The ZEBRA protein activates expression of Epstein-Barr virus early-lytic-cycle genes in human B lymphocytes .


Here it is shown that ZEBRA also behaves as a sequence-specific transcriptional activator in Saccharomyces cerevisiae .


Deletional mutagenesis defined three regions of ZEBRA that participate in activation in S. cerevisiae .


These regions are designated YI ( amino acids [ aa ] 1 to 25 ) , YII ( aa 51 to 102 ) , and YIII ( aa 228 to 245 ) .


Two of the three regions of the native ZEBRA protein act together to mediate activation when assayed on ZEBRA binding sites .


However , when fused to the DNA binding domain of GAL 4 and assayed on GAL4 binding sites , regions YII and YIII were each sufficient to confer activation in S. cerevisiae .


Regions of ZEBRA which affected activation in S. cerevisiae were also required in human B lymphocytes .


The amino-terminal region of ZEBRA ( aa 1 to 98 ) was required for activation both in S. cerevisiae and in human B cells ; deletion of the carboxy-terminal 18 aa also significantly reduced activation in both cell types .


Thus , the behavior of ZEBRA in human B cells and S. cerevisiae suggests that the protein contains universal activation motifs that interact with conserved components of the transcription machinery .


However , certain deletion mutants of ZEBRA containing mutations in the N-terminal region exhibited discordant behaviors in S. cerevisiae and in B cells .


For example , deletion of ZEBRA aa 26 to 51 impaired activation to a great extent in B cells but had little or no effect in S. cerevisiae .


The discordant mutants may reflect interactions with a variable domain of a conserved component or unique interactions with specialized components of the basal transcription apparatus in different cells .


Functional interaction of the v-Rel and c-Rel oncoproteins with the TATA-binding protein and association with transcription factor IIB .


Rel family proteins regulate the expression of genes linked to kappa B-binding motifs .


Little is known , however , of the mechanism by which they enhance transcription .


We have investigated the ability of the v-Rel and c-Rel oncoproteins to interact with components of the basal transcription machinery .


Here we report that both the acidic transcription activation domain mapping to the unique C terminus of chicken c-Rel and the F9 cell-specific activation region common to both v-Rel and c-Rel interact with the TATA-binding protein ( TBP ) and transcription factor IIB ( TFIIB ) in vitro and in vivo .


We also demonstrate that TPB interaction with Rel activation regions leads to synergistic activation of transcription of a kappa B-linked reporter gene .


Combined with the observation that the mouse c-Rel and human RelA proteins also interact with TBP and TFIIB in vitro , these results suggest that association with basal transcription factors is important for the transcriptional activities of Rel family proteins .


p21ras and calcineurin synergize to regulate the nuclear factor of activated T cells .


In T lymphocytes , triggering of the T cell receptor ( TCR ) induces several signaling cascades which ultimately synergize to induce the activity of the nuclear factor of activated T cells ( NFAT ) , a DNA binding complex critical to the inducibility and T cell specificity of the T cell growth factor interleukin 2 .


One immediate consequence of T cell activation via the TCR is an increase in cytosolic calcium .


Calcium signals are important for NFAT induction , and recent studies have identified calcineurin , a calcium-calmodulin dependent serine-threonine phosphatase , as a prominent component of the calcium signaling pathway in T cells .


A second important molecule in TCR signal transduction is the guanine nucleotide binding protein , p21ras , which is coupled to the TCR by a protein tyrosine kinase dependent mechanism .


The experiments presented here show that expression by transfection of mutationally activated calcineurin or activated p21ras alone is insufficient for NFAT transactivation .


However , coexpression of the activated calcineurin with activated p21ras could mimic TCR signals in NFAT induction .


These data identify calcineurin and p21ras as cooperative partners in T cell activation .


Expression of the chicken GATA factor family during early erythroid development and differentiation .


The DNA motif WGATAR has been identified within transcriptional regulatory domains of globin and other erythroid-specific genes and the activator proteins that bind to this regulatory element , the GATA factors , belong to a multi-gene family that is expressed in chicken erythroid cells .


Here we show that , as in chickens , multiple members of the GATA factor family are expressed in human and murine erythroid cells .


During the early stages of chicken embryogenesis ( well before blood island formation ) , each of the GATA family members is transcribed with a unique temporal and spatial pattern .


