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A Truneh

Publications and source records attributed to A Truneh.

At least 37 records · Page 2Linked to original sources
PubMed1997

Carvedilol, a multiple-action neurohumoral antagonist, inhibits mitogen-activated protein kinase and cell cycle progression in vascular smooth muscle cells.

Recent findings that the multiple-action neurohumoral antagonist carvedilol inhibits the mitogenic effects of a broad variety of mitogens and produces marked protection against neointima formation after balloon angioplasty injury prompted further study into the molecular and biochemical mechanism of action. In the present study, the effects of carvedilol on mitogen-activated protein (MAP) kinase activity and cell cycle progression were evaluated. Carvedilol produced significant concentration-dependent inhibition of mitogen-induced MAP kinase activity in rat smooth muscle cells. Furthermore, when MAP kinase was purified from mitogen-stimulated cells by FPLC Mono Q chromatography, carvedilol produced direct enzyme inhibition. In the cell-free assay, carvedilol (10 microM) produced 50% inhibition of MAP kinase activity. Cell flow cytometry studies revealed that quiescent rat aortic smooth muscle cells showed 96% of the cell population in the G0/G1 phase of the cell cycle. The addition of serum (10%) increased the number of cells in S and G2/M phases 20% to 40%, respectively. Carvedilol (10 microM) significantly decreased (30-50%) the number of cells in S and G2/M phase. In addition, carvedilol significantly inhibited (>70%) serum-induced stimulation of the S phase-specific marker thymidine kinase. These data suggest that the antimitogenic actions of carvedilol on vascular smooth muscle may be in part due to the inhibition of MAP kinase activity and regulation of cell cycle progression.

Animals↗
PubMed1996

Characterization of CD4-gp120 activation intermediates during human immunodeficiency virus type 1 syncytium formation.

The mechanism by which cells expressing HIV envelope glycoproteins progress from binding CD4+ cells to syncytia formation is not entirely understood. The purpose of these investigations was to use physical and biochemical tools (temperature shifts, soluble CD4, protease inhibitors, and a battery of anti-CD4 monoclonal antibodies) to isolate discrete steps during syncytia formation. Previously (Fu et al., J Virol 1993;67:3818), we found that preincubation of cells stably expressing HIV-1 gp 160 (TF228.1.16) with CD4+ SupT1 cells at 16 degrees C, a temperature that is nonpermissive for syncytia formation, resulted in an increased rate of syncytia formation when the cocultures were shifted to the syncytia-permissive temperature of 37 degrees C. We have since found that syncytia formation is further enhanced by shifting the cocultures from 16 to 4 degrees C prior to incubation at 37 degrees C. Together, these data suggest that two discrete states, which we term the first and second activation intermediates (FAI and SAI), are involved in syncytia formation. We have found that acquisition of the FAI (by preincubation at 16 degree C) is sensitive to some serine protease inhibitors (PI), soluble CD4 (sCD4), shedding of gp120, and anti-CD4 monoclonal antibodies (MAb) directed toward the CDR-1/2 and CDR-3 regions of domain 1 on CD4. Expression of the FAI (formation of syncytia by shifting from 16 to 37 degrees C) remains sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2 but is less sensitive to MAb that bind CDR-3 and is insensitive to PI. Similarly, acquisition of the SAI (shifting cocultures from 16 to 4 degrees C), is sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2. In contrast, expression of the SAI (shifting cocultures from 16 to 4 to 37 degrees C) is sensitive only to MAb directed toward CDR-1/2 and cannot be blocked by sCD4, shedding of gp120, or PI. These data allow us to propose that syncytia formation, mediated by HIV-1 envelope glycoproteins, proceeds by a multistep cascade.

Antibodies, Monoclonal↗
PubMed1996

Simple differentiation between core-fucosylated and nonfucosylated glycans by capillary electrophoresis.

