Cholinesterase inhibition and toxicokinetics in immature and adult rats after acute or repeated exposures to chlorpyrifos or chlorpyrifos-oxon.

The effect of age or dose regimen on cholinesterase inhibition (ChEI) from chlorpyrifos (CPF) or CPF-oxon (CPFO) was studied in Crl:CD(SD) rats. Rats were exposed to CPF by gavage in corn oil, rat milk (pups), or in the diet (adults) or to CPFO by gavage in corn oil. Blood CPF/CPFO levels were measured. With acute exposure, ChEI NOELs were 2 mg/kg CPF for brain and 0.5 mg/kg CPF for red blood cells (RBCs) in both age groups. In pups, ChEI and blood CPF levels were similar using either milk or corn oil vehicles. Compared to gavage, adults given dietary CPF (12 h exposure) had greater RBC ChEI, but lower brain ChEI at corresponding CPF doses, indicating an effect of dose rate. With repeated CPF exposures, ChEI NOELs were the same across ages (0.5 and 0.1 mg/kg/day for brain and RBCs, respectively). With CPFO dosing, the ChEI NOELs were 0.1 mg/kg (acute) and 0.01 mg/kg/day (repeated doses) for RBCs with no ChEI in brain at CPFO doses up to 0.5 (pup) or 10 mg/kg (adult) for acute dosing or 0.5 mg/kg/day for both ages with repeat dosing. Thus, there were no age-dependent differences in CPF ChEI via acute or repeated exposures. Pups had less ChEI than adults at comparable blood CPF levels. Oral CPFO resulted in substantial RBC ChEI, but no brain ChEI, indicating no CPFO systemic bioavailability to peripheral tissues.

The analysis of costimulatory receptor signaling cascades in normal T lymphocytes using in vitro gene transfer and reporter gene analysis.

Ligation of the antigen receptor and costimulatory receptors on the surface of T lymphocytes initiates intracellular signals that regulate cell-cycle progression and cell differentiation. To effectively manipulate the activation of T cells for immunotherapeutic applications, it will be important to understand how these signaling pathways are integrated to control specific gene transcription events. Here we describe a novel transient transfection procedure that efficiently introduces DNA into non-dividing normal human and murine T lymphocytes while maintaining high cell viability. Using this technique, reporter genes can be introduced to characterize intracellular signaling pathways that regulate specific gene transcription events in normal T-lymphocyte populations. We show that the CD28 receptor can be differentially coupled to downstream signaling pathways in different T-lymphocyte populations. In addition, we demonstrate that a gene encoding a tagged constitutively active mitogen-activated kinase kinase-1 protein can be transfected and rapidly expressed to regulate the expression of Bcl-2 in normal thymocytes.

Molecular cloning of the human eosinophil peroxidase. Evidence for the existence of a peroxidase multigene family.

Human eosinophil peroxidase (EPO) was purified from eosinophil granules derived from the peripheral blood of patients with eosinophilia. The molecular mass of the H and L subunits was determined by gel filtration to be 57,000 and 11,000 daltons, respectively. The partial amino acid sequences of both subunits were used to construct oligonucleotides for the screening of several cDNA libraries, including one derived from human-induced umbilical cord mononuclear cells. A cDNA clone was isolated corresponding to EPO. The nucleotide sequence revealed an open reading frame of 2,106 bp, corresponding to a prosequence, L chain, and H chain, in this order. Comparison of the EPO nucleotide sequence with other peroxidases, such as myeloperoxidase, suggests the existence of a multigene family.

A beta polymorphic residues responsible for class II molecule recognition by alloreactive T cells.

In an effort to characterize the ligand that is bound by T helper lymphocyte antigen receptors, we have begun to identify class II polymorphic residues that comprise part of the allospecific TCR binding sites. Site-directed mutagenesis was used to construct mutant Ak beta (Ak beta*) genes that encode polypeptides into which single or multiple residues of the Ad beta polypeptide have been substituted in the beta 1 domain. A panel of cloned cell lines expressing the mutant Ak beta* Ak alpha or Ak beta* Ad alpha molecules was analyzed for the ability to stimulate Ak or Ad alloreactive T cell hybridomas. Substitution of d for k residues at specific positions in the beta 1 domain resulted not only in the loss of epitopes recognized by Ak-reactive T cells but, more importantly, in the gain of epitopes recognized by Ad-reactive T cells. Some of the polymorphic residues identified as contributing to the T cell epitopes are the same residues that contribute to the serologically immunodominant epitope. Other T cell epitopes map to positions predicted to be located either in an alpha-helix forming one side, or in a beta-pleated sheet forming the bottom of the putative antigen binding site. Thus, unlike serologic epitopes, TCR epitopes can be determined by A beta polymorphic residues in many different regions of the beta 1 domain and frequently depend upon contributions of A alpha polymorphic residues.