In the primitive erythroid lineage , transcription of the embryonic epsilon-globin gene parallels GATA-1 expression while the switch to beta-globin transcription in definitive erythroid cells is directly preceded by a pronounced increase in GATA-3 accumulation .


The timing and pattern of expression of these different mRNAs during avian erythroid development and differentiation suggests that temporally regulated changes in GATA factor expression are required for vertebrate hematopoiesis .


Molecular regulation of human interleukin 2 and T-cell function by interleukin 4 .


Distinct functional T-cell subsets , differing in the patterns of lymphokines produced , regulate cell-mediated and humoral immune responses .


The two major types and their principal products , interleukin 4 and interferon gamma ( IL-4 and IFN-gamma ) , are reciprocally negatively interactive .


To analyze the molecular mechanism of IL-4-mediated suppression of cell-mediated immunity we studied its effects on expression of interleukin 2 ( IL-2 ) and IFN-gamma .


IL-4 pretreatment of Jurkat cells prior to stimulation resulted in a decrease in transcription of the IL2 gene .


IL-4 suppressed IL-2 and IFN-gamma mRNA levels in primary human T cells , and addition of anti-CD28 antibodies relieved this suppression .


Using enhancer-reporter constructs , IL-4 specifically down-regulated the NFIL-2B element .


Electrophoretic mobility shift assays using a DNA oligomer containing the NFIL-2B binding site indicated that IL-4 inhibited the NFIL-2B complex and that the NFIL-2B DNA binding factor is distinct from AP- 1 .


These results suggest that IL-4 may regulate development and function of T-cell subsets involved in cell-mediated immunity in part by inhibiting factors required for transcription of the IL2 gene


Immunochemical differences between glucocorticoid receptors from corticoid-sensitive and -resistant malignant lymphocytes .


We have explored the possibility of using antibodies against purified rat liver glucocorticoid receptors to study the immunochemical properties of glucocorticoid receptors from murine and human malignant lymphocytes .


For this purpose , purified immune immunoglobulin G was covalently linked to Sepharose CL-4B .


We then examined the ability of the affinity gel to recognize cytosolic [ 3H ] triamcinolone acetonide-receptor complexes from the corticoid-sensitive ( CS ) and -resistant strains of mouse lymphoma P1798 , from CS lymphocytes of patients with chronic lymphatic leukemia , and from a CS clone of human leukemic lymphoblasts in tissue culture ( CH6 ) .


Mouse thymus was used as a source of glucocorticoid receptor from normal CS lymphocytes .


Whereas the immunoaffinity column retained 70 to 84 % of the 58- to 62-A ( Stokes radius ) [ 3H ] triamcinolone acetonide-receptor complexes characteristic of the CS mouse and human lymphocytes , it failed to recognize the 27- to 28-A ( Stokes radius ) glucocorticoid receptor present in corticoid-resistant mouse lymphoma P1798 cells .


Therefore , under appropriate experimental conditions , it was possible to demonstrate cross-reactivity between the antiserum against rat liver glucocorticoid receptor and the 58- to 62-A ( Stokes radius ) glucocorticoid receptor from species as diverse as mouse and humans .


Heterogeneity of the in vitro responses to glucocorticoids in acute leukemia .


In leukocyte population freshly isolated from the blood of 26 patients with acute leukemia , we have measured several parameters including glucocorticoid receptors , nucleoside incorporation , percentage of cells in S phase , and steroid-induced cell lysis .


In addition , in some cases , the short-term response to steroid therapy was determined .


Although , in all the patients studied , leukocytes were found to contain glucocorticoid receptors , we failed to demonstrate any correlation between the level of binding sites and the in vitro or in vivo response to glucocorticoids .


This absence of correlation could be in part explained by the marked heterogeneity of the steroid response demonstrated in leukocyte subpopulations .


It appears , however , that the degree of steroid action in vitro as well as the extent of spontaneous and dexamethasone-induced cell death may be related to the number of cells in the S phase of the cell cycle .


Glucocorticoid receptors in cytosol and nuclear extract of human leukocytes .


Cortisol binding by cytosol and 0.4 M KCl extract of the nuclear fraction of human leukocytes were studied by gel chromatography and ion exchange filtration on DEAE cellulose .