Core-fucosylated glycans, derivatized with 2-aminoacridone, consistently migrate slower than the corresponding oligosaccharides which lack this fucose residue, using the micellar electrophoretic capillary chromatography conditions outlined in this study. alpha-Fucosidase digestion of glycans followed by CE analysis has allowed facile differentiation of these two classes of oligosaccharides and this methodology has been applied to obtain preliminary information on the carbohydrate content from two glycoproteins, a monoclonal IgG antibody and the soluble complement receptor type 1 (sCR1).

Aminoacridines↗
PubMed1996

Analysis of the site of interaction of CD28 with its counter-receptors CD80 and CD86 and correlation with function.

CD28 and CTLA-4 are homologue members of the Ig superfamily of molecules, containing a single V-like domain, transmembrane, and cytoplasmic regions. Both receptors associate with the counter-receptors CD80 and CD86, but the avidity of interaction for CD28 is about 20-fold lower than for CTLA-4. The interaction between CD28 and its cognate receptors provides a costimulatory signal for optimal T cell activation. Our previous mutational analysis of CD28 defined the highly conserved "MYPPPY" motif in the CDR3-region of the V-like domain as a key site of common and selective recognition. We have extended our analysis to cover all residues in the membrane distal loops of the V region, examining their effect on association with CD80/CD86 in cell adhesion and novel protein-based binding assays, and determining correlation between binding and functional response. Conservative F substitutions at either Y residue in the MYPPPY motif selectively reduced binding to CD86, but mutation of the three amino acids immediately C-terminal to Y 104 equivalently reduced binding to both co-receptors. The conservative F substitution of Y 26 in the CDR1-like region also reduced binding to CD80 and CD86. Other substitutions in the CDR1 loop and mutations spanning the CDR2 and DE loops had no effect. We conclude that the CDR1 and CDR3 regions contribute to a common binding site for CD80/CD86, and that the CDR3 region also carries determinants for selective recognition of these counter-receptors within the MYPPPY motif. Furthermore, for CD28, the strength of functional response, as measured by IL-2 production, directly correlates with binding avidity.

Amino Acid Sequence↗
PubMed1996

Two distinct intracytoplasmic regions of the T-cell adhesion molecule CD28 participate in phosphatidylinositol 3-kinase association.

Through the interaction with its ligands, CD80/B7-1 and CD86/B7-2 or B70, the human CD28 molecule plays a major functional role as a costimulator of T cells along with the CD3-TcR complex. We and others have previously reported that phosphatidylinositol 3-kinase inducibly associates with CD28. This association is mediated by the SH2 domains of the p85 adaptor subunit interacting with a cytoplasmic YMNM consensus motif present in CD28 at position 173-176. Disruption of this binding site by site-directed mutagenesis abolishes CD28-induced activation events in a murine T-cell hybridoma transfected with human CD28 gene. Here we show that the last 10 residues of the intracytoplasmic domain of CD28 (residues 193-202) are required for its costimulatory function. These residues are involved in interleukin-2 secretion, p85 binding, and CD28-associated phosphatidylinositol 3-kinase activity. In contrast, the CD28/CD8O interaction is unaffected by this deletion, as is the induction of other second messengers such as the rise in intracellular calcium and tyrosine phosphorylation of CD28-specific substrates. Furthermore, we also demonstrate that, within these residues, the tyrosine at position 200 is involved in p85 binding, probably together with the short proline-rich motif present between residues 190 and 194 (PYAPP).

Amino Acid Sequence↗
PubMed1996

Signal transduction by CD28 costimulatory receptor on T cells. B7-1 and B7-2 regulation of tyrosine kinase adaptor molecules.

This study compares the biochemical responses in T cells activated with the CD28 ligands B7-1 and B7-2. The patterns of tyrosine phosphorylation induced in T cells by these two CD28 ligands are identical, but clearly different from the tyrosine phosphorylation induced by the T cell receptor (TCR). The TCR regulates protein complexes mediated by the adapter Grb2 both in vivo and in vitro. In contrast, there is no apparent regulation of in vivo Grb2 complexes in response to B7-1 or B7-2. Rather, B7-1 and B7-2 both induce tyrosine phosphorylation of a different adaptor protein, p62. The regulation of p62 is a unique CD28 response that is not shared with the TCR. These data indicate that B7-1 and B7-2 induce identical tyrosine kinase signal transduction pathways. The data show also that the TCR and CD28 couple to different adapter proteins, which could explain the divergence of TCR and CD28 signal transduction pathways during T cell activation.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Comparison of CD28-B7.1 and B7.2 functional interaction in resting human T cells: phosphatidylinositol 3-kinase association to CD28 and cytokine production.

CD28 is a 44kDa homodimer present on T cells providing an important costimulatory signal for T cell proliferation, cytokine production and cytokine receptor expression. CD28 activation is mediated by interaction with its counter-receptors, B7.1/CD80 and B7.2/B70/CD86. The biochemical basis of these co-stimulatory signals are still poorly understood, particularly in resting T cells. However, various biochemical pathways such as tyrosine phosphorylation, phospholipase C, sphingomyelinase and phosphatidylinositol 3-kinase (PI3-K) activation have been reported to play a role in CD28 signaling in tumor T cell lines and CD28-transfected cells or pre-activated T cells. In addition, recent reports propose that CD28-B7.1 and B7.2 interaction could be involved in the production of Th1 and Th2 cytokines, respectively, but the putative biochemical basis for these different functions is still unknown. We have analyzed the functional and molecular consequences of CD28 activation by B7.1 and B7.2 in human resting T cells. We demonstrate in this report that both CD28-B7.1 and CD28-B7.2 interactions induce the association of PI3-K to CD28 in the CD4 subpopulation, whereas it was barely detectable in CD8 cells. This association involves the binding of the src homology domain 2 (SH2) of p85 to tyrosine-phosphorylated CD28 and does not require pre-activation by CD3-T cell receptor. Worthmannin, a specific inhibitor of PI3-K enzymatic activity within the nanomolar range also inhibits the interleukin-2 production induced by costimulation mediated by either the B7.1- and B7.2-transfected cells or CD28 monoclonal antibodies. The only slight difference between B7.1 and B7.2 costimulation is the IC50 of worthmannin being 25 and 110 nM, respectively, which could suggest differences in their activation of the T cell PI3-K.

Androstadienes↗
PubMed1996

Differential recognition by CD28 of its cognate counter receptors CD80 (B7.1) and B70 (B7.2): analysis by site directed mutagenesis.

CD28, which is a member of the immunoglobulin superfamily of molecules (IgSF), is a homodimer of two polypeptides containing a single V-like domain with short transmembrane and cytoplasmic regions. It serves as a co-signalling molecule for T cell activation through binding to its cognate counter-receptors CD80 and B70, expressed on antigen presenting cells. In the current study, we investigated the regions of CD28 which are involved in its interactions with CD80 and B70, using site directed mutagenesis, CD28 mAb epitope mapping, receptor based adhesion assays and direct binding of Ig-fusion proteins to cell surface receptors. Truncation or substitution of a stretch of a proline rich "hallmark" sequence, "MYPPPY", abrogates binding to CD80 or B70, while retaining CD28 mAb epitopes and cell surface expression. On an Ig-fold model of the CD28 V-domain, this fully conserved motif localizes to a CDR3-like region. Mutations introduced into other loops, including the CDRI-like and CDR2-like regions, had very little effect on CD80 or B70 binding. Mutations introduced within the predicted beta-strand regions caused loss of receptor expression. Conservative substitution of both the flanking tyrosine residues within the "MYPPPY" motif with phenylalanine, caused loss of binding to B70 but not to CD80. These results show that, although the same overall region on CD28 may be involved in the interactions with CD80 and B70, subtle but important differences distinguish recognition by the two molecules. These finding, along with previous observations on the differential pattern of expression and tissue distribution of CD80 and B70, support the contention that these molecules play distinct roles in the regulation of immune responses in vivo.

Abatacept↗
PubMed1996

CD28 signal transduction pathways. A comparison of B7-1 and B7-2 regulation of the map kinases: ERK2 and Jun kinases.

The present study compares the mitogen-activated protein (MAP) kinase responses in T cells activated with the CD28 ligands B7-1 (CD80) and B7-2/B70 (CD86). Ligands B7-1 and B7-2 do not activate the Raf-1/ERK2 cascade, but share the ability to activate related Jun kinases. These natural ligands for CD28 had no stimulatory effect alone on Jun kinase activation, but the data show that B7-1 and B7-2 could both co-operate with intracellular Ca2+ increase and protein kinase C (PKC) activation to stimulate Jun kinases. The present study shows that the interaction of CD28 with its ligands B7-1 and B7-2 can induce identical signal transduction through the MAP kinase cascades.

Antigens, CD↗
PubMed1995

Identification of residues in the V domain of CD80 (B7-1) implicated in functional interactions with CD28 and CTLA4.

The CD80 (B7-1) molecule is a 45-60-kD member of the immunoglobulin superfamily that is expressed on a variety of cell types of haematopoietic origin. CD80 can provide a critical costimulatory signal to T cells by interacting with the T cell surface molecule CD28. CD80 also binds to the CD28-related molecule CTLA4, which is expressed on activated T cells, Recently, additional ligands of CD28 and CTLA4 have been described in mice and humans. One of them, CD86 (B-70 or B7-2) was characterized at the molecular level. Although similar in predicted structure to CD80, it is distantly related in amino acid sequence. In this study, human CD80 mutants were generated and tested for their ability to maintain the interaction with CD28 leading to adhesion and enhanced IL-2 production. Two hydrophobic residues in the V-like domain of CD80 were identified as critical for binding to CD28 and are also important for the interaction with CTLA4. These residues are adjacent to the epitope of the BB1 antibody, which inhibits CD28-CD80 interactions. One of these residues, Y87, is conserved in all CD80 and CD86 cloned from various species. These results being to unravel the structural requirements for binding to CD28 and CTLA4.

Abatacept↗
PubMed1994

The role of p21ras in CD28 signal transduction: triggering of CD28 with antibodies, but not the ligand B7-1, activates p21ras.

CD28 is a 44-kD homodimer expressed on the surface of the majority of human T cells that provides an important costimulus for T cell activation. The biochemical basis of the CD28 accessory signals is poorly understood. Triggering of the T cell antigen receptor (TCR) activates the p21ras proteins. Here we show that ligation of CD28 by a monoclonal antibody (mAb) also stimulates p21ras and induces Ras-dependent events such as stimulation of the microtubule-associated protein (MAP) kinase ERK2 and hyperphosphorylation of Raf-1. One physiological ligand for CD28 is the molecule B7-1. In contrast to the effect of CD28 mAb, the present studies show that interactions between CD28 and B7-1 do not stimulate p21ras signaling pathways. Two substrates for TCR-regulated protein tyrosine kinases (PTKs) have been implicated in p21ras activation in T cells: p95vav and a 36-kD protein that associates with a complex of Grb2 and the Ras exchange protein Sos. Triggering CD28 with both antibodies and B7-1 activates cellular PTKs, and we have exploited the differences between antibodies and B7-1 for p21ras activation in an attempt to identify critical PTK-controlled events for Ras activation in T cells. The data show that antibodies against TCR or CD28 induce tyrosine phosphorylation of both Vav and p36. B7-1 also induces Vav tyrosine phosphorylation but has no apparent effect on tyrosine phosphorylation of the Grb2-associated p36 protein. The intensity of the Vav tyrosine phosphorylation is greater in B7-1 than in TCR-stimulated cells. Moreover the kinetics of Vav tyrosine phosphorylation is prolonged in the B7-1-stimulated cells. These studies show that for CD28 signaling, the activation of p21ras correlates more closely with p36 tyrosine phosphorylation than with Vav tyrosine phosphorylation. However, the experiments demonstrate that Vav is a major substrate for B7-activated PTKs and hence could be important in CD28 signal transduction pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Binding of phosphatidylinositol-3-OH kinase to CD28 is required for T-cell signalling.

The engagement of CD28 with its ligand B7.1/CD80 results in potent costimulation of T-cell activation initiated through the CD3/T-cell receptor complex. The biochemical basis of CD28 costimulatory function is poorly understood. The signalling pathways used by CD28 are unlike those used by the CD3/T-cell receptor in that they are resistant to cyclosporin A and independent of changes in cyclic AMP concentrations. These differences suggest that each pathway provides unique biochemical information which is required for T-cell activation. We report here that CD28 becomes tyrosine-phosphorylated following interaction with B7.1/CD80, which induces formation of a complex with phosphatidylinositol-3-OH kinase, mediated by the SH2 domains of the p85 subunit of the kinase. Phosphatidylinositol-3-OH kinase is a heterodimer of this 85K regulatory subunit and a 110K catalytic subunit, and is a common substrate for most receptor tyrosine kinases and some cytokine receptors, binding through its SH2 domain to phosphotyrosine in the motif Tyr-X-X-Met in the CD28 sequence, which is highly conserved between human, mouse and rat and lies in the intracellular domain. We show that CD28 mutants that have their kinase-binding site deleted or the tyrosine at position 173 substituted by phenylalanine do not associate with the kinase after CD28 stimulation and cannot stimulate production of interleukin-2. Our results suggest that phosphatidylinositol-3-OH kinase is critical for signalling by CD28.

Animals↗
PubMed1994

Structures of an HIV and MHC binding fragment from human CD4 as refined in two crystal lattices.

BACKGROUND: The T-cell surface glycoprotein CD4 interacts with class II molecules of the major histocompatibility complex (MHC) enhancing the signal for T-cell activation. Human CD4 also interacts, at high affinity, with the HIV envelope glycoprotein, gp120, to mediate T-cell infection by HIV. Crystal structures of amino-terminal two-domain (D1D2) fragments of human CD4, which contain the residues implicated in HIV and MHC interactions, have been reported earlier. RESULTS: We have determined the crystal structure of a new D1D2 construct by molecular replacement from a previously described crystal structure of D1D2. This structure has more uniform lattice contacts than are in the first. This gives an improved image of domain D2, which in turn has permitted further refinement of the initial structure at 2.3 A resolution against a more complete data set. The structure of the second crystal form was also refined at 2.9 A resolution. In both models, all residues from 1 to 178 are now well defined, including the loop regions in D2. CONCLUSIONS: Similarities of the molecular structure in the two lattices suggest that the D1D2 fragment works as a unit, with segmental flexibility largely restricted to the junction between domains D2 and D3. Variability of conformation in loops, including those implicated in MHC and HIV binding, requires an 'induced fit' in these interactions. Well defined density for the exposed side chain of Phe43 in both crystals confirms a prominent role for this residue in gp120 binding.

Amino Acid Sequence↗
PubMed1994

Functional expression of human CD28 in murine T cell hybridomas.

CD28 is a 44 kDa Ig superfamily cell surface molecule expressed on most mature T cells. Through its interaction with the recently identified B7/BB1 counter-receptor, it is believed to play an important role as a co-stimulator of T cells along with the TCR-CD3 complex. Activation of T cells with CD28 mAbs synergizes with TCR-CD3 and CD2 stimulation, resulting in long term T cell proliferation, differentiation of cytotoxic T cells and production of large amounts of cytokines. In order to further delineate the role of CD28 in signal transduction and T cell activation, human CD28 was transfected into CD3+ murine T cell hybridomas. High levels of cell surface CD28 expression was achieved by protoplast fusion. The transfected molecule retained all the native CD28 mAb epitopes found on human T cells. In these transfectants, CD28 mAbs, similarly to CD3 mAbs, were able to induce Ca2+ mobilization, IL-2 promoter induction (measured as beta-galactosidase activity in T cells hybridomas pre-transfected with the IL-2-lac Z reporter gene), IL-2 secretion, TNF alpha production and apoptosis (observed as growth arrest and genome fragmentation). The parental host cells, or cells transfected with vector alone, responded only to mAbs to CD3. IL-2 secretion in the transfectants was obtained using either an IgM mAb to CD28 or IgG mAbs presented on the surface of IgG-FcR+ B lymphoma cells. Optimal activation via CD28 was inhibited by suboptimal concentrations of soluble CD3 mAb, suggesting an interaction between the two pathways. The immunosuppressive drugs Cyclosporin A and FK506 completely blocked CD28 and CD3 mediated IL-2 production in these transfectants whereas rapamycin had only a partial inhibitory effect. Finally, since the transfected human CD28 molecule confers full functional responsiveness to the murine T cell hybridomas without the need for costimulators such as PMA, this model is ideal for studying the structure-function relationships of the CD28 molecule as well as the transmembrane and cytoplasmic associations implied in CD28 signaling.

Animals↗
PubMed1994

Pyridinyl imidazoles inhibit the inflammatory phase of delayed type hypersensitivity reactions without affecting T-dependent immune responses.

The effects of pyridinyl imidazoles, specifically SK&F 105809 and its metabolite, on normal T-cell and B-cell mediated immune responses were examined and compared to the fungal macrolide immunosuppressives, cyclosporin A, FK506 and rapamycin and to the corticosteroid, dexamethasone. The orally active prodrug SK&F 105809 [2-(4-methylsulfinylphenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo [1,2-a] imidazole[ and its metabolite, SK&F 105561 [2-(4-methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2 -a] imidazole] are dual 5-lipoxygenase (5-LO) and cycloxygenase (CO) inhibitors with potent anti-inflammatory and cytokine (IL-1/TNF) suppressive activities. The anti-inflammatory activity of SK&F 105809 and its metabolite were evaluated in an antigen-specific murine model of delayed type hypersensitivity (DTH) response, where they were found to affect only the inflammatory and not the induction phase of this response. In contrast, these compounds and other pyridinyl imidazoles (SK&F 86002 and its analog, SK&F 104351) exhibited no immunosuppressive activity under conditions where the macrolide rapamycin and the corticosteroid dexamethasone abrogated both the cellular and humoral immune responses. Thus, the ability of pyridinyl imidazoles to attenuate independently the inflammatory components of the disease without causing generalized immunosuppression enhances their profile as candidates for therapy of chronic inflammatory diseases, specifically those mediated by cytokines (e.g. IL-1, TNF) and eicosanoids.

Animals↗
PubMed1993

Oligodeoxynucleotides interact with recombinant CD4 at multiple sites.

Phosphodiester oligodeoxynucleotides bearing the 5'-alkylating moiety 4-(N-2-chloroethyl-N-methyl)aminobenzylamine specifically modify recombinant soluble CD4 (rsCD4) in solution. This reaction is saturable with respect to the alkylating oligonucleotide reagent. The existence of at least two binding sites, with different affinities, on the rsCD4 molecule, were demonstrated. The values of apparent Kd for the sites are approximately 0.1 and 1 microM. The existence of two sites was confirmed by electrophoretic analysis of the modified protein, in which two distinct gel bands were seen. The modification is inhibited by excess non-alkylating oligonucleotide, as well as by phosphorothioate oligonucleotides. Quantitative estimates of the competition constants (Kc), for the binding of these competitors of the binding of the alkylating oligonucleotide reagent with rsCD4, have been made. By use of this method, several anionic dyes as well as potential anti-HIV therapeutic agents were also demonstrated to interact with rsCD4. Phosphorothioate oligonucleotides also inhibit binding of rsCD4 with the monoclonal antibody L71.1.1 This monoclonal antibody recognizes the CDR3-like loop (D1 domain) of the rsCD4 molecule. Thus, oligonucleotide binding sites exist on two remote regions (i.e. both the CDR2- and CDR3-like loops) of the D1 domain of CD4.

Alkylating Agents↗