Regulation of murine MHC class II molecule expression. Identification of A beta residues responsible for allele-specific cell surface expression.

A panel of mutant class II genes have been constructed using site-directed mutagenesis and DNA-mediated gene transfer. Using this technique, Ak beta polypeptides have been altered by substituting one or more Ad beta-specific residues at polymorphic positions in the beta 1 domain. Transfection of M12.C3 B lymphoma cells with most mutant Ak beta* genes results in the expression of Ak beta* Ad alpha molecules on the cell surface. However, the substitution of a single d allele residue at position 78 or 86 in the Ak beta polypeptide results in either the complete absence or very low levels, respectively, of cell surface expression of the Ak beta* Ad alpha molecule, but does not alter Ak beta* Ak alpha expression. The T.86 Ak beta* Ad alpha is expressed primarily in an intracellular compartment while the T.78 Ak beta* molecule does not appear to be produced. The core-glycosylated T.78 Ak beta* polypeptide does, however, form a complex intracellularly with the core-glycosylated Ii polypeptide. Substitution of the combination of d allele residues at Ak beta polymorphic positions 9, 12, 13, 14, and 17 results in the absence of Ak beta* Ak alpha cell surface expression but does not alter the expression of this mutant Ak beta* polypeptide with the Ad alpha polypeptide. These allele-specific expression mutants demonstrate that substitution at certain beta 1 domain positions may result in the alteration of Ia cell surface expression and that the transport of Ia molecules from the Golgi apparatus to the cell surface may be regulated by signals that are determined by the interaction of polymorphic residues in both the alpha and beta polypeptides.

DNA sequence analysis of I-Ak beta mutants reveals serologically immunodominant region.

We have produced a series of in vitro serologically selected cell lines that express mutant I-Ak molecules. In this report we describe the DNA sequence analysis of the Ak beta gene of four cell lines that express serologically altered Ak beta polypeptides in association with wild-type Ak alpha polypeptides. Each of the major serologic epitopes on the Ak beta polypeptide has been altered in one or more of the four mutants. In addition, the four mutants exhibit a broad spectrum of functional defects when used to stimulate a panel of T hybridomas of various specificities. The DNA sequence analysis revealed that each mutant had sustained a single nucleotide substitution resulting in a single amino acid substitution. All four independent substitutions occurred within or near the third of the four variable regions defined in the beta 1 domain of the A beta polypeptide by allelic comparisons. These data strongly suggest that the third variable region is the major determinant of alloantigenicity on the Ak beta polypeptide.

Identification of an immunodominant region on the I-A beta chain using site-directed mutagenesis and DNA-mediated gene transfer.

To identify which polymorphic residues determine the allospecific antibody binding sites on A beta polypeptides, mutant Ak beta genes were constructed encoding single or multiple amino acids of the d allele at 14 polymorphic positions in the beta 1 domain. Cell lines expressing these genes were analyzed by quantitative immunofluorescence using 16 mAbs reactive to Ak beta or Ad beta. Substitution of d allele residues at positions 63 and 65-67 in the Ak beta polypeptide resulted in the loss of binding of all Ak beta-reactive antibodies and the gain of binding of most Ad beta-reactive antibodies. Two Ad beta-reactive mAbs bound to the mutant Ak beta polypeptide containing d allele-characteristic residue at position 40. In contrast, substitution of the other polymorphic residues in the NH2-terminal and COOH-terminal regions of the beta 1 domain did not alter antibody binding.

Effects of p56lck deficiency on the growth and cytolytic effector function of an interleukin-2-dependent cytotoxic T-cell line.

The growth, differentiation, and functional activities of antigen-stimulated T lymphocytes are regulated by the interaction of the T-cell-derived cytokine, interleukin-2 (IL-2), with the high-affinity IL-2 receptor (IL-2R). IL-2R occupancy initiates a rapid increase in intracellular protein tyrosine phosphorylation, suggesting that a receptor-coupled protein tyrosine kinase (PTK) serves as a proximal signaling element for the IL-2R. Previous studies implicated the src-family kinase, p56lck, as a potential IL-2R-linked signal transducer. In this study, we have characterized a spontaneous variant of the IL-2-dependent cytotoxic T-cell line, CTLL-2, which contains no detectable lck-derived mRNA transcripts, protein, or PTK activity. The p56lck-deficient CTLL-2 cells retained strict dependence on IL-2 for both viability and growth, indicating that p56lck activity was not required for the transduction of IL-2-mediated mitogenic signals. However, the p56lck-deficient cells exhibited a moderate decrease in their rate of IL-2-dependent proliferation. In contrast to this relatively modest proliferative defect, the p56lck-deficient cell line displayed a profound reduction in T-cell antigen receptor-dependent cytolytic effector functions. Both the proliferative and the cytolytic defects observed in the p56lck-deficient cells were completely reversed by transfection of these cells with a wild-type lck expression vector. These results indicate that p56lck expression is not obligatory for IL-2-mediated T-cell growth stimulation; however, this PTK plays a central role in the generation T-cell-mediated cytotoxic responses.

Biochemical characterization of I-Ak proteins from mutant antigen-presenting B-cell--B-lymphoma hybridomas.

Chemically induced mutants of an I-Ak,d expressing antigen-presenting B-cell--B-lymphoma hybridoma have recently been generated by immunoselection in vitro and were found to possess alterations in some of their serologically and functionally defined I-Ak region dependent functions. In order to identify at the structural level the origin of the differences in serological and functional properties of these mutants, I-Ak molecules from several of these mutant hybridomas were compared biochemically to wild-type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic tryptic peptide analyses. Two-dimensional gel electrophoresis indicated that no major structural alterations, resulting in changes in mol. wt or charge, had occurred in the Ak alpha or Ak beta polypeptides from the mutant cells. Likewise, Ak alpha peptide maps of the mutants were indistinguishable from the normal Ak alpha peptide maps. However, two of the three mutants studied did exhibit one additional peptide in their Ak beta peptide maps. These results suggest that the major deficiencies in T-cell-activating functions of these mutants are a result of a limited alteration in the Ak beta polypeptide primary structure.

Forming, changing, and acting on attitude toward affirmative action programs in employment: a theory-driven approach.

A model of attitude toward affirmative action programs (AAPs) was applied in 4 studies involving 1,622 participants. In Study 1, attributes people tacitly associate with AAPs were identified by open-ended elicitation. Using those attributes, an instrument was developed and administered in Studies 2, 3, and 4. In those studies, a multiplicative composite of beliefs and evaluations about the AAP attributes predicted AAP attitude, consistent with M. Fishbein and I. Ajzen's (1975) theory of reasoned action. Demographic effects on AAP attitude were partially mediated by this composite. In Studies 3 and 4, an experimental manipulation of AAP information was successful in changing AAP attitude, but in a way that polarized existing demographic differences. Study 4 also showed that AAP attitude and subjective norm jointly and uniquely predicted intentions to perform AAP-related behaviors. Intentions predicted the actual behavior of mailing postcards to political representatives reflecting participants' support for AAPs.

Disruptions to women's social identity: a comparative study of workplace stress experienced by women in three geographic regions.

Drawing on social identity theory (P. J. Burke, 1991) and the current status of women and equal opportunity legislation, the authors tested several factors associated with distress in working women in the People's Republic of China (PRC), Hong Kong, and the United States. Women in Hong Kong experienced significantly greater levels of life stress than PRC and U.S. women. Reports of negative attitudes toward women, gender evaluation, and avoidance coping were greater for Hong Kong and PRC women than for U.S. women. Hong Kong women reported more use of positive/confrontational coping mechanisms. Negative attitudes toward women had an important influence on life stress across regions. Moderator tests resulted in 2 significant findings: The effect of negative attitudes toward women on life stress was stronger for PRC and Hong Kong women, and the relationship between nervous/self-destructive coping and life stress was stronger for U.S. women.

The structure-function relationship of I-A molecules: a biochemical analysis of I-A polypeptides from mutant antigen-presenting cells and evidence of preferential association of allelic forms.

