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Biomedical subjects

E Gulbins

Publications and source records attributed to E Gulbins.

At least 91 records · Page 5Linked to original sources
PubMed1996

Tyrosine phosphorylation-dependent suppression of a voltage-gated K+ channel in T lymphocytes upon Fas stimulation.

Selective cell death plays a critical role in the development of the immune system and in the elimination of target cells expressing foreign antigens. Most of programmed cell death occurs by apoptosis. Apoptotic cell death of lymphocytes can be triggered by ligation of APO-1/Fas (CD95) antigen (Suda, T., and Nagata, S. (1994) J. Exp. Med. 179, 873-879; Nagata, S., and Golstein, P. (1995) Science 267, 1449-1456). We find that activation of Fas leads to the inhibition of the voltage-dependent n-type K+ channels (Kv1.3) studied by patch clamp technique in Jurkat T lymphocytes. Tyrosine kinases have been shown to be crucial in Fas-induced cell death (Eischen, C. M., Dick, C. J., and Leibson, P. J. (1994) J. Immunol. 153, 1947-1954). The inhibition of the current is correlated with the tyrosine phosphorylation of immunoprecipitated and blotted K+ channel protein. We show, that the Src-like protein-tyrosine kinase inhibitor herbimycin A and the deficiency of the p56(lck) tyrosine kinase in mutant Jurkat cells abolished the channel inhibition and phosphorylation by anti-Fas antibody, while reconstitution of the p56(lck) kinase partly restored these effects of Fas receptor triggering. These results suggest a regulation of n-type K+ channels by tyrosine kinases upon Fas receptor triggering, which might be important for apoptosis.

Apoptosis↗
PubMed1996

Activation of Src-family tyrosine kinases during Fas-induced apoptosis.

Stimulation of several human and murine hematopoietically derived cell lines with anti-Fas antibodies induced increased tyrