, or more specifically, what words should be grouped with “red” to define a first sense, a second sense, etc.\",\n \"This is achieved in steps 16-24, as discussed below.\",\n \"A first term from the list of trigger terms is then selected, as shown at step 16 of FIG.\",\n \"1.For example, the trigger term having the highest importance score may be selected first, if the trigger terms have been assigned importance scores.\",\n \"Subsequent trigger terms may then be selected in order of decreasing importance score.\",\n \"Accordingly, consider that “wine” is selected as the first trigger.\",\n \"A list of important terms having high relevance to a combination of the source term and the trigger term is then identified, as shown at step 18 of FIG.\",\n \"1.This may be carried out using any suitable method.\",\n \"For example, the source term and trigger term may be combined in a search query with a Boolean AND operator, and the search query may be executed to search reference text, the search results being used as the sample text, and the affinity set technique described above may be used to identify an affinity set including important terms of the sample text.\",\n \"Alternatively, the important terms may be the terms that appear most frequently, the terms appearing with a frequency above a threshold, the top X most frequently appearing terms, etc.\",\n \"Other suitable methods for determining important terms will be apparent to those skilled in the art and any suitable method may be used.\",\n \"This list of important terms is referred to herein as a “sense” of the source term.\",\n \"More specifically, the sense is a list of terms that often co-occur with the source term when the source term is used in an associated sense, with a certain meaning, in a certain context, etc.\",\n \"To illustrate step 18 of FIG.\",\n \"1, consider that a query including “red” and “wine” is executed to identify relevant documents.\",\n \"The relevant documents are then analyzed, e.g., using the affinity set technique described in U.S.\",\n \"Provisional Application No.\",\n \"60/271,962, to identify important terms including “cabernet, merlot, blush, grape, vineyard, tasting”.\",\n \"These important terms define a first sense for the term “red”, namely, a sense associated with wine.\",\n \"Accordingly, any documents containing these terms, and the term “red”, is probably using “red” in the context of wine.\",\n \"Additionally, any document containing the term “red”, and being significantly similar to this sense, i.e.\",\n \"list of important terms, is probably using “red” in the context of wine.\",\n \"Various methods are known for determining similarity of documents, and any suitable method may be used, such as a cosine measure, a probabilistic measure, etc.\",\n \"According to the present invention, the list of terms of the sense is treated as a document and a known similarity technique is used to determine similarity to assign a sense, as discussed in greater detail below with reference to FIG.\",\n \"2.Optionally, as shown in FIG.\",\n \"1, each new sense determined in step 18 is compared to each previously determined sense for a given source term to determine similarity, as shown at step 20.Various techniques are known for determining such similarity and any suitable technique may be used.\",\n \"For example, such techniques include cosine measures, probabilistic measures, and other techniques well known in the art.\",\n \"If a high degree of similarity exists (according to user or system preferences), it may be advantageous to skip to the next trigger term (e.g.\",\n \"by skipping to step 24, as shown), or to stop the process of FIG.\",\n \"1 (i.e.\",\n \"to end by skipping to step 27) and/or to repeat the process of FIG.\",\n \"1 for a next source term.\",\n \"Such similarity indicates that the same or very similar senses are being repeatedly identified.\",\n \"It has been determined that once this begins to happen, it happens very frequently, and relatively few new senses will likely be found, at least during the present iteration of FIG.\",\n \"1.Optionally, the sense for the source term determined in step 18 is stored as a first sense, e.g.\",\n \"in a memory of an information processing system, as shown at step 22 of FIG.\",\n \"1.This sense may be referenced later as necessary, e.g.\",\n \"when assigning a sense to a term of a document, as discussed below with reference to FIG.\",\n \"4.Although a first sense of the source term has been identified, it is likely that additional senses exist.\",\n \"Accordingly, it is useful to repeat the process to identify additional senses for the source term.\",\n \"To enhance efficiency of the process, and to prevent identification of highly similar senses, the terms from the first sense are first deleted, removed, or otherwise flagged as used or unusable, etc., from the list of trigger terms identified in step 14, as shown at step 24.In this manner, terms associated with the first sense will not be used as trigger terms when trying to find a next (different) sense.\",\n \"Then, if the end of the list of trigger terms has not yet been reached, a next term is selected from the list of trigger terms and steps 16-26 are repeated, as shown.\",\n \"The process of FIG.\",\n \"1 ends when the end of the list of trigger terms is reached, as shown at steps 26 and 27.At this point, multiple senses have likely been determined for the source term.\",\n \"To illustrate, consider that the terms “cabernet” and “merlot” (the only terms appearing in the sense list of important terms and the trigger term list) are deleted from the trigger term list in step 24.The next trigger term, “cross” is then selected in step 16 and is combined with “red” to form a query used to identify another set of search-relevant documents.\",\n \"Important terms of these documents are identified as “blood, donations, international” in step 18 and define a second sense for the term red, namely a sense associated with the Red Cross organization.\",\n \"Accordingly, any documents containing these terms, and the term red, is probably using “red” in the context of the Red Cross.\",\n \"Preferably, the method of FIG.\",\n \"1 is repeated for subsequent source terms until multiple senses have been identified for all source terms.\",\n \"This creates a “library” of senses for use in assigning senses to document terms as discussed below with reference to FIG.\",\n \"2.FIG.\",\n \"2 is a flow diagram 30 illustrating an exemplary method for assigning a sense to a term of a document according to a second aspect of the present invention.\",\n \"This method considers that a “library” of senses has already been established.\",\n \"As shown in FIG.\",\n \"2, the method starts with the identifying of a term of a document, as shown at steps 31 and 32.The term may be identified in any suitable manner.\",\n \"For example, the document may be indexed to find a frequency of occurrence of each term within the document and the terms may be identified in order of decreasing frequency.\",\n \"Alternatively, the terms may be identified in order of their first appearance in a document.\",\n \"A set of senses for the term is next identified, as shown at step 34.For example, this step may include referencing a database of senses stored in a memory of an information processing system, e.g.\",\n \"those stored in step 20 of FIG.\",\n \"1.In the example of FIG.\",\n \"2, each sense is compared to the document and assigned a respective similarity score according to its degree of similarity to the document, as shown at step 36.Many suitable techniques are known in the art for determining document similarity and assigning a similarity score.\",\n \"Any suitable method for determining similarity may be used.\",\n \"In accordance with the present invention, the sense, i.e.\",\n \"the list of terms, is treated as a document for the purpose of determining similarity to another document.\",\n \"In the example of FIG.\",\n \"2, the term is assigned the sense having the highest respective similarity score, as shown at step 38, and the method ends, as shown at step 39.Suitable alternatives for assigning a sense having sufficient similarity will be apparent to those skilled in the art.\",\n \"For example, senses may be identified and compared sequentially until a similarity score above a desired threshold is obtained, at which point the corresponding sense may be assigned to the term.\",\n \"It should be noted that the method of FIG.\",\n \"2 may be used to assign a single sense to all occurrences of the term in the document, which is generally acceptable.\",\n \"Alternatively, the document may be divided into smaller units (e.g.\",\n \"sentences, or the n terms preceding and the n terms following an occurrence of a source term), and senses may be assigned to each such unit separately.\",\n \"In some embodiments, if a term is not sufficiently similar to any known (previously identified) sense, the term is assigned a generic sense indicating that the sense is presently unknown or cannot yet be classified.\",\n \"Terms assigned to such a class may be later assigned a sense as additional senses are identified (e.g.\",\n \"as the steps of FIGS.\",\n \"1 and 2 are repeated) and/or through sense-refinement, as discussed below with respect to FIG.\",\n \"4.Preferably, but not exclusively, the method of FIG.\",\n \"2 is repeated for each term (or selected term) of the document, and for each document of the group of documents.\",\n \"Accordingly, each document is provided with sense-relevant information that may be used for sense-relevant information retrieval purposes, cross-language translations, etc.\",\n \"Accordingly, after step 2, terms that have been assigned a sense according to FIG.\",\n \"2 are distinguishable from one another.\",\n \"In particular, the terms may be distinguishable by indexing software.\",\n \"Various indexing software and techniques are well known in the art.\",\n \"Various approaches can be used to cause standard/known indexing software to distinguish between terms to which senses have been assigned.\",\n \"In one embodiment, sense-distinctive string equivalents may be inserted into the text.\",\n \"For example, consider that the term “bank” is determined to have ten different senses, and that “bank” in a certain document has the fourth sense.\",\n \"Accordingly, the sense-distinctive string “bank#4” may be substituted into the document for each occurrence of “bank” so that when a usual indexing method is performed on the document “bank#4” appears distinct from “bank#3”, “bank#2”, etc.\",\n \"appearing in other documents.\",\n \"When a search query is performed for “bank#4”, or a user provides a search term of “bank” and it is specified by the user or determined by a system that the fourth sense is sought, appropriate documents may be identified in a straightforward manner using known information retrieval techniques, as will be apparent to those skilled in the art.\",\n \"Exemplary, but not limiting, methods for identifying a sense for a search query are provided below.\",\n \"In an alternate embodiment, sense determinations are made dynamically as a document is indexed.\",\n \"More specifically, as each term of the document is identified for indexing purposes as known in the art, the term's sense is determined according to the method of FIG.\",\n \"2.Then, a reference representing that sense of the word may be created or specified for further processing in the same way that the system represents a new, distinct term, e.g.\",\n \"the string “bank#1” or an integer not already used to represent any other term or sense.\",\n \"This requires straightforward modification of known indexing techniques to incorporate the method of FIG.\",\n \"2 where appropriate, as will be apparent to those skilled in the art.\",\n \"In yet another alternative embodiment, senses are assigned to an internal representation of each document, not to the document itself.\",\n \"For example, an information retrieval system may internally represent a document as a document identifier, followed by a list of terms or term identifiers (collectively “term identifiers”) contained in the document, and an occurrence count for each of those term identifiers.\",\n \"A sensed version, i.e.\",\n \"a version in which senses have been assigned to term identifiers, of such an internal representation may be created by performing the process of FIG.\",\n \"2 for each term identifier to determine which sense of the term identifier is most similar to the document, and replacing the original term identifier with a new term identifier representing the desired sense, e.g.\",\n \"to replace “bank” with “bank#3”.\",\n \"This process may be applied to each document representation.\",\n \"Other data structures (e.g., an ‘inverted index’) that are part of the index can then be rebuilt by referring to the new document representations including the new term identifiers.\",\n \"Alternatively, other data structures may be modified to reflect the identified senses.\",\n \"For example, an inverted index typically includes a pairing of each term identifier with a list of document identifiers indicating the documents that contain that term.\",\n \"To create a sense-indicative index, each such pairing is replaced by a set of pairings, one for each term sense, each of which associates the term identifier for a certain sense with a list of document identifiers indicating the documents associated with that sense.\",\n \"The sense-indicative index may then be used for information retrieval purposes in a straightforward manner, provided that a sense of a search term and/or query may be discerned or is provided.\",\n \"By way of further example, FIG.\",\n \"3 is a flow diagram 40 illustrating an exemplary method for creating a sense-indicative index according to the present invention that may be used, for example, for sense relevant information retrieval.\",\n \"As shown in FIG.\",\n \"3, the method starts with creating an index for a group of documents using any well-known indexing method, as shown at steps 41 and 42.In this example, the index associates term identifiers with document identifiers to indicate that a term corresponding to a given term identifier may be found in documents corresponding to the associated document identifiers.\",\n \"For each term identifier, a corresponding term is identified, and the term is assigned a sense for each document in which it appears, using the method of FIG.\",\n \"2 as described above.\",\n \"This is repeated for all document identifiers (and corresponding documents) for a given term identifier (and corresponding term), as shown at steps 44-52.When all document identifiers have been considered, and sense assigned, all documents corresponding to the document identifiers associated with the given term identifier are grouped by sense to create sense subgroups, as shown at step 54.For example, if term identifier A is associated with document identifiers 1,2 and 3, and the term is assigned sense Y for documents 1 and 3 and sense Z for document 2, documents 1 and 3 are grouped for sense Y and document 2 is grouped for sense Z.\",\n \"A sensed term identifier is then created for each sense subgroup, e.g.\",\n \"TermIdentifierY, TermIdentifierZ, as shown at step 56.The sensed term identifier is then stored in association with all document identifiers corresponding to the documents in the sense subgroup, as shown at step 58.For example, TermIdentifierY would be stored in correspondence with the document identifiers for documents 1 and 3.This is repeated for all term identifiers as shown at step 60.From this information, a sensed index is created as shown at step 62 to associate the sensed term identifiers with corresponding document identifiers.\",\n \"In this manner, a new index has been created that is sense-indicative, and each term identifier from the index is replaced with one or more sense-indicative term identifiers, each of which is associated with a different sense of the associated term.\",\n \"Each of the sense-indicative identifiers can then be treated as a unique term by an information retrieval system referencing the sense-indicative index to allow for sense relevant information retrieval when a sense for a search term and/or query is known.\",\n \"Because the sense differentiation is performed at the document and/or index level, known information retrieval techniques may be used with little or no modification, as will be apparent to those skilled in the art.\",\n \"A sense for a search term and/or query may be determined in various ways.\",\n \"For example, a search query provided by a user is treated as a document and the method of FIG.\",\n \"2 is used to identify a sense for each term of the search query.\",\n \"Alternatively, a user entering a term into a search engine is asked to specify a sense, e.g.\",\n \"by picking from a pick list displayed to the user and showing terms for each sense, using prior knowledge of the user or a user-profile to determine a likely sense, etc.\",\n \"Alternatively, the search query may be executed on an unsensed group of documents, i.e.\",\n \"a group to which senses have not been assigned, to retrieve documents, the senses of terms in those documents may be identified “on the fly”, e.g.\",\n \"as part of the document retrieval process, or in a pre-search processing step, and a most common sense for each of the search term(s) may be used as the query that is executed against the sensed collection of documents.\",\n \"Various other techniques will be apparent to those skilled in the art and any suitable technique may be used.\",\n \"It should be noted that sensing of the documents and/or an index of the documents may be refined by repeating the steps of FIGS.\",\n \"1 and 2 for a given body of documents.\",\n \"In effect, the first iteration applies sense to an unsensed collection of documents, while the second iteration applies senses to a sensed collection of documents.\",\n \"By identifying senses in a collection of documents to which senses have already been assigned, cross-pollution of senses is reduced, and more subtle senses may be detected.\",\n \"The resulting sense may be applied either to the sensed document collection, or to the original unsensed collection.\",\n \"FIG.\",\n \"4 is a flow diagram 70 illustrating an exemplary method for iteratively refining a sense for a source term according to the present invention.\",\n \"Optionally, this method for refining senses for a source term may be used to implement step 18 of FIG.\",\n \"1.It may be desirable to use this method because a sense initially identified may include terms that actually represent more than one sense.\",\n \"This method helps to refine the senses so that multiple “senses” of a term are not conflated in a single sense.\",\n \"As shown in FIG.\",\n \"4, the method starts with creation of an initially empty set of subtrigger lists, as shown at steps 71 and 72.An initial subtrigger list is identified that includes subtrigger terms having high relevance to a combination of the source term and the trigger term, as shown at step 74.As used herein, a “subtrigger term” is a term that is selected for use to identify senses for a source term, and preferably is a term related to the source term.\",\n \"Such terms may be identified using the affinity set technique described in U.S.\",\n \"Provisional Application No.\",\n \"60/271,962 and as discussed above.\",\n \"More specifically, the source term and trigger term are used to identify documents used as the sample text, and important terms of such documents (e.g.\",\n \"as determined using the affinity set technique) are used as the subtrigger terms.\",\n \"The initial subtrigger list is then added to the set of subtrigger lists and all subtrigger lists are combined to form a unified subtrigger list including all subtrigger terms from all subtrigger lists, as shown at steps 76 and 78.The terms of the unified subtrigger list are preferably ranked in order of importance, e.g.\",\n \"in order of decreasing importance score.\",\n \"For example, the importance score for a term in the unified subtrigger list that appears on one or more subtrigger lists may be determined to be equal to its worst rank on any individual substrigger list, or equal to its worst score on any individual subtrigger list.\",\n \"A first subtrigger term is then selected from the unified subtrigger list, as shown at step 80.This subtrigger term is a term that has not already been selected as a trigger term (e.g.\",\n \"in FIG.\",\n \"1) or as a subtrigger term (e.g.\",\n \"in FIG.\",\n \"4, as discussed further below).\",\n \"A new subtrigger list is then identified that includes terms having high relevance to a combination of the source term and the first subtrigger term, as shown at step 82.For example, such terms can be identified using the affinity set technique, wherein the source term and first subtrigger term are used to identify documents used as the sample text, and wherein the important terms of such documents are used as the new subtrigger terms.\",\n \"All terms from the new subtrigger list are then deleted from the list of trigger terms identified in step 14 of FIG.\",\n \"1, as shown at step 84.The new subtrigger list is then added to the set of subtrigger lists as shown at step 86.These steps (78 to 86) may be repeated for as many iterations as desired, as shown at step 88.More iterations generally providing a higher degree of sense refinement but an increased processing costs.\",\n \"It has been found that between 1 and 3 iterations provide adequate results.\",\n \"If the desired number of iterations has been completed, the sense refinement process ends with combination of all subtrigger lists to form a list of important terms, as shown at steps 88, 90 and 91.The list of important terms is then stored as a sense for the source term, as shown at step 22 of FIG.\",\n \"1.By way of example, each important term appearing on multiple term lists may be assigned a respective importance score equal to its worst rank or its worst score on any individual subtrigger list before those lists are combined and ordered according to importance score.\",\n \"It should be noted that the present invention is useful for various purposes beyond automated information retrieval discussed above for illustrative purposes.\",\n \"For example, the present invention may be used in the context of a thesaurus, a dictionary for cross-language translation, for document translation, or to compose a search query in a foreign language to search foreign documents as a function of a search query in a primary (native) language.\",\n \"For example, consider that English is the primary language and translations in French are desired to be searched.\",\n \"Further consider that an English language large text sample and a French language large text sample that is an aligned, parallel collection of the English language large text sample, meaning that it has a one-to-one correspondence of documents.\",\n \"For example the Hansards of the Canadian legislature are published in both French and English and may be used as aligned, parallel collections of text.\",\n \"Such a collection may be used to find an appropriate French translation for an English word by searching the English collection for documents relevant to the word to be translated, selecting the set of French documents that are translations of the retrieved English documents, extracting a set of important terms from the French documents so selected, and identifying the set's term or terms having the greatest importance as possible translation(s).\",\n \"When this method is applied to an unsensed collection, a word that has many different translations (such as ‘bank,’ which may be translated into French either as ‘banque’ for a financial bank or as ‘rive’ for a river bank) may lead to an inappropriate translation.\",\n \"If, however, the English documents have senses assigned to their words, then each such sense may be translated to different, sense appropriate, terms.\",\n \"More specifically, a first French term of the set of important terms may be used as a trigger term and combined with the English search term to find a plurality of important terms relating to their combination, those terms defining a sense.\",\n \"This can be performed for multiple French terms of the set of important terms to define multiple senses that may define alternative French “translations” for the corresponding English term.\",\n \"FIG.\",\n \"5 is a block diagram of an information processing system in accordance with the present invention.\",\n \"As is well known in the art, the information processing system of FIG.\",\n \"5 includes a general purpose microprocessor (CPU) 202 and a bus 204 employed to connect and enable communication between the microprocessor 202 and the components of the information processing system 200 in accordance with known techniques.\",\n \"The information processing system 200 typically includes a user interface adapter 206, which connects the microprocessor 202 via the bus 204 to one or more interface devices, such as a keyboard 208, mouse 210, and/or other interface devices 212, which can be any user interface device, such as a touch sensitive screen, digitized entry pad, etc.\",\n \"The bus 204 also connects a display device 214, such as an LCD screen or monitor, to the microprocessor 202 via a display adapter 216.The bus 204 also connects the microprocessor 202 to memory 218 and long-term storage 220 (collectively, “memory”) which can include a hard drive, diskette drive, tape drive, etc.\",\n \"The information processing system 200 may communicate with other computers or networks of computers, for example via a communications channel, network card or modem 222.The information processing system 200 may be associated with such other computers in a local area network (LAN) or a wide area network (WAN), or the information processing system 200 can be a client or server in a client/server arrangement with another computer, etc.\",\n \"All of these configurations, as well as the appropriate communications hardware and software, are known in the art.\",\n \"Software programming code for carrying out this inventive method is typically stored in memory.\",\n \"Accordingly, the information processing system 200 stores in its memory microprocessor executable instructions.\",\n \"When the information processing system 200 is configured for determining a sense of a source term of a document, the system stores in its memory a first program for identifying a trigger term relating to a source term, and a second program for identifying a plurality of important terms relating to a combination of the source term and the trigger term.\",\n \"When the information processing system 200 is configured for assigning a sense to a document's term for facilitating sense-relevant retrieval of the document by an information retrieval system, it stores in its memory a first program for identifying a term of the document, a second program stored for identifying a first sense for the term, a third program for comparing the first sense to the document to determine similarity, and a fourth program for assigning the first sense to the term if the first sense and the document are sufficiently similar.\",\n \"When the information processing system 200 is configured for preparing a group of documents for sense-relevant retrieval by an information retrieval system, it stores in its memory a first program for creating an index for the group of documents.\",\n \"The index associates each of a plurality of term identifiers with a corresponding set of document identifiers.\",\n \"Each of the plurality of term identifiers is associated with a term and each of the sets of document identifiers is associated with a document.\",\n \"It further stores in its memory a second program for identifying, for each of the term identifiers, a sense corresponding to a respective term and a respective document associated with a respective one of the corresponding set of document identifiers.\",\n \"The sense includes a plurality of important terms.\",\n \"It also stores a third program for creating, for each of the term identifiers, at least one sensed term identifier, each sensed term identifier corresponding to a respective term identifier and a corresponding sense; and a fourth program stored for creating a sensed index for the group of documents.\",\n \"The sensed index associates each of the sensed term identifiers with a corresponding set of document identifiers.\",\n \"Each of the sensed term identifiers is associated with a term and a sense.\",\n \"Having thus described particular embodiments of the invention, various alterations, modifications, and improvements will readily occur to those skilled in the art.\",\n \"Such alterations, modifications and improvements as are made obvious by this disclosure are intended to be part of this description though not expressly stated herein, and are intended to be within the spirit and scope of the invention.\",\n \"Accordingly, the foregoing description is by way of example only, and not limiting.\",\n \"The invention is limited only as defined in the following claims and equivalents thereto.\"\n 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"Production of genetically modified plants with puroindolines",
"A new method for increasing expression of a puroindoline protein in a plant, said puroindoline protein modifying grain hardness, notably breadmaking quality, said method comprising the steps of: (e) preparing a DNA construct which will allow... | Patent_10469922 |
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"Apparatus for processing waste with distribution/mixing chamber for oxidising fluid",
"A waste processing apparatus for processing a column of waste in a processing chamber having one or more gas outlets at an upper part thereof and one or more plasma torches an output end thereof extending into a lower ... | Patent_10469926 |
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"Gene panel participating in liver regeneration",
"A gene panel comprising a group of genes of which expression levels change in hepatocytes during liver regeneration as compared with those in a normal state is produced by the following steps: (a) the step of measuring expression levels of various genes i... | Patent_10469938 |
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"TARGET VISIBILITY ENHANCEMENT SYSTEM",
"A method and apparatus (10) for enhancing target visibility comprises producing pulse returns in response to a sequence of received pulses.",
"The pulse returns are processed to produce primary returns which vary from pulse to pulse unlike unmodulated clutter r... | Patent_10469980 |
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"Furniture with screen",
"In order to achieve a reduction in weight and convenience in terms of handling, a piece of furniture with a screen according to the present invention comprises a main body and a drawer provided therein.",
"A screen is rolled up and stored in the interior of the drawer.",
... | Patent_10469988 |
[
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"Method for identification isolation and production of antigens to a specific pathogen",
"Described is a method for identification, isolation and production of hyperimmune serum-reactive antigens from a specific pathogen, a tumor, an allergen or a tissue or host prone to autoimmunity, said antigens being ... | Patent_10470048 |
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"Computer-based creation of a document in a markup language",
"A document in a markup language containing text elements between first markers and second markers is created by: loading a master table containing formatting for a plurality of columns, the columns including a left-hand column containing the f... | Patent_10470051 |
[
[
"Device for ducting electrical lines through the wall of a fuel tank",
"The invention relates to a device for ducting electrical lines through the wall of a fuel tank (1), whereby a steel support (3) is materially connected to the wall of the fuel tank (1).",
"Contacts (4) are introduced though the su... | Patent_10470056 |
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[
"Method for evolving a cell having desired phenotype and evolved cells",
"The present invention relates to evolution of a cell or a composition of cells having a desired property or functionally.",
"The principle behind the evolution of cells according to the invention is to produce a great diversity ... | Patent_10470062 |
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"Method and apparatus for managing publication and sharing of data",
"A first user is generally designated as being eligible to operate as a publisher of data publications.",
"The first user specifically defines a first data publication.",
"A second user is generally designated as being eligible t... | Patent_10470066 |
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[
"Interactive system for enabling tv shopping",
"An improved interactive system for enabling TV shopping and similar purposes from a central provider by remote customers without the customers having to leave their remote locations.",
"The system has a complex interactive network of co-operating element... | Patent_10470071 |
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"Polynucleotides for use as tags and tag complements, manufacture and use thereof",
"A family of minimally cross-hybridizing nucleotide sequences, methods of use, etc.",
"A specific family of 1168 24mers is described."
],
[
"1.A composition comprising molecules for use as tags or tag complemen... | Patent_10470073 |
[
[
"Texturing nozzle and method for the texturing of endless yarn",
"The invention relates to a method for the texturing of endless yarn by means of a texturing nozzle having a continuous yarn duct into which compressed air at a pressure higher than 4 bar is blown in the direction of the yarn conveyance, whe... | Patent_10470078 |
[
[
"Binary mixtures of biodegradable aliphatic polyesters and products obtained from these",
"A biodegradable mixture obtained by melt mixing polyesters comprising (A) an aliphatic polyester obtained from aliphatic diacids selected from the group consisting of azelaic acid, sebacic acid, brassilic acid, mixt... | Patent_10470098 |
[
[
"Polynucleotides for use as tags and tag complements, manufacture and use thereof",
"A family of minimally cross-hybridizing nucleotide sequences, methods of use, etc.",
"A specific family of 210 24mers is described."
],
[
"1.A composition comprising molecules for use as tags or tag complement... | Patent_10470107 |
[
[
"Communication device and method",
"The present invention relates to a communication apparatus and method that allow setting for forming a wireless link to be automatically executed without requiring a special setting operation.",
"A personal computer 1 forms a wireless link with an access-point devic... | Patent_10470127 |
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"Method and device for the subjective determination of abberations of higher order",
"The invention relates to a device for the subjective determination of aberrations of higher orders Xi in an optical system, in particular in an eye (5), comprising at least one observation channel (15) into which defined... | Patent_10470149 |
[
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"ARRANGEMENT FOR DEMAGNETIZING A TRANSFORMER",
"To demagnetize a transformer (TR) in a single-ended forward DC/DC converter with self-driven synchronized rectifiers (V1, V2) connected across the secondary winding (N2) of the transformer (TR), a diode (D1) is connected in series with a capacitor (C1) acros... | Patent_10470160 |
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"Combined pen and knife implement",
"A combined writing instrument and knife comprises a tubular body, a mount disposed within the body, an elongate writing element (e.g., pen or pencil) projecting in one direction from the mount, and a knife projecting in the opposite direction from the mount.",
"The... | Patent_10470175 |
[
[
"Fast all-optical switches and attenuators",
"A polarizing beam-splitter apparatus, comprising: an input port through which an input beam of lights is provided; a first polarizing beam splitter that receives the input beam and splits the beam into at least a first and second beam, said first beam having s... | Patent_10470177 |
[
[
"Sirna expression system and process for producing functional gene knockdown cell or the like using the same",
"The in vivo siRNA expression system according to this invention is a system that intracellularly expresses small interfering (si) RNAs and comprises antisense and sense code DNAs coding for anti... | Patent_10470197 |
[
[
"Partially interconnected networks",
"A partially interconnected network has a plurality of topological nodes, each of the topological nodes having at least three direct point-to-point topological links connected to other topological nodes.",
"Each of a proportion of the plurality of topological nodes... | Patent_10470204 |
[
[
"Partially interconnected networks",
"A partially interconnected network comprises a plurality of allocated nodes, each allocated to one of a number of areas, a plurality of star nodes, and point-to-point interconnections between the allocated nodes and the star nodes.",
"The number of areas with allo... | Patent_10470205 |
[
[
"Arrangement and a method relating to access of applications/services",
"The present invention relates to a portal structure for providing end user stations (5) with access to services/applications.",
"It comprises a portal core (1) a connectivity layer via which end user station access is provided an... | Patent_10470224 |
[
[
"Edge transfer lithography",
"A method for applying a nanoscale resolution pattern of a molecular link onto a surface of a substrate is disclosed.",
"The method referred to as edge transfer lithography, comprises providing a stamp structure (10) having a surface (11) with at least one protruding featu... | Patent_10470230 |
[
[
"Transition metal complexes with proton sponges as ligands",
"Transition metal complexes with quino[7,8-h]quinolines and cyclopentadieno[1,2-h:4,3-h′]diquinolines as proton sponge ligands and a process for their preparation.",
"These complexes are suitable as catalysts, e.g.",
"the palladium compl... | Patent_10470260 |
[
[
"Method for the production of plant seed with modified fibre content and modified seed coat",
"The present invention relates to a nucleotide sequences commonly designated CJAS1 comprising a novel gene from plants.",
"The novel gene encodes a protein that is involved in seed formation and is associated... | Patent_10470264 |
[
[
"Veterinary dermatologic composition",
"This invention relates to the use of an active compound selected from the group comprising a sphingoid base, a sphingoid base derivative or a mixture of two or more these compounds for manufacturing a cosmetic composition suitable for topical application with animal... | Patent_10470299 |
[
[
"Information processing method and apparatus program storage medium, program and information recording medium",
"The characteristic playback domain of an AV stream is to be reproduced simply and reliably without complicating the management.",
"As marks representing characteristic points of an AV strea... | Patent_10470312 |
[
[
"Process for preparing a laminated, thermoformed film",
"A process for preparing a laminated, thermoformed film which comprises: a. blowing a film-forming composition to form a bubble; b. collapsing the bubble to form a film stack comprising a plurality of layers; and c. heating said film stack in a mould... | Patent_10470315 |
[
[
"Scheduling and decision system",
"The inventive subject matter herein is directed toward improved scheduling and planning system (200) in which computer implemented software uses a hierarchical selection list to select at least one of a plurality of unconnected users and contact the selected user(s) as a... | Patent_10470354 |
[
[
"Expander device",
"Apparatus (9) for radially expanding an expandable member, the apparatus including a plurality of radially moveable fingers (22).",
"The radially moveable fingers (22) can move between two positions; in a first position, the moveable fingers (22) are radially extended to a form a c... | Patent_10470393 |
[
[
"Self cleaning shredding device having movable cleaning rings",
"A self cleaning cutting assembly is provided, having an array of counter rotating cutters in which a plurality of cutters rotating in a first direction are mounted on a first shaft and a plurality of second cutters are mounted on a second sh... | Patent_10470510 |
[
[
"Induction of tolerance by apoptotic and/or necrotic cells",
"The present invention is directed to a method of inducing tolerance to self-antigens in a subject having an autoimmune diseases.",
"In particular, the invention provides a pharmaceutical composition and method of use thereof for the modulat... | Patent_10470536 |
[
[
"Superior molecular vaccine based on self-replicating rna, suicidal dna or naked dna vector, that links antigen with polypeptide that promotes antigen presentation",
"Improved molecular vaccines comprise nucleic acid vectors that encode a fusion polypeptide that includes polypeptide or peptide physically ... | Patent_10470543 |
[
[
"Burner unit and method for operation thereof",
"Arranged upstream of each swirl-stabilized premix burner (1) of a burner system there is respectively an adjustable distributing device (5) with control valves (V1, .",
".",
".",
",V8) and/or on/off valves (V″1, .",
".",
".",
", V″16... | Patent_10470556 |
[
[
"Method for the production of a burner unit",
"In the case of swirl-stabilized premix burners (1), an axial mass flow distribution of the fuel introduced which has especially favorable values with respect to characteristics such as NOx emission and maximum amplitudes of pulsations occurring is used.",
... | Patent_10470557 |
[
[
"Fabricating a semiconductor device using fully cured bisbenzocyclobutene",
"The present invention is a method of fabricating a semiconductor device using a fully cured BCB layer and removing the same using wet etching.",
"The first step is selecting a substrate.",
"The second step of the method i... | Patent_10470621 |
[
[
"Absorbent articles having improved fastening system",
"A disposable absorbent article such as, e.g., a T-shaped diaper comprising two portions, a lateral portion (backwaist) having two lateral segments or wings for wrapping around the waist of a wearer of the diaper, and a second vertical portion or inse... | Patent_10470625 |
[
[
"Apparatus comprising a button and a part adjustable by the button",
"In an apparatus (1) having an adjustable adjustment slider (13) and having a second part (9) provided for adjusting the adjustment slider (13), provision is made for at least one coupling counterpart (16) to be present in the adjustment... | Patent_10470626 |
[
[
"DVD and entertainment distribution system and method",
"A method for placing advertising on a DVD or comparable format such as “pay per view,” comprising the following steps: determining a demographic criterion for a DVD; choosing an advertisement related to that criterion and affixing it to the DVD; pro... | Patent_10470628 |
[
[
"Monitoring system for land-based navigational and landing systems",
"The present invention relates to a monitoring system for land-based navigational and landing systems.",
"A monitoring system of this type monitors the functions that are carried out by the navigational and landing system, especially... | Patent_10470630 |
[
[
"Pump for supplying a gas to a sensor and methods therefor",
"A pump for a gas sampling or analyzing system includes a pump body having walls impervious to the gas and an electrical coil within the body.",
"The pump is coupled in series with a gas sensor and a gas volume containing the gas to be sampl... | Patent_10470636 |
[
[
"Reduced weight control stage for a high temperature steam turbine",
"The control stage buckets for a steam turbine have airfoils which are hollowed out to form an interior cavity within the airfoil.",
"The cavity opens through the radial inner platform of the bucket and terminates short of the tip of... | Patent_10470637 |
[
[
"Media keying for updateable content distribution",
"A method and system for keying media allows for the distribution of updateable content to an end user.",
"The media are any type of processor readable, portable apparatus containing a preinstalled Keying Application and unique Source ID.",
"A pa... | Patent_10470638 |
[
[
"Thermoforming system",
"In the case of a thermoforming system, it is provided, for the purpose of changing over the moulds (11, 12) in a station (1) of this system, that one (4) of the two transporting devices (3, 4), for transporting the sheet (7) from one station to the other, can be displaced in the d... | Patent_10470640 |
[
[
"Collapsible headgear",
"Headgear which can be formed into a compact unit such as a carrying case comprising a headband portion and a bill portion, the bill portion being comprised of at least two bill sections which are hingedly connected to one another, the bill sections being made of a semi-rigid plast... | Patent_10470642 |
[
[
"River card poker",
"A casino game comprising one or more decks of standard playing cards and a layout which designates a first area for placing player cards and a second area composed of a plurality of betting areas The object of the game being to make a good poker hand of five cards being composed of pl... | Patent_10470644 |
[
[
"Capture and detection of microbes by membrane methods",
"Methods and systems for detecting the presence of analytes using a membrane based detection system are described.",
"A fluid sample is passed through a membrane based detection system (100).",
"Particulate analytes (e.g., microbes) are capt... | Patent_10470646 |
[
[
"Fast disintegrating meloxicam tablet",
"The present invention relates to a fast disintegrating tablet comprising meloxicam or a pharmaceutically acceptable salt thereof, starch or various starches, glidant and at least one additional excipient."
],
[
"1.A fast disintegrating in water tablet consi... | Patent_10470647 |
[
[
"High speed combination multi-mode ionization source for mass spectrometers",
"The present invention combines ionization modes produced by, for example, electrospray (ESI), atmospheric pressure chemical ionization (APCI), and thermospray for analysis of molecules.",
"Specifically, this invention relat... | Patent_10470648 |
[
[
"Hand truck/forklift aparatus",
"The hand truck/forklift as disclosed in this application is new and useful as an improvement in manually operated lifting trolleys in that it is versatile, fast, safe, practical and user friendly for transporting and lifting loads commonly carried on a hand truck, and with... | Patent_10470653 |
[
[
"Method for detecting, screening and/or montoring a cancer in individual",
"The invention relates to a method for screening and/or detecting and/or monitoring a cancer in an individual, said method comprising determining a first parameter represented by the concentration of TIMP-1 in at least one excreta,... | Patent_10470658 |
[
[
"Method and apparatus for application route discovery",
"A method and system for determining a route of an application in a network.",
"The network includes a number of interconnected nodes, between a source node and a destination node.",
"A sequence of application packets, being transmitted over ... | Patent_10470660 |
[
[
"Gear shifter pen assembly",
"A gear shifter pen assembly comprises an upper assembly and a lower assembly.",
"The upper assembly comprises a cartridge-receiving shaft housing and a writing element.",
"The writing element is received in the shaft housing.",
"The lower assembly comprises an inn... | Patent_10470662 |
[
[
"Branched polyalkylene glycols",
"The present invention provides a branched polyalkylene glycol wherein three or more single-chain polyalkylene glycols and a group having reactivity with an amino acid side chain, the N-terminal amino group or the C-terminal carboxyl group in a polypeptide or a group conve... | Patent_10470680 |
[
[
"Metering of catalysts",
"To meter catalysts into a reactor, the catalyst is firstly suspended in a hydrocarbon in a reservoir and the suspension obtained is kept in motion by stirring and then fed via a three-way metering valve and an ejector into the actual reactor, wherein the suspension containing the... | Patent_10470686 |
[
[
"Oral drug delivery system",
"There is disclosed an oral delivery system, which comprises: a sachet at least partially formed from at least one microporous permeable membrane, and defining a cavity; a physiologically active substance dissolved or dispersed in a liquid or gel, within the cavity, the microp... | Patent_10470711 |
[
[
"Methods of identifying agents that mediate polypeptide aggregation",
"The present invention is based, in part, on the discovery of methods for identifying compounds that mediate (by promoting or inhibiting) protein-protein interaction (e.g., aggregation, dimerization, or other physiologially significant ... | Patent_10470765 |
[
[
"Systems of transferring embryos and managing recipients",
"Provided herein are methods of managing embryo transfer programs, for instance bovine embryo transfer programs.",
"Examples of such methods are referred to as “bred recipient transfer” programs, in which embryos (e.g., clonal embryos) are tra... | Patent_10470785 |
[
[
"Artificial reef formation to shape sea waves",
"A method of forming artificial reefs in which a set of blocks having internal cavities full of air are interconnected in the desired shape of the artificial reef.",
"The blocks of the set float when full of air and the thus defined reef is towed to the ... | Patent_10470823 |
[
[
"Substituted oxazolidinones and their use in the field of blood coagulation",
"The invention relates to the field of blood coagulation, more particularly, to novel compounds of general formula (I), to a method for producing said compounds and to their use as active ingredients in medicaments for the preve... | Patent_10470861 |
[
[
"Seal for assembling fluid-circuit tubular elements",
"The invention relates to an annular seal used for the telescopic mounting of a male end (10) of a first tubular element (1) in a socket (20) of a second tubular element (2).",
"Said seal comprises an elastically-deformable annular body (3) designe... | Patent_10470905 |
[
[
"Cutting tool",
"The invention relates to a milling tool 1, in particular a tubular drill, comprising a shaft 2 and a working piece 3, wherein at least one cutting edge 8 is provided at the face of the working piece 3 and wherein at least on bore 4 is introduced into the face, the axis of which extends ax... | Patent_10470908 |
[
[
"Crystallized hnf4 gamma ligand binding domain polypeptide and screening methods employing same",
"A solved three-dimensional crystal structure of an HNF4g ligand binding domain polypeptide is disclosed, along with a crystal form of the HNF4g ligand binding domain.",
"Methods of designing modulators o... | Patent_10470958 |
[
[
"Methods and compositions for modulating the immune system of animals",
"Methods and compositions are disclosed for modulating the immune system of animals.",
"Applicant has identified that oral administration of immunoglobulins purified from animal blood can modulate serum IgG, TNF-α or other nonspec... | Patent_10470982 |
[
[
"Delivery of biologically active agents",
"A method of delivering a biologically active agent to the cervix, the method comprising using a needleless injector.",
"Typically, the biologically active agent is a cervical ripening agent.",
"The needleless injector may be a powder injector or a liquid ... | Patent_10470984 |
[
[
"System and method for fabricating bragg gratings",
"A novel method and apparatus for fabrication of blazed and slanted fiber Bragg gratings is disclosed.",
"The method comprises the step of simultaneously exposing the fiber with two mutually coherent light beams so as to create an interference patter... | Patent_10471027 |
[
[
"Non-isothermal method for fabricating hollow composite parts",
"A process for making hollow composite structures or vessels which includes the steps of: A) heating a mixture of thermoplastic matrix and reinforcing fibres wrapped over a rigid or semi-rigid thermoplastic liner or bladder above the melting ... | Patent_10471054 |
[
[
"Methods and devices for treating and/or processing data",
"At the inputs and/or outputs, memories are assigned to a reconfigurable module to achieve decoupling of internal data processing and in particular decoupling of the reconfiguration cycles from the external data streams (to/from peripherals, memor... | Patent_10471061 |
[
[
"Integration of e-mail with instant messaging services",
"A system for instant communication in real time is described wherein it is determined if a user with a given e-mail address is available for on-line messaging.",
"The system takes in a group of e-mail addresses and automatically looks for scree... | Patent_10471068 |
[
[
"Large touch-sensitive area with time-controlled and location-controlled emitter and receiver modules",
"The invention enables the realization of an optical and electronic structure of a touch-sensitive area on any flat display surface such as a monitor.",
"The optical structure does not require any a... | Patent_10471069 |
[
[
"Method for the amplification and detection of hbv dna using a transcription based amplification",
"The present invention provides a method for the transcription based amplification of a target HBV nucleic acid sequence starting from HBV DNA optionally present in a sample, comprising the steps of,—incubat... | Patent_10471073 |
[
[
"Adenosine receptor selective modulators",
"The invention relates to the compounds of formula (I), to a method for producing the same and to the use thereof as medicaments."
],
[
"1.A compound of the formula (I) in which R1, R2 and R3 independently of one another represent (C1-C8)-alkyl which may ... | Patent_10471079 |
[
[
"Helicomimetics and stabilized lxxll peptidomimetics",
"This invention pertains to the design and synthesis of molecules that can act as protein mimics.",
"In particular this disclosure teaches the preparation of short, cyclic peptide sequences that can adopt a helical conformation and display a parti... | Patent_10471120 |
[
[
"Novel fungicide compositions based on pyridylmethylbenzamide and propamocarb derivative",
"1) Fungicidal compositions comprising: a) at least one pyridylmethylbenzamide derivative of formula (I): in which the various radicals are as defined in the description, and b) at least one compound (II), which is ... | Patent_10471125 |
[
[
"Automated immunohistochemical and in situ hybridization assay formulations",
"The present invention provides reagents for use in an automated environment for cell conditioning of biological samples wherein the cells or tissues are predisposed for access by reagent molecules for histochemical and cytochem... | Patent_10471126 |
[
[
"Tightening device for cables and similar",
"The aim of the invention is to produce a tightening device for cables or similar for controlling the transmission of force.",
"Said device comprises a clamping disk (6) and a deflector (10) engaging therebetween.",
"As a result, the cable (4) is guided ... | Patent_10471143 |
[
[
"Quantitative analysis, visualization and movement correction in dynamic processes",
"The invention relates to the quantitative analysis and/or visualization (virtual or real) of moving processes and to the detection, description, correction and the comparison of global movements inside the image space.",... | Patent_10471153 |
[
[
"Mechanical seal without elastomers",
"A seal that provides sealing between a rotatable shaft and a housing has a stationary part for connection to the housing and a rotary part for rotation with the shaft.",
"The rotary part includes a sleeve for mounting on the shaft.",
"One end of the sleeve is... | Patent_10471158 |
[
[
"COOLING OF MILK IN AN AUTOMATIC MILKING SYSTEM",
"The present invention relates to a method for cooling milk in a milk storage tank of an automatic milling system comprising the steps of (i) measuring an amount of extracted milk by means of a milk flow meter, (ii) determining a cooling need for milk stor... | Patent_10471172 |
[
[
"Drainage and irrigation approach and structure as well as its implementation",
"This Invention relates to a drainage and irrigation approach, which primarily includes the initial stage of green belt irrigation and the later stage of rainwater drainage.",
"The drainage and irrigation structures employ... | Patent_10471198 |
[
[
"Apparatus and method for displaying virtual endoscopy display",
"An apparatus and method for displaying a three-dimensional virtual endoscopic image are provided.",
"In the method, information on a virtual endoscopic image is input in the form of volume data expressed as a function of three-dimension... | Patent_10471228 |
[
[
"Modulation of gene expression associated with inflammation proliferation and neurite outgrowth using nucleic acid based technologies",
"The present invention relates to nucleic acid molecules, including antisense, enzymatic nucleic acid molecules, and RNA interference molecules, such as hammerhead ribozy... | Patent_10471271 |
[
[
"Glazing laminates",
"There is provided a glazing laminate comprising a polyvinyl butyral core layer having polyvinyl butyral/IR dye layers on each side.",
"The dye containing layers are solvent cast from methanol onto the core layer.",
"The dyes in each layer are different comprising BN 18 type P... | Patent_10471272 |
[
[
"Micromechanical capacitive acceleration sensor",
"A micromechanical capacitive acceleration sensor is described for picking up the acceleration of an object in at least one direction.",
"The sensor includes a frame structure (110), a sensor inertia mass (101) made of a wafer and movably mounted relat... | Patent_10471296 |
[
[
"Metal sheet coated with thermoplastic resin and can obtained therefrom",
"A thermoplastic-resin-coated metal sheet in which the thermoplastic resin has been applied to the metal sheet without through an adhesive primer and which, even after having been formed into a can, is excellent in impact resistance... | Patent_10471317 |
[
[
"Method for Protecting a Logic or Mathematical operator Installed in an Electronic Module with a Microprocessor, as well as the Associated Embarked Electronic Module and the System",
"The method for protecting a logic or mathematical operator of the NOR operator type, able to be used for executing a progr... | Patent_10471323 |
[
[
"Diagnosis and treatment of blood disorders",
"Based on the discovery of the nucleotide and amino acid differences which distinguish the Gova and Govb allelic forms of the membrane glycoprotein CD109, and which comprise the biallelic Gov platelet alloantigen system, compositions and methods are provided f... | Patent_10471327 |
[
[
"Alkylated c-sugars and uses thereof",
"The present invention relates to alkylated C-sugars which are new carbohydrate derivatives based on the “C-Sugar” platform.",
"These alkylated C-sugars are converted from hydrophilic, hydrogen-bonded saccaride derivatives and are very stable, highly soluble and ... | Patent_10471328 |
[
[
"Circuit arrangement for switching a current of and off without the occurrence of overcurrent",
"There is provided a circuit arrangement for switching a current on and off without overcurrent.",
"The circuit arrangement includes a current source, a load associated with the current source, a switching ... | Patent_10471342 |
[
[
"Novel genes for conditional cell ablation",
"A Novel deacetylase genes for use in conditional cell ablation are described.",
"These genes are particularly useful for the production of transgenic plants with plant parts which can be destroyed upon treatment with N-acetyl-PPT."
],
[
"1.A DNA mo... | Patent_10471370 |
[
[
"Bushing with controlled elastic absorption of radial stress",
"Bushing, in particular of the kind with controlled elastic absorption of radial stress, includes a cylindrical tubular element (1) out of self-lubricating material onto which a ring (2) is freely fitted out of an elastically buckling material... | Patent_10471426 |
[
[
"Method and compositions for evaluating risk of developing type 2 diabetes in people of chinese descent",
"Methods and compositions for identifying mutations and polymorphisms in mutant genes encoding gene product involved in insulin secretion, for example, hepatocyte nuclear factor-1∝, glucokinase, amyli... | Patent_10471446 |
[
[
"Propellant powder charge for barrel weapon",
"The present invention relates to a method for producing propellant powder charges intended for heavy barrel weapons, such as canons and howitzers, and with a high degree of filling and a high energy content, and also to propellant powder charges produced in a... | Patent_10471457 |
[
[
"Method of synchronizing fin fold-out on a fin-stabilized artillery shell, and an artillery shell designed in accordance therewith",
"The present invention relates to a method of as far as possible limiting the yawing motion on the trajectory of an artillery shell(1), provided during the firing phase with... | Patent_10471458 |
[
[
"Method for pumping fluids",
"The production rate, useful life and operating efficiency of electric submergible pumping systems (ESP) is improved by operating the system so that the motor (220, 320) is cooled by two passes of production.",
"Production fluids remove waste heat from the motor (220, 320)... | Patent_10471462 |
[
[
"Receptacle for a programmable, electronic processing device",
"A mount for a programmable electronic processing device is disclosed.",
"It is divided into at least a first functional unit (7) and a second functional unit (8) which represent separate, particularly monolithic integrated, components.",
... | Patent_10471467 |
[
[
"Noise cancellation",
"A noise cancellation circuit has a first and second input for a first signal having a signal element and a noise element, and a second signal comprising at least a smaller amplitude of the said signal element, a first inverter arrangement for producing an inverted signal output that... | Patent_10471470 |
[
[
"Method for determining synthetic term senses using reference text",
"A method for determining term senses by identifying terms having multiple “senses” or meanings.",
"For a given source term and trigger term, a list of important terms having high relevance to a combination of the source term and tri... | Patent_10471483 |