Chemically induced mutants of an I-Ak,d-expressing, antigen-presenting B cell-B lymphoma hybridoma have recently been generated by immunoselection in vitro with I-Ak-specific monoclonal antibodies, and were found to possess alterations in some of the I-Ak region-dependent functions. The mutants were categorized as alpha-polypeptide mutants or beta-polypeptide mutants on the basis of the patterns of reactivity with anti I-Ak alpha and anti I-Ak beta monoclonal antibodies. To delineate the structural alterations underlying the differences in serologic and functional properties of these mutants, I-A molecules from several of these mutant hybridomas were compared biochemically with wild type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic (HPLC) tryptic peptide analyses. These results suggest that the marked alterations in antibody reactivity and T cell-activating functions of the beta-polypeptide mutants G1, K2, and LD3, as well as the Ia alpha-polypeptide mutant JE50, may be due to very limited alterations in the Ia polypeptides. The functional deficiencies of the alpha-polypeptide mutant JE67 could be attributed to the change in net charge exhibited by its Ak alpha polypeptide. HPLC tryptic peptide analysis of I-A molecules isolated from the alpha-polypeptide mutant J4 indicates that the functional deficiencies exhibited by this mutant are due to a complete loss of expression of the Ak alpha polypeptide. The inability to detect significant amounts of Ad alpha Ak beta and Ak alpha Ad beta hybrid molecules in immunoprecipitates from some of these cell lines suggests that some hybrid molecules may be expressed at low levels due to preferential Ia polypeptide chain association. Together, these results indicate that most serologically defined epitopes are localized on either one or the other Ia polypeptide, whereas T cell-defined epitopes are determined by a combination of both Ia polypeptides. The results of these analyses also enable us to evaluate different immunoselection strategies for the most efficient production of mutants expressing limited alterations in Ia polypeptides.

Biochemical and amino acid sequence analysis of human eosinophil granule major basic protein.

Eosinophil granule major basic protein (MBP) is a relatively low molecular weight cationic (pI greater than 10) protein present in the crystalloid core of the eosinophil granule. Amino acid sequence analysis of this protein was undertaken as part of an analysis of the structural basis of the potent cytotoxic activities of MBP on parasites and mammalian cells. Many conventional sequencing strategies were unworkable because of the unusual amino acid composition of MBP and its insolubility in solutions buffered at neutral pH. Less conventional chemical reactions, including cyanogen bromide-induced cleavage at tryptophan and acid-induced cleavage at aspartic acid, were used successfully to obtain peptides which allowed definition of the amino acid sequence of MBP. Characterization of MBP by reverse-phase high pressure liquid chromatography and two-dimensional gel analysis showed no microheterogeneity that might be attributed to post-translational modifications. Comparison of the MBP sequence with a protein sequence data base showed that MBP has no significant sequence homology with other characterized proteins. The basicity (pI 10.9) and hydrophobicity predicted from the MBP sequence are likely responsible for the observed affinity of this cytotoxic molecule for cell surfaces and some serum proteins.

The isolation, characterization and amino terminal sequence of the vitamin D-binding protein (group specific component) from mouse plasma.

1. In order to establish a homologous system in which to study the interaction of mouse vitamin D-binding protein (MVDBP) with mouse T-cell lymphocytes, we purified MVDBP from mouse plasma. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that purified MVDBP had an apparent relative molecular weight of 49,000. 3. Previous work in our laboratory has shown that purified rat vitamin D-binding protein (RVDBP) has an apparent relative molecular weight of 52,000. 4. The amino terminal amino acid sequence of MVDBP is shown below and compared with that of RVDBP. MVDBP: LeuGluArgGlyArgAspTyrGluLysAspLysValCysAsnGluLeuAlaMetLeuGlyLysGlu RVDBP: LeuGluArgGlyArgAspTyrGluLysAspLysValCysGlnGluLeuSerThrLeuGlyLysAsp AspPhe AspPhe While 21 out of 24 residues (87.5%) of the amino terminus of MVDBP are the same as those in RVDBP, residues 14, 17, 18 and 22 (underlined) are different. 5. The sedimentation coefficient of the protein, determined by sucrose density gradient ultracentrifugation, is 3.8 for MVDBP and 4.1 for the rat VDBP. 6. The MVDBP purified in this study exhibits only one isoform on isoelectric focusing; the isoelectric point was 4.87 as determined on pH 4.0-6.5 isoelectric focusing gels (IEF). 7. The binding of vitamin D3, 25-hydroxyvitamin D3 and three other analogs was investigated with a charcoal dextran assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Delta-opioid receptors expressed by Jurkat T cells enhance IL-2 secretion by increasing AP-1 complexes and activity of the NF-AT/AP-1-binding promoter element.

Recent molecular evidence points to transient and/or stage-specific expression of delta- and kappa-opioid receptors by thymic and peripheral T lymphocytes. Since medical treatments or stress commonly increase opioid levels, it is important to understand the mechanisms by which opioids affect T lymphocyte functions. We therefore created and studied a T cell line expressing the cloned delta-opioid receptor (DOR1). DOR1 ligation by a specific DOR1 agonist, deltorphin, augmented IL-2 secretion by synergizing with signals from TCR-CD3 and CD28. Reporter gene constructs were used to map this effect of deltorphin to the AP-1- and NF-AT/AP-1-binding sites of the IL-2 promoter. Although DOR1 signaling increased [Ca2+]i, deltorphin enhanced transcriptional activity of the NF-AT/AP-1-binding site via a mechanism independent of calcineurin and distinct from the effects of elevated [Ca2+]i. Deltorphin also increased accumulation of AP-1 transcription factor complexes, suggesting that DOR1 augments IL-2 secretion by increasing the AP-1 component of the NF-AT/AP-1 transcription factor. These results advance the molecular understanding of opioid effects on lymphocytes, and in addition, demonstrate regulation of IL-2 synthesis and secretion by the novel mechanism of receptor-mediated AP-1 induction.

Class II histocompatibility molecules associate with calnexin during assembly in the endoplasmic reticulum.

Class II histocompatibility antigens are composed of polymorphic alpha and beta polypeptides which associate in the endoplasmic reticulum (ER) with a third, non-polymorphic invariant polypeptide (Ii). The alpha beta Ii complexes are subsequently transported through the Golgi to the endosomes, where the Ii chain dissociates before the alpha beta complex is transported to the cell surface. Results from transport-defective class II expression variant studies suggest that class II intracellular transport is regulated in multiple intracellular compartments. Consistent with this, a large number of studies have demonstrated that protein folding and/or oligomerization is facilitated in the ER by a class of proteins collectively known as molecular chaperones. In this report, we show that the ER-resident protein calnexin associates with human and murine class II antigens. Specifically, calnexin associates in the ER with free Ii polypeptides and partially assembled wild-type class II complexes, including A alpha and/or A alpha Ii complexes, as well as with alpha beta dimers isolated from class II transport-defective cells. Calnexin also physically associates with alpha beta Ii complexes, but not with mature alpha beta dimers. These results suggest that calnexin may regulate class II intracellular transport by facilitating the production of transport competent molecules out of the ER. In addition, we report that the nucleotide sequence of the gene encoding murine calnexin shows a high degree of homology to human IP90 and dog calnexin at both the nucleotide and deduced amino acid sequence level. The isolation of cDNA fragments encoding murine calnexin will allow us to further evaluate the functional consequences of calnexin-class II interaction.

Biochemical and functional similarities between human eosinophil-derived neurotoxin and eosinophil cationic protein: homology with ribonuclease.

Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) were isolated from lysates of human eosinophil granules by gel filtration and ion exchange chromatography on heparin-Sepharose. Radioimmunoassay, using monoclonal antibodies, of fractions from the heparin-Sepharose chromatography showed one peak of EDN activity and two peaks of ECP activity (termed ECP-1 and ECP-2). EDN, ECP-1, and ECP-2 each exhibited heterogeneity in charge and molecular weight when analyzed by two-dimensional nonequilibrium pH gradient electrophoresis and NaDodSO4/PAGE. Digestion of EDN with endoglycosidase F (endo F) decreased its molecular weight and charge heterogeneity. Thus, END likely contains a single complex oligosaccharide. Endo F digestion of ECP-1 and ECP-2 decreased the molecular weight of both polypeptides, indicating that both likely contain at least one complex oligosaccharide. Amino acid sequence analyses showed that ECP-1 and ECP-2 are identical from residue 1 through residue 59 and that the sequences of EDN and ECP are highly homologous (37 of 55 residues identical). Both EDN and ECP NH2-terminal sequences showed significant homology to RNase, especially in regions of the RNase molecule involved in ligand binding. EDN, ECP-1, and ECP-2 had neurotoxic activity, causing the Gordon phenomenon at doses down to 0.15 micrograms when injected into the cisterna magna; the proteins were comparable in their activities. These results indicate that EDN and ECP are related proteins and suggest that they derived from genes associated with the RNase family.

Gi proteins use a novel beta gamma- and Ras-independent pathway to activate extracellular signal-regulated kinase and mobilize AP-1 transcription factors in Jurkat T lymphocytes.

Receptors coupled to pertussis toxin (PTX)-sensitive Gi proteins regulate T lymphocyte cytokine secretion, proliferation, and chemotaxis, yet little is known about the molecular mechanisms of Gi protein signaling in mammalian lymphocytes. Using the Jurkat T lymphocyte cell line, we found that a stably expressed Gi protein-coupled receptor (the delta-opioid receptor (DOR1)) stimulates MEK-1 and extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) and transcriptional activity by an ERK target, Elk-1, via a mechanism requiring a PTX-sensitive Gi protein. Levels of beta-adrenergic receptor kinase-1 C-terminal fragment that inhibited signaling by Gi protein beta gamma subunits in these cells had no effect on DOR1 stimulation of either MEK-1- or Elk-1-dependent transcription, indicating that this pathway is independent of beta gamma. Analysis of this betagamma-independent pathway indicates a role for a herbimycin A-sensitive tyrosine kinase. Unlike beta gamma-mediated pathways, the beta gamma-independent pathway was insensitive to RasN17, inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), and constitutive PI 3-kinase activity. The beta gamma-independent pathway regulates downstream events, since blocking it abrogated both Elk-1-dependent transcription and mobilization of the mitogenic transcription factor, AP-1, in response to DOR1 signaling. These results characterize a novel, Ras- and PI 3kinase-independent pathway for ERK activation by Gi protein signaling that is distinct from ERK activation by beta gamma and may therefore be mediated by the alphai subunit.

Maturation versus death of developing double-positive thymocytes reflects competing effects on Bcl-2 expression and can be regulated by the intensity of CD28 costimulation.

Immature double-positive (DP) thymocytes mature into CD4(+)CD8(-) cells in response to coengagement of TCR with any of a variety of cell surface "coinducer" receptors, including CD2. In contrast, DP thymocytes are signaled to undergo apoptosis by coengagement of TCR with CD28 costimulatory receptors, but the molecular basis for DP thymocyte apoptosis by TCR plus CD28 coengagement is not known. In the present study, we report that TCR plus CD28 coengagement does not invariably induce DP thymocyte apoptosis but, depending on the intensity of CD28 costimulation, can induce DP thymocyte maturation. We demonstrate that distinct but interacting signal transduction pathways mediate DP thymocyte maturation signals and DP thymocyte apoptotic signals. Specifically, DP maturation signals are transduced by the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and up-regulate expression of the antiapoptotic protein Bcl-2. In contrast, the apoptotic response stimulated by CD28 costimulatory signals is mediated by ERK/MAPK-independent pathways. Importantly, when TCR-activated thymocytes are simultaneously coengaged by both CD28 and CD2 receptors, CD28 signals can inhibit ERK/MAPK-dependent Bcl-2 protein up-regulation. Thus, there is cross-talk between the signal transduction pathways that transduce apoptotic and maturation responses, enabling CD28-initiated signal transduction pathways to both stimulate DP thymocyte apoptosis and also negatively regulate maturation responses initiated by TCR plus CD2 coengagement.

Inhibition of interleukin-1-stimulated NF-kappaB RelA/p65 phosphorylation by mesalamine is accompanied by decreased transcriptional activity.

Nuclear factor kappaB (NF-kappaB) is an inducible transcription factor that regulates genes important in immunity and inflammation. The activity of NF-kappaB is highly regulated: transcriptionally active NF-kappaB proteins are sequestered in the cytoplasm by inhibitory proteins, IkappaB. A variety of extracellular signals, including interleukin-1 (IL-1), activate NF-kappaB by inducing phosphorylation and degradation of IkappaB, allowing nuclear translocation and DNA binding of NF-kappaB. Many of the stimuli that activate NF-kappaB by inducing IkappaB degradation also cause phosphorylation of the NF-kappaB RelA (p65) polypeptide. The transactivating capacity of RelA is positively regulated by phosphorylation, suggesting that in addition to cytosolic sequestration by IkappaB, phosphorylation represents another mechanism for control of NF-kappaB activity. In this report, we demonstrate that mesalamine, an anti-inflammatory aminosalicylate, dose-dependently inhibits IL-1-stimulated NF-kappaB-dependent transcription without preventing IkappaB degradation or nuclear translocation and DNA binding of the transcriptionally active NF-kappaB proteins, RelA, c-Rel, or RelB. Mesalamine was found to inhibit IL-1-stimulated RelA phosphorylation. These data suggest that pharmacologic modulation of the phosphorylation status of RelA regulates the transcriptional activity of NF-kappaB, independent of nuclear translocation and DNA binding. These findings highlight the importance of inducible phosphorylation of RelA in the control of NF-kappaB activity.