The cytoplasmic cortisol binding protein has a molecular weight 95 000 and the soluble nuclear binding protein 50 000 .


The absence of the uptake of radioactive cortisol by isolated nuclei and the apparent requirement of the cytosol for glucocorticoid specific binding in nuclear receptor sites was observed .


The association constant characterising the binding of cortisol to cytosol was KA = 3.5 . 10 ( 9 ) l/mol .


Corticosteroid-induced lymphopenia , immunosuppression , and body defense .


The apparent paradox of heightened adrenal corticosteroid levels associated with reduction in the competence of the body 's defensive apparatus to cope with exposure to new microbial antigens is considered .


The question is asked how this lowered defensive capability , which occurs in the face of a threat to body integrity , is consistent with Cannon 's principals of the `` wisdom of the body. ''


The suggestion is offered that the immunologic response to self-antigens exposed by disease or trauma may be suppressed by corticosteroid to offset the likelihood of autoimmune attack .


Protein tyrosine kinase activation is required for lipopolysaccharide induction of cytokines in human blood monocytes .


Bacterial LPS induce production of cytokines such as IL-1 , IL-6 , and TNF in mononuclear phagocytes , and this represents a central component in the pathogenesis of septic shock syndrome .


However , the mechanisms by which LPS activates these cells to express cytokines are not completely characterized .


The present study addressed the role of different protein kinases in the LPS induction of cytokines .


It is shown that LPS induced a 12- to 16-fold increase in IL-1 beta , IL-6 , and TNF-alpha mRNA levels , and this was completely or more than 80 % blocked by the protein tyrosine kinase specific inhibitors herbimycin A and genistein at the concentrations of 1.7 and 37 microM , respectively .


Protein kinase C inhibition by staurosporine reduced LPS induction of TNF-alpha , whereas it had no effects on IL-6 and IL-1 beta .


Inhibition of protein kinase A by H89 reduced IL-6 mRNA levels but did not detectably change IL-1 beta or TNF-alpha mRNA levels .


In contrast , LPS did not increase leukemia inhibitory factor mRNA , which was constitutively expressed and not significantly reduced by these inhibitors .


In addition to cytokine mRNA levels , LPS-induced IL-6 protein synthesis and IL-6 bioactivity were also reduced to baseline levels by the PTK inhibitors herbimycin A and genistein .


Both PTK inhibitors also reduced the LPS activation of nuclear factor-kappa B ( NF-kappa B ) , which is a transcription factor involved in the expression of cytokine genes such as IL-6 and TNF-alpha .


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


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


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


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 .


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 .


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 .


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


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 .


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 .


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


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 ) .


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 .


The region between residues -411 and -566 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 .


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 .


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


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


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


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 .


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


Stimulation of primary human T-lymphocytes via CD2 and CD28 adhesion molecules 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 .


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 .


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 .


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 .


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


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 .


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 .


Vitamin D receptor quantitation in human blood mononuclear cells 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 .


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


Following activation with phytohemagglutinin , patients 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 .


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 .


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 .


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 .


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 .


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 .


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


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 .


The combined inhibition of phosphatases and activation of kinases 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


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


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 .


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


Increased glucocorticoid receptor levels in antigen-stimulated lymphocytes .


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 .


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 .


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 .


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 .


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 .


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 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 .


Nuclear glucocorticoid binding in chronic lymphatic leukemia lymphocytes .


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 .


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 .


Multiple closely-linked NFAT/octamer and HMG I ( Y ) binding sites 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 .


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 .


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


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 .


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


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


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


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 ) .


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 .


Reversibility of the differentiated state in somatic cells .


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 .


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 .


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 .


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 .


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


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 .


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 .


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


NF-kappa B activation promoted by addition of exogenous PC-PLC 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 .


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


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 .


Novel mechanism for inhibition of human T cells by glucocorticoids .


Glucocorticoids inhibit signal transduction through IL-2 receptor .


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 .


Glucocorticoids inhibit IL-2 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 .


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 ) .


Dex inhibited the IL-2-dependent proliferation of PMA blasts 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 .


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 .


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 .


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


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


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


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 .


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 .


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 .


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 .


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 .


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 .


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 .


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 .


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 .


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


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 .


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 .


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 .


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 .


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


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+