The activation of NF-kappa B was also reduced by H89 , whereas staurosporine had no effect on this response .

The activation of <protein>NF-kappa B</protein> was also reduced by H89 , whereas staurosporine had no effect on this response .


In summary , these findings suggest that protein kinase C and protein kinase A appear to have selective effects in the LPS induction of cytokines , whereas PTK is required for LPS induction of a broad spectrum of cytokines and NF-kappa B activation in monocytes .

In summary , these findings suggest that <protein>protein kinase C</protein> and <protein>protein kinase A</protein> appear to have selective effects in the LPS induction of <protein>cytokines</protein> , whereas <protein>PTK</protein> is required for LPS induction of a broad spectrum of <protein>cytokines</protein> and <protein>NF-kappa B</protein> activation in <cell_type>monocytes</cell_type> .


In vivo control of NF-kappa B activation by I kappa B alpha .

In vivo control of <protein>NF-kappa B</protein> activation by <protein>I kappa B alpha</protein> .


The transcription factor NF-kappa B is stored in the cytoplasm in complexes with the inhibitor protein I kappa B alpha .

The <protein>transcription factor</protein> <protein>NF-kappa B</protein> is stored in the cytoplasm in complexes with the <protein>inhibitor protein I kappa B alpha</protein> .


It has been shown in vitro that dissociation of I kappa B alpha from these complexes results in active NF-kappa B .

It has been shown in vitro that dissociation of <protein>I kappa B alpha</protein> from these complexes results in <protein>active NF-kappa B</protein> .


In this report we show that lipopolysaccharide ( LPS ) -induced activation of B or pre-B cells results in loss of I kappa B alpha from NF-kappa B complexes in vivo .

In this report we show that lipopolysaccharide ( LPS ) -induced activation of <cell_type>B or pre-B cells</cell_type> results in loss of <protein>I kappa B alpha</protein> from <protein>NF-kappa B complexes</protein> in vivo .


Many liberated NF-kappa B dimers reached the nucleus , where increased c-rel , p65 and p50 were detected by immunoblotting and by DNA binding assays .

Many liberated <protein>NF-kappa B dimers</protein> reached the nucleus , where increased <protein>c-rel</protein> , p65 and p50 were detected by immunoblotting and by DNA binding assays .


Some liberated dimers were retained in the cytoplasm , however , through binding to newly synthesized I kappa B alpha , a finding which strongly suggests ( i ) that the LPS-induced signal causes dissociation of complexes rather than preventing their association and ( ii ) that dissociation results from modification of I kappa B alpha and not of c-rel or p65 .

Some <protein>liberated dimers</protein> were retained in the cytoplasm , however , through binding to newly synthesized <protein>I kappa B alpha</protein> , a finding which strongly suggests ( i ) that the LPS-induced signal causes dissociation of complexes rather than preventing their association and ( ii ) that dissociation results from modification of <protein>I kappa B alpha</protein> and not of <protein>c-rel</protein> or <protein>p65</protein> .


No effect of LPS treatment was detected on p105 or p100 , which also retain rel family members in the cytoplasm .

No effect of LPS treatment was detected on <protein>p105</protein> or <protein>p100</protein> , which also retain <protein>rel family members</protein> in the cytoplasm .


Quite unexpectedly , we also found that in unstimulated cells there is a constant ongoing process of degradation and replacement of complexed I kappa B alpha .

Quite unexpectedly , we also found that in <cell_type>unstimulated cells</cell_type> there is a constant ongoing process of degradation and replacement of <protein>complexed I kappa B alpha</protein> .


We propose that this turnover results in the low level of active NF-kappa B presumably necessary even in the unstimulated cell , and that the high rate of synthesis of I kappa B alpha provides the ability to turn off NF-kappa B activity rapidly as soon as the activating signal ceases .

We propose that this turnover results in the low level of <protein>active NF-kappa B</protein> presumably necessary even in the unstimulated cell , and that the high rate of synthesis of <protein>I kappa B alpha</protein> provides the ability to turn off <protein>NF-kappa B</protein> activity rapidly as soon as the activating signal ceases .


Identification of a killer cell-specific regulatory element of the mouse perforin gene : an Ets-binding site-homologous motif that interacts with Ets-related proteins .

Identification of a <dna>killer cell-specific regulatory element</dna> of the <dna>mouse perforin gene</dna> : an Ets-binding site-homologous motif that interacts with <protein>Ets-related proteins</protein> .


The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes .

The gene encoding the <protein>cytolytic protein</protein> <protein>perforin</protein> is selectively expressed by <cell_type>activated killer lymphocytes</cell_type> .


To understand the mechanisms underlying the cell-type-specific expression of this gene , we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene ( Pfp ) .

To understand the mechanisms underlying the cell-type-specific expression of this gene , we have characterized the regulatory functions and the DNA-protein interactions of the <dna>5'-flanking region</dna> of the <dna>mouse perforin gene</dna> ( <dna>Pfp</dna> ) .


A region extending from residues +62 through -141 , which possesses the essential promoter activity , and regions further upstream , which are able to either enhance or suppress gene expression , were identified .

A region extending from <dna>residues +62 through -141</dna> , which possesses the essential promoter activity , and regions further upstream , which are able to either enhance or suppress gene expression , were identified .


The region between residues -411 and -566 was chosen for further characterization , since it contains an enhancer-like activity .

The region between <dna>residues -411 and -566</dna> was chosen for further characterization , since it contains an enhancer-like activity .


We have identified a 32-mer sequence ( residues -491 to -522 ) which appeared to be capable of enhancing gene expression in a killer cell-specific manner .

We have identified a 32-mer sequence ( <dna>residues -491 to -522</dna> ) which appeared to be capable of enhancing gene expression in a killer cell-specific manner .


Within this segment , a 9-mer motif ( 5'-ACAGGAAGT-3 ' , residues -505 to -497 ; designated NF-P motif ) , which is highly homologous to the Ets proto-oncoprotein-binding site , was found to interact with two proteins , NF-P1 and NF-P2 .

Within this segment , a 9-mer motif ( 5'-ACAGGAAGT-3 ' , <dna>residues -505 to -497</dna> ; designated NF-P motif ) , which is highly homologous to the <dna>Ets proto-oncoprotein-binding site</dna> , was found to interact with two proteins , <protein>NF-P1</protein> and <protein>NF-P2</protein> .


NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells .

<protein>NF-P2</protein> appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in <cell_type>killer cells</cell_type> .


Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits .

Electrophoretic mobility shift assay and UV cross-linking experiments revealed that <protein>NF-P1</protein> and <protein>NF-P2</protein> may possess common <protein>DNA-binding subunits</protein> .


However , the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit ( s ) .

However , the larger native molecular mass of <protein>NF-P1</protein> suggests that <protein>NF-P1</protein> contains an additional <protein>non-DNA-binding subunit</protein> ( s ) .


In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites , it is postulated that NF-P1 and NF-P2 belong to the Ets protein family .

In view of the homology between the <protein>NF-P motif</protein> and other <protein>Ets proto-oncoprotein-binding sites</protein> , it is postulated that <protein>NF-P1</protein> and <protein>NF-P2</protein> belong to the <protein>Ets protein family</protein> .


Results obtained from the binding competition assay , nevertheless , suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins , e.g. , Ets-1 , Ets-2 , and NF-AT/Elf-1 , known to be expressed in T cells .

Results obtained from the binding competition assay , nevertheless , suggest that <protein>NF-P1</protein> and <protein>NF-P2</protein> are related to but distinct from <protein>Ets proteins</protein> , e.g. , <protein>Ets-1</protein> , <protein>Ets-2</protein> , and <protein>NF-AT/Elf-1</protein> , known to be expressed in <cell_type>T cells</cell_type> .


The role of NF-kappa B1 ( p50/p105 ) gene expression in activation of human blood T-lymphocytes via CD2 and CD28 adhesion molecules .

The role of <dna>NF-kappa B1 ( p50/p105 ) gene</dna> expression in activation of <cell_type>human blood T-lymphocytes</cell_type> via <protein>CD2 and CD28 adhesion molecules</protein> .


Stimulation of primary human T-lymphocytes via CD2 and CD28 adhesion molecules induces a long-lasting proliferation ( > 3 weeks ) .

Stimulation of <cell_type>primary human T-lymphocytes</cell_type> via <protein>CD2 and CD28 adhesion molecules</protein> induces a long-lasting proliferation ( > 3 weeks ) .


This potent activation does not require accessory cells , such as monocytes , but depends on persistent interleukin 2 ( IL-2 ) secretion and receptivity , which is associated with high and prolonged expression of the inducible CD25/IL-2 receptor alpha ( IL-2R alpha ) chain gene .

This potent activation does not require accessory cells , such as <cell_type>monocytes</cell_type> , but depends on persistent <protein>interleukin 2</protein> ( <protein>IL-2</protein> ) secretion and receptivity , which is associated with high and prolonged expression of the inducible <dna>CD25/IL-2 receptor alpha ( IL-2R alpha ) chain gene</dna> .


The transcription factor NF-kappa B participates in the regulation of both IL-2 and IL-2R alpha genes , as well as multiple cellular genes involved in T-cell proliferation .

The <protein>transcription factor NF-kappa B</protein> participates in the regulation of both <dna>IL-2 and IL-2R alpha genes</dna> , as well as <dna>multiple cellular genes</dna> involved in T-cell proliferation .


To evaluate the role of NF-kappa B in human peripheral blood T-lymphocytes , we previously analyzed the activation of NF-kappa B-related complexes in response to CD2+CD28 costimulation .

To evaluate the role of <protein>NF-kappa B</protein> in <cell_type>human peripheral blood T-lymphocytes</cell_type> , we previously analyzed the activation of <protein>NF-kappa B-related complexes</protein> in response to CD2+CD28 costimulation .


We demonstrated a long-term induction of p50/p65 heterodimer , a putative p65/c-Rel heterodimer , and a constitutive nuclear expression of KBF1/p50 homodimers .

We demonstrated a long-term induction of <protein>p50/p65 heterodimer</protein> , a <protein>putative p65/c-Rel heterodimer</protein> , and a constitutive nuclear expression of <protein>KBF1/p50 homodimers .</protein>


As the role of p50 remains unclear , we focused our present study on NF-kappa B1 ( p50/p105 ) gene regulation .

As the role of <protein>p50</protein> remains unclear , we focused our present study on <protein>NF-kappa B1</protein> ( <protein>p50/p105</protein> ) gene regulation .


Using electrophoretic mobility shift assays and Western and Northern blot analyses , we studied NF-kappa B1 gene expression during T-cell stimulation via CD2+CD28 .

Using electrophoretic mobility shift assays and Western and Northern blot analyses , we studied <dna>NF-kappa B1 gene</dna> expression during T-cell stimulation via <protein>CD2+CD28</protein> .


We observed a transient 4- to 5-fold increase of NF-kappa B1 gene expression at both the mRNA and protein levels , lasting for at least 24 h .


p50 DNA-binding activity apparently stays highly controlled when p105 expression is enhanced by a physiological stimulus of peripheral blood T-cells .

p50 DNA-binding activity apparently stays highly controlled when p105 expression is enhanced by a physiological stimulus of <cell_type>peripheral blood T-cells</cell_type> .


Partial inhibition of p50 and p105 expression by NF-kappa B1 antisense oligonucleotides significantly reduced T-cell proliferation and CD25/IL-2R alpha cell surface expression .

Partial inhibition of p50 and p105 expression by <protein>NF-kappa B1</protein> antisense oligonucleotides significantly reduced T-cell proliferation and <protein>CD25/IL-2R alpha</protein> cell surface expression .


( ABSTRACT TRUNCATED AT 250 WORDS )


Vitamin D receptor quantitation in human blood mononuclear cells in health and disease .

<protein>Vitamin D receptor</protein> quantitation in <cell_type>human blood mononuclear cells</cell_type> in health and disease .


Vitamin D receptor ( VDR ) concentration was quantitated in human peripheral blood mononuclear cells ( PBMC ) from patients with absorptive hypercalciuria ( AH ) and patients with high 1 , 25 ( OH ) 2D3 due to acquired or transient disease states and the results compared to those in normal subjects .

<protein>Vitamin D receptor</protein> ( VDR ) concentration was quantitated in <cell_type>human peripheral blood mononuclear cells</cell_type> ( <cell_type>PBMC</cell_type> ) from patients with absorptive hypercalciuria ( AH ) and patients with high 1 , 25 ( OH ) 2D3 due to acquired or transient disease states and the results compared to those in normal subjects .


VDR concentration in resting cells was not different between the three groups and represented constitutive receptor expression of monocytes .

VDR concentration in <cell_type>resting cells</cell_type> was not different between the three groups and represented constitutive receptor expression of <cell_type>monocytes</cell_type> .


Following activation with phytohemagglutinin , patients with hypercalcitriolemia demonstrated significantly greater VDR concentrations .

Following activation with <protein>phytohemagglutinin</protein> , <cell_type>patients</cell_type> with hypercalcitriolemia demonstrated significantly greater VDR concentrations .


Patients with AH demonstrated a normal value for the group , but 6 patients had significantly greater concentrations of VDR despite normal plasma 1 , 25 ( OH ) 2D3 in four of the patients .

Patients with AH demonstrated a normal value for the group , but 6 patients had significantly greater concentrations of <protein>VDR</protein> despite normal plasma 1 , 25 ( OH ) 2D3 in four of the patients .


Proliferation , as assessed from [ 3H ] thymidine incorporation was inversely correlated with serum 1 , 25 ( OH ) 2D3 and was significant ( R = -0.299 , p = 0.048 ) .


Taken together , the results suggest that PBMC provide a useful system for studying VDR status in transient or acquired states of hypercalcitriolemia .

Taken together , the results suggest that <cell_type>PBMC</cell_type> provide a useful system for studying <protein>VDR</protein> status in transient or acquired states of hypercalcitriolemia .


Furthermore , the studies in patients with absorptive hypercalciuria disclosed it to be a heterogeneous disorder , characterized by both vitamin D-dependent and D-independent forms of receptor up-regulation .


Reactive oxygen intermediates activate NF-kappa B in a tyrosine kinase-dependent mechanism and in combination with vanadate activate the p56lck and p59fyn tyrosine kinases in human lymphocytes .

Reactive oxygen intermediates activate <protein>NF-kappa B</protein> in a tyrosine kinase-dependent mechanism and in combination with vanadate activate the <protein>p56lck and p59fyn tyrosine kinases</protein> in <cell_type>human lymphocytes</cell_type> .


We have previously observed that ionizing radiation induces tyrosine phosphorylation in human B-lymphocyte precursors by stimulation of unidentified tyrosine kinases and this phosphorylation is substantially augmented by vanadate .

We have previously observed that ionizing radiation induces tyrosine phosphorylation in <cell_type>human B-lymphocyte precursors</cell_type> by stimulation of unidentified <protein>tyrosine kinases</protein> and this phosphorylation is substantially augmented by vanadate .


Ionizing radiation generates reactive oxygen intermediates ( ROI ) .


Because H2O2 is a potent ROI generator that readily crosses the plasma membrane , we used H2O2 to examine the effects of ROI on signal transduction .


We now provide evidence that the tyrosine kinase inhibitor herbimycin A and the free radical scavenger N-acetyl-cysteine inhibit both radiation-induced and H2O2-induced activation of NF-kappa B , indicating that activation triggered by ROI is dependent on tyrosine kinase activity .

We now provide evidence that the tyrosine kinase inhibitor herbimycin A and the free radical scavenger N-acetyl-cysteine inhibit both radiation-induced and H2O2-induced activation of <protein>NF-kappa B</protein> , indicating that activation triggered by ROI is dependent on tyrosine kinase activity .


H2O2 was found to stimulate Ins-1 , 4 , 5-P3 production in a tyrosine kinase-dependent manner and to induce calcium signals that were greatly augmented by vanadate .

H2O2 was found to stimulate <protein>Ins-1 , 4 , 5-P3</protein> production in a tyrosine kinase-dependent manner and to induce calcium signals that were greatly augmented by vanadate .


The synergistic induction of tyrosine phosphorylation by H2O2 plus vanadate included physiologically relevant proteins such as PLC gamma 1 .

The synergistic induction of tyrosine phosphorylation by H2O2 plus vanadate included physiologically relevant proteins such as <protein>PLC gamma 1</protein> .


Although treatment of cells with H2O2 alone did not affect the activity of src family kinases , treatment with H2O2 plus vanadate led to activation of the p56lck and p59fyn tyrosine kinases .

Although treatment of cells with H2O2 alone did not affect the activity of <protein>src family kinases</protein> , treatment with H2O2 plus vanadate led to activation of the <protein>p56lck and p59fyn tyrosine kinases</protein> .


The combined inhibition of phosphatases and activation of kinases provides a potent mechanism for the synergistic effects of H2O2 plus vanadate .

The combined inhibition of <protein>phosphatases</protein> and activation of <protein>kinases</protein> provides a potent mechanism for the synergistic effects of H2O2 plus vanadate .


Induction of tyrosine phosphorylation by ROI may thus lead to many of the pleiotropic effects of ROI in lymphoid cells , including downstream activation of PLC gamma 1 and NF-kappa B

Induction of tyrosine phosphorylation by ROI may thus lead to many of the pleiotropic effects of ROI in <cell_type>lymphoid cells</cell_type> , including downstream activation of <protein>PLC gamma 1</protein> and <protein>NF-kappa B</protein>


Influence of sex hormone binding globulin and serum albumin on the conversion of androstenedione to testosterone by human erythrocytes .

Influence of <protein>sex hormone binding globulin</protein> and <protein>serum albumin</protein> on the conversion of androstenedione to testosterone by <cell_type>human erythrocytes</cell_type> .


The influence of human serum albumin and sex hormone binding globulin ( SHBG ) on the enzymic conversion of androstenedione to testosterone in human erythrocytes was investigated in vitro .

The influence of <protein>human serum albumin</protein> and <protein>sex hormone binding globulin</protein> ( <protein>SHBG</protein> ) on the enzymic conversion of androstenedione to testosterone in <cell_type>human erythrocytes</cell_type> was investigated in vitro .


Total plasma and albumin delayed the conversion rate of androstenedione , while SHBG increased it markedly .

Total plasma and <protein>albumin</protein> delayed the conversion rate of androstenedione , while <protein>SHBG</protein> increased it markedly .


The effect of SHBG was largely abolished by heating to 60 degrees C for 1 h and by saturating its binding sites by DHT .

The effect of <protein>SHBG</protein> was largely abolished by heating to 60 degrees C for 1 h and by saturating its binding sites by DHT .


The effect of both proteins was found to be related to their concentration .


It appears that the binding sites of albumin provide a mechanism for retarding androstenedione uptake by the erythrocytes and that the high binding affinity of SHBG for testosterone facilitates the diffusion of this steroid out of the cell and thus , displaces the chemical equilibrium within the cell .

It appears that the binding sites of <protein>albumin</protein> provide a mechanism for retarding androstenedione uptake by the <cell_type>erythrocytes</cell_type> and that the high binding affinity of <protein>SHBG</protein> for testosterone facilitates the diffusion of this steroid out of the cell and thus , displaces the chemical equilibrium within the cell .


Glucocorticoids and lymphocytes .

Glucocorticoids and <cell_type>lymphocytes</cell_type> .


I .


Increased glucocorticoid receptor levels in antigen-stimulated lymphocytes .

Increased <protein>glucocorticoid receptor</protein> levels in <cell_line>antigen-stimulated lymphocytes</cell_line> .


Recently a 2- to 3-fold increase in the number of glucocorticoid receptors in human peripheral lymphocytes has been noted after in vitro mitogen stimulation .

Recently a 2- to 3-fold increase in the number of <protein>glucocorticoid receptors</protein> in <cell_type>human peripheral lymphocytes</cell_type> has been noted after in vitro mitogen stimulation .


Here , we extend these observations to in vivo immunization .


After unilateral immunization of adrenalectomized male rats , a 50 % increase in glucocorticoid receptor sites per cell , determined by binding of dexamethasone , was observed in cell suspensions of homolateral lymph nodes over those from the contralateral nonimmunized side of the same animal .


The association constant for dexamethasone was similar in both groups , as was the stereospecificity for various steroids , the time course of cytoplasmic and nuclear association , and cytoplasmic-to-nuclear translocation .


Despite a 50 % increase in the number of glucocorticoid receptor sites per cell , the cells from the homolateral and controlateral lymph nodes were equally sensitive to the inhibitory effects of dexamethasone , as determined by measurements of the incorporation of radiolabeled precursors of protein , RNA , and DNA , or measurements of in vitro cell survival .


Glucocorticoid receptors and actions in rat thymocytes and immunologically stimulated human peripheral lymphocytes .

Glucocorticoid receptors and actions in <cell_type>rat thymocytes</cell_type> and immunologically stimulated <cell_type>human peripheral lymphocytes</cell_type> .


After reviewing briefly our earlier studies on glucocorticoid receptors and mechanisms in thymus cells , we have outlined results from the following two areas of current interest in our laboratories : the `` life-cycle '' of glucocorticoid receptors and complexes in thymus cells , and the levels of glucocorticoid receptors and sensitivity in immunologically stimulated human peripheral lymphocytes .

After reviewing briefly our earlier studies on glucocorticoid receptors and mechanisms in <cell_type>thymus cells</cell_type> , we have outlined results from the following two areas of current interest in our laboratories : the `` life-cycle '' of glucocorticoid receptors and complexes in <cell_type>thymus cells</cell_type> , and the levels of glucocorticoid receptors and sensitivity in <cell_type>immunologically stimulated human peripheral lymphocytes</cell_type> .


Several of our results on energetics and kinetics of hormone binding to glucocorticoid receptors in rat thymus cells seem to require extension of the simplest model of hormone-receptor transformations in intact cells .

Several of our results on energetics and kinetics of hormone binding to <protein>glucocorticoid receptors</protein> in <cell_type>rat thymus cells</cell_type> seem to require extension of the simplest model of hormone-receptor transformations in <cell_type>intact cells</cell_type> .


ATP-depletion experiments suggest the existence of a nonbinding form of the receptor ; `` chase '' experiments suggest reaction of hormone directly with nuclear-bound receptor ; experiments on depletion and replenishment of cytoplasmic receptor using cortisol and dexamethasone suggest the existence of at least two subpopulations of nuclear-bound hormone-receptor complex .

ATP-depletion experiments suggest the existence of a nonbinding form of the receptor ; `` chase '' experiments suggest reaction of hormone directly with nuclear-bound receptor ; experiments on depletion and replenishment of <protein>cytoplasmic receptor</protein> using cortisol and dexamethasone suggest the existence of at least two subpopulations of <protein>nuclear-bound hormone-receptor complex</protein> .


We have found that mitogen or immunologic stimulation of human peripheral lymphocytes in culture leads within 24 h or so to a striking increase in the number of glucocorticoid receptor sites per cell .

We have found that mitogen or immunologic stimulation of <cell_type>human peripheral lymphocytes</cell_type> in culture leads within 24 h or so to a striking increase in the number of <protein>glucocorticoid receptor</protein> sites per cell .


We believe this increase may be due to partial synchronization of the cell population in a phase of the cell cycle in which receptor content is high .


Contrary to the widely held view that mitogen-stimulated cells become insensitive to glucocorticoids , our experiments show that with respect to inhibition of thymidine and uridine incorporation and glucose uptake , the cells are highly sensitive to dexamethasone at 24 , 48 , and 72 h after stimulation with concanavalin A .

Contrary to the widely held view that <cell_type>mitogen-stimulated cells</cell_type> become insensitive to glucocorticoids , our experiments show that with respect to inhibition of thymidine and uridine incorporation and glucose uptake , the cells are highly sensitive to dexamethasone at 24 , 48 , and 72 h after stimulation with <protein>concanavalin A</protein> .


Nuclear glucocorticoid binding in chronic lymphatic leukemia lymphocytes .

Nuclear glucocorticoid binding in <cell_type>chronic lymphatic leukemia lymphocytes</cell_type> .


A reliable procedure is described for isolating 3H-triamcinolone acetonide ( 3H-TA ) receptor complexes from purified chronic lymphatic leukemia ( CLL ) lymphocyte nuclei , based on the use of carbobenzoxy-L-phenylalanine ( CBZ-L-phe ) to prevent breakdown of hormone-receptor complexes during extraction of nuclei with 0.6M KCl .

A reliable procedure is described for isolating <protein>3H-triamcinolone acetonide ( 3H-TA ) receptor complexes</protein> from purified chronic <cell_type>lymphatic leukemia ( CLL ) lymphocyte</cell_type> nuclei , based on the use of carbobenzoxy-L-phenylalanine ( CBZ-L-phe ) to prevent breakdown of <protein>hormone-receptor complexes</protein> during extraction of nuclei with 0.6M KCl .


Using this method , specific nuclear glucocorticoid binding was demonstrated in 14/14 patients with untreated CLL .


No correlation was found between levels of nuclear-associated 3H-TA and peripheral white blood cell count or rosetting ability of circulating lymphocytes .

No correlation was found between levels of nuclear-associated 3H-TA and peripheral white blood cell count or rosetting ability of <cell_type>circulating lymphocytes</cell_type> .


Multiple closely-linked NFAT/octamer and HMG I ( Y ) binding sites are part of the interleukin-4 promoter .

Multiple closely-linked <dna>NFAT/octamer</dna> and <dna>HMG I ( Y ) binding sites</dna> are part of the interleukin-4 promoter .


We show here that the immediate upstream region ( from position -12 to -270 ) of the murine interleukin 4 ( Il-4 ) gene harbors a strong cell-type specific transcriptional enhancer .

We show here that the <dna>immediate upstream region</dna> ( from position -12 to -270 ) of the <dna>murine interleukin 4 ( Il-4 ) gene</dna> harbors a strong cell-type specific <dna>transcriptional enhancer</dna> .


In T lymphoma cells , the activity of the Il-4 promoter/enhancer is stimulated by phorbol esters , Ca++ ionophores and agonists of protein kinase A and inhibited by low doses of the immunosuppressant cyclosporin A .

In <cell_type>T lymphoma cells</cell_type> , the activity of the <dna>Il-4 promoter/enhancer</dna> is stimulated by phorbol esters , Ca++ ionophores and agonists of <protein>protein kinase A</protein> and inhibited by low doses of the immunosuppressant cyclosporin A .


The Il-4 promoter/enhancer is transcriptionally inactive in B lymphoma cells and HeLa cells .

The <dna>Il-4 promoter/enhancer</dna> is transcriptionally inactive in <cell_type>B lymphoma cells</cell_type> and <cell_type>HeLa cells</cell_type> .


DNase I footprint protection experiments revealed six sites of the Il-4 promoter/enhancer to be bound by nuclear proteins from lymphoid and myeloid cells .

DNase I footprint protection experiments revealed six sites of the <dna>Il-4 promoter/enhancer</dna> to be bound by <protein>nuclear proteins</protein> from <cell_type>lymphoid and myeloid cells</cell_type> .


Among them are four purine boxes which have been described to be important sequence motifs of the Il-2 promoter .

Among them are four purine boxes which have been described to be important sequence motifs of the <dna>Il-2 promoter</dna> .


They contain the motif GGAAA and are recognized by the inducible and cyclosporin A-sensitive transcription factor NFAT-1 .

They contain the motif GGAAA and are recognized by the <protein>inducible and cyclosporin A-sensitive transcription factor NFAT-1</protein> .


Three of the Il-4 NFAT-1 sites are closely linked to weak binding sites of Octamer factors .

Three of the <dna>Il-4 NFAT-1 sites</dna> are closely linked to weak binding sites of <protein>Octamer factors</protein> .


Several purine boxes and an AT-rich protein-binding site of the Il-4 promoter are also recognized by the high mobility group protein HMG I ( Y ) .

Several purine boxes and an <dna>AT-rich protein-binding site</dna> of the Il-4 promoter are also recognized by the <protein>high mobility group protein</protein> <protein>HMG I ( Y</protein> ) .


Whereas the binding of NFAT-1 and Octamer factors enhance the activity of the Il-4 promoter , the binding of HMG I ( Y ) suppresses its activity and , therefore , appears to be involved in the suppression of Il-4 transcription in resting T lymphocytes .

Whereas the binding of <protein>NFAT-1</protein> and <protein>Octamer factors</protein> enhance the activity of the <dna>Il-4 promoter</dna> , the binding of HMG I ( Y ) suppresses its activity and , therefore , appears to be involved in the suppression of <protein>Il-4</protein> transcription in <cell_type>resting T lymphocytes</cell_type> .


Reversibility of the differentiated state in somatic cells .

Reversibility of the differentiated state in <cell_type>somatic cells</cell_type> .


Analysis of de novo gene activation in multinucleated heterokaryons has shown that the differentiated state , although stable , is not irreversible , and can be reprogrammed in the presence of appropriate combinations of trans-acting regulatory molecules .

Analysis of de novo gene activation in <cell_type>multinucleated heterokaryons</cell_type> has shown that the differentiated state , although stable , is not irreversible , and can be reprogrammed in the presence of appropriate combinations of <protein>trans-acting regulatory molecules</protein> .


These properties have been exploited to design strategies for identifying novel regulators of cellular differentiation .


Phosphatidylcholine hydrolysis activates NF-kappa B and increases human immunodeficiency virus replication in human monocytes and T lymphocytes .

Phosphatidylcholine hydrolysis activates <protein>NF-kappa B</protein> and increases human immunodeficiency virus replication in <cell_type>human monocytes</cell_type> and <cell_type>T lymphocytes</cell_type> .


We have tested whether breakdown of phosphatidylcholine ( PC ) initiated by exogenous addition of a PC-specific phospholipase C ( PC-PLC ) from Bacillus cereus or by endogenous overexpression of PC-PLC induces functional activation of NF-kappa B and increases human immunodeficiency virus ( HIV ) enhancer activity .

We have tested whether breakdown of phosphatidylcholine ( PC ) initiated by exogenous addition of a <protein>PC-specific phospholipase C</protein> ( <protein>PC-PLC</protein> ) from Bacillus cereus or by endogenous overexpression of <protein>PC-PLC</protein> induces functional activation of <protein>NF-kappa B</protein> and increases <dna>human immunodeficiency virus ( HIV ) enhancer</dna> activity .


PC-PLC-activated hydrolysis of PC was found to induce bona fide p50/p65 NF-kappa B binding activity in three different cell lines of human or murine origin .

PC-PLC-activated hydrolysis of PC was found to induce bona fide <protein>p50/p65 NF-kappa B</protein> binding activity in three different <cell_line>cell lines</cell_line> of human or murine origin .


No significant changes in the turnover of other cellular phospholipids were detected in PC-PLC-treated cells .

No significant changes in the turnover of other cellular phospholipids were detected in <cell_line>PC-PLC-treated cells</cell_line> .


Induction of NF-kappa B by PC-PLC did not depend on de novo synthesis of proteins or autocrine secretion of either tumor necrosis factor or interleukin 1 .

Induction of <protein>NF-kappa B</protein> by <protein>PC-PLC</protein> did not depend on de novo synthesis of proteins or autocrine secretion of either <protein>tumor necrosis factor</protein> or <protein>interleukin 1</protein> .


In human monocytic and lymphoblastoid T-cell lines , induction of NF-kappa B by PC-PLC resulted in clear induction of luciferase expression vectors placed under the control of synthetic kappa B enhancers or wild type , but not kappa B-mutated , HIV long terminal repeat constructs .

In <cell_line>human monocytic and lymphoblastoid T-cell lines</cell_line> , induction of <protein>NF-kappa B</protein> by <protein>PC-PLC</protein> resulted in clear induction of <protein>luciferase</protein> expression vectors placed under the control of <dna>synthetic kappa B enhancers</dna> or wild type , but not kappa B-mutated , <dna>HIV long terminal repeat constructs</dna> .


HIV replication was increased by PC-PLC in chronically infected monocytes and T lymphocytes .

HIV replication was increased by <protein>PC-PLC</protein> in chronically infected <cell_type>monocytes</cell_type> and T <cell_type>lymphocytes</cell_type> .


NF-kappa B activation promoted by addition of exogenous PC-PLC correlated with an intense production of diacylglycerol .

NF-kappa B activation promoted by addition of <protein>exogenous PC-PLC</protein> correlated with an intense production of diacylglycerol .


However , addition of a phosphatidylinositol-specific PLC from B.cereus also induced diacylglycerol but did not activate kappa B enhancer-directed vectors .

However , addition of a <protein>phosphatidylinositol-specific PLC</protein> from B.cereus also induced diacylglycerol but did not activate <protein>kappa B enhancer-directed vectors</protein> .


PC-PLC-induced NF-kappa B activation could not be blocked by a specific inhibitor of phorbol ester-inducible protein kinases C .

PC-PLC-induced NF-kappa B activation could not be blocked by a specific inhibitor of <protein>phorbol ester-inducible protein kinases C</protein> .


These results indicate that a cellular transduction pathway , dependent on specific PC breakdown , is functional in T lymphocytes and monocytes and may be used by various transmembrane receptors to activate HIV transcription through NF-kappa B-dependent induction of the HIV enhancer .

These results indicate that a cellular transduction pathway , dependent on specific PC breakdown , is functional in <cell_type>T lymphocytes</cell_type> and <cell_type>monocytes</cell_type> and may be used by various transmembrane receptors to activate HIV transcription through NF-kappa B-dependent induction of the <dna>HIV enhancer</dna> .


Novel mechanism for inhibition of human T cells by glucocorticoids .

Novel mechanism for inhibition of <cell_type>human T cells</cell_type> by glucocorticoids .


Glucocorticoids inhibit signal transduction through IL-2 receptor .

Glucocorticoids inhibit signal transduction through <protein>IL-2 receptor</protein> .


Interaction of IL-2 with its high affinity membrane receptor complex ( IL-2R ) present on activated T lymphocytes induces cell proliferation and mediates effector functions .

Interaction of <protein>IL-2</protein> with its <protein>high affinity membrane receptor complex</protein> ( <protein>IL-2R</protein> ) present on activated <cell_type>T lymphocytes</cell_type> induces cell proliferation and mediates effector functions .


Glucocorticoids inhibit IL-2 production by inhibiting TCR-mediated signal transduction .

Glucocorticoids inhibit <protein>IL-2</protein> production by inhibiting TCR-mediated signal transduction .


We asked whether they also inhibit the action of IL-2 by inhibiting signal transduction through IL-2R .

We asked whether they also inhibit the action of <protein>IL-2</protein> by inhibiting signal transduction through <protein>IL-2R</protein> .


Human peripheral blood T cells , stimulated with PMA for 48 h ( PMA blasts ) , were incubated with IL-2 in the presence of incremental dosages of dexamethasone ( Dex ; 10 ( -5 ) -10 ( -9 ) M ) .

<cell_type>Human peripheral blood T cells</cell_type> , stimulated with PMA for 48 h ( <cell_line>PMA blasts</cell_line> ) , were incubated with <protein>IL-2</protein> in the presence of incremental dosages of dexamethasone ( Dex ; 10 ( -5 ) -10 ( -9 ) M ) .


Dex inhibited the IL-2-dependent proliferation of PMA blasts in a dose-dependent fashion ( IC50 , 5 x 10 ( -8 ) M ) .

Dex inhibited the IL-2-dependent proliferation of <cell_line>PMA blasts</cell_line> in a dose-dependent fashion ( IC50 , 5 x 10 ( -8 ) M ) .


Cell surface expression of IL-2R alpha- and beta-chains as determined by immunofluorescence analysis was not affected by Dex .

Cell surface expression of <protein>IL-2R alpha- and beta-chains</protein> as determined by immunofluorescence analysis was not affected by Dex .


In addition , Scatchard plot analysis of 125I-labeled IL-2 showed that Dex did not affect the binding of IL-2 , thus suggesting that inhibition is due to a postreceptor effect .

In addition , Scatchard plot analysis of <protein>125I-labeled IL-2</protein> showed that Dex did not affect the binding of <protein>IL-2</protein> , thus suggesting that inhibition is due to a postreceptor effect .


Inhibition of T cell proliferation by Dex was associated with decreased IL-2-dependent tyrosine phosphorylation of several intracellular proteins and decreased phosphorylation of the retinoblastoma gene product Rb , a protein essential for controlling the progression of cells through the cell cycle .

Inhibition of T cell proliferation by Dex was associated with decreased IL-2-dependent tyrosine phosphorylation of several <protein>intracellular proteins</protein> and decreased phosphorylation of the <protein>retinoblastoma gene product Rb</protein> , a protein essential for controlling the progression of cells through the cell cycle .


IL-2-dependent IL-2R alpha expression in PMA blasts and NF-kB induction in resting human T cells were also inhibited by Dex .

IL-2-dependent IL-2R alpha expression in <cell_line>PMA blasts</cell_line> and <protein>NF-kB</protein> induction in <cell_type>resting human T cells</cell_type> were also inhibited by Dex .


These results demonstrate that glucocorticoids inhibit preactivated T cells by down-regulating signal transduction through IL-2R .

These results demonstrate that glucocorticoids inhibit <cell_line>preactivated T cells</cell_line> by down-regulating signal transduction through <protein>IL-2R</protein> .


Chronic human immunodeficiency virus type 1 infection stimulates distinct NF-kappa B/rel DNA binding activities in myelomonoblastic cells .

Chronic human immunodeficiency virus type 1 infection stimulates distinct NF-kappa B/rel DNA binding activities in <cell_type>myelomonoblastic cells</cell_type> .


The relationship between human immunodeficiency virus type 1 ( HIV-1 ) infection and the induction of NF-kappa B binding activity was examined in a myeloid cell model of HIV-1 infection derived from the PLB-985 cell line .

The relationship between human immunodeficiency virus type 1 ( HIV-1 ) infection and the induction of NF-kappa B binding activity was examined in a <cell_line>myeloid cell model</cell_line> of HIV-1 infection derived from the <cell_line>PLB-985 cell line</cell_line> .


Chronic infection of PLB-985 cells led to increased monocyte-specific surface marker expression , increased c-fms gene transcription , and morphological alterations consistent with differentiation along the monocytic pathway .

Chronic infection of <cell_line>PLB-985 cells</cell_line> led to increased monocyte-specific surface marker expression , increased c-fms gene transcription , and morphological alterations consistent with differentiation along the monocytic pathway .


PLB-IIIB cells displayed a constitutive NF-kappa B-like binding activity that was distinct from that induced by tumor necrosis factor alpha or phorbol 12-myristate 13-acetate treatment of the parental PLB-985 cell line .

<cell_line>PLB-IIIB cells</cell_line> displayed a constitutive NF-kappa B-like binding activity that was distinct from that induced by <protein>tumor necrosis factor alpha</protein> or phorbol 12-myristate 13-acetate treatment of the <cell_line>parental PLB-985 cell line</cell_line> .


This unique DNA binding activity consisted of proteins of 70 , 90 , and 100 kDa with a high degree of binding specificity for the NF-kappa B site within the PRDII domain of beta interferon .

This unique DNA binding activity consisted of proteins of <protein>70 , 90 , and 100 kDa</protein> with a high degree of binding specificity for the <protein>NF-kappa B site</protein> within the <protein>PRDII domain</protein> of <protein>beta interferon</protein> .


In this report , we characterize the nature of these proteins and demonstrate that binding of these proteins is also induced following Sendai paramyxovirus infection .


The 70-kDa protein corresponds to the NF-kappa B RelA ( p65 ) subunit , which is activated in response to an acute paramyxovirus infection or a chronic HIV-1 infection .

The <protein>70-kDa protein</protein> corresponds to the <protein>NF-kappa B RelA ( p65 ) subunit</protein> , which is activated in response to an acute paramyxovirus infection or a chronic HIV-1 infection .


Virus infection does not appear to alter the amount of RelA ( p65 ) or NFKB1 ( p50 ) but rather affects the capacity of I kappa B alpha to sequester RelA ( p65 ) , therefore leading to constitutive levels of RelA DNA binding activity and to increased levels of NF-kappa B-dependent gene activity .

Virus infection does not appear to alter the amount of <protein>RelA</protein> ( <protein>p65</protein> ) or <protein>NFKB1</protein> ( <protein>p50</protein> ) but rather affects the capacity of <protein>I kappa B alpha</protein> to sequester <protein>RelA ( p65 )</protein> , therefore leading to constitutive levels of RelA DNA binding activity and to increased levels of NF-kappa B-dependent gene activity .


The virally induced 90- to 100-kDa proteins have a distinct binding specificity for the PRDII domain and an AT-rich sequence but do not cross-react with NF-kappa B subunit-specific antisera directed against NFKB1 ( p105 or p50 ) , NFKB2 ( p100 or p52 ) , RelA ( p65 ) , or c-rel .

The virally induced <protein>90- to 100-kDa proteins</protein> have a distinct binding specificity for the <dna>PRDII domain</dna> and an <dna>AT-rich sequence</dna> but do not cross-react with NF-kappa B subunit-specific antisera directed against <protein>NFKB1</protein> ( <protein>p105</protein> or <protein>p50</protein> ) , <protein>NFKB2</protein> ( <protein>p100</protein> or <protein>p52</protein> ) , <protein>RelA</protein> ( <protein>p65</protein> ) , or <protein>c-rel</protein> .


DNA binding of the 90- to 100-kDa proteins was not inhibited by recombinant I kappa B alpha/MAD-3 and was resistant to tryptic digestion , suggesting that these proteins may not be NF-kappa B related .

DNA binding of the <protein>90- to 100-kDa proteins</protein> was not inhibited by <protein>recombinant I kappa B alpha/MAD-3</protein> and was resistant to tryptic digestion , suggesting that these proteins may not be <protein>NF-kappa B related</protein> .


Transient cotransfection experiments demonstrated that RelA and NFKB1 expression maximally stimulated HIV-1 LTR- and NF-kappa B-dependent reporter genes ; differences in NF-kappa B-like binding activity were also reflected in higher constitutive levels of NF-kappa B-regulated gene expression in HIV-1-infected myeloid cells .

Transient cotransfection experiments demonstrated that RelA and NFKB1 expression maximally stimulated <dna>HIV-1 LTR- and NF-kappa B-dependent reporter genes</dna> ; differences in NF-kappa B-like binding activity were also reflected in higher constitutive levels of NF-kappa B-regulated gene expression in <cell_line>HIV-1-infected myeloid cells</cell_line> .


Presence of estrogen-binding sites on macrophage-like synoviocytes and CD8+ , CD29+ , CD45RO+ T lymphocytes in normal and rheumatoid synovium .

Presence of estrogen-binding sites on <cell_type>macrophage-like synoviocytes</cell_type> and <cell_line>CD8+ , CD29+ , CD45RO+ T lymphocytes</cell_line> in normal and rheumatoid synovium .


OBJECTIVE .


To study the presence of estrogen-binding sites ( EBS ) in the synovial tissues of male and female patients with rheumatoid arthritis ( RA ) and in age- and sex-matched healthy controls .


METHODS .


Both type 1 ( high affinity , low binding capacity ) and type 2 ( reduced affinity , higher binding capacity ) EBS were investigated in both soluble and nuclear fractions of homogenized synovial tissue samples by a dextran-coated charcoal method .


To determine what type of synovial cell was positive for EBS , cryosections of synovial tissues were immunostained with a specific monoclonal anti-estrogen receptor antibody ( anti-ER MAb ) using both immunofluorescence and immunoperoxidase techniques .

To determine what type of <cell_type>synovial cell</cell_type> was positive for EBS , cryosections of synovial tissues were immunostained with a specific <protein>monoclonal anti-estrogen receptor antibody</protein> ( <protein>anti-ER MAb</protein> ) using both immunofluorescence and immunoperoxidase techniques .


Double immunostaining with the anti-ER MAb and with specific MAb to detect different macrophage antigens ( Ber-MAC3 , MAC387 , CD68 ) and CD8+ T cell subsets ( CD29+ , CD45RO+ and CD29- , CD45RO- ) was performed .

Double immunostaining with the <protein>anti-ER MAb</protein> and with <protein>specific MAb</protein> to detect different <protein>macrophage antigens</protein> ( <protein>Ber-MAC3</protein> , <protein>MAC387</protein> , <protein>CD68</protein> ) and <cell_line>CD8+ T cell subsets</cell_line> ( <cell_line>CD29+</cell_line> , <cell_line>CD45RO+</cell_line> and <cell_line>CD29-</cell_line> , <cell_line>CD45RO-</cell_line> ) was performed .


RESULTS .


Higher affinity EBS were found mostly in nuclear cell fractions of either RA or control synovial tissues ( 28 of the 33 ) .


These EBS were present to a lesser extent in soluble cell fractions ( 11 of the 33 ) .


Immunostaining showed the estrogen receptor-positive cells to be the macrophage-like synoviocytes and the CD8+ , CD29+ T cells both in RA and in control synovial tissues .

Immunostaining showed the <cell_type>estrogen receptor-positive cells</cell_type> to be the <cell_type>macrophage-like synoviocytes</cell_type> and the <cell_line>CD8+</cell_line> , <cell_line>CD29+ T cells</cell_line> both in RA and in control synovial tissues .


Higher nuclear content of EBS was consistent with more intense nuclear staining of synoviocytes and T cells .

Higher nuclear content of EBS was consistent with more intense nuclear staining of <cell_type>synoviocytes</cell_type> and <cell_type>T cells</cell_type> .


CONCLUSION .


It is conceivable that the immunomodulatory activity exerted by estrogens is at least partly mediated through their interaction with EBS that are present on macrophage-like synoviocytes , functioning as antigen-processing and antigen-presenting cells , and on antigen-experienced ( memory ) CD8+ T lymphocytes ( CD29+ , CD45RO+

It is conceivable that the immunomodulatory activity exerted by estrogens is at least partly mediated through their interaction with EBS that are present on <cell_type>macrophage-like synoviocytes</cell_type> , functioning as <cell_type>antigen-processing and antigen-presenting cells</cell_type> , and on <cell_line>antigen-experienced ( memory ) CD8+ T lymphocytes</cell_line> ( <cell_line>CD29+</cell_line> , <cell_line>CD45RO+</cell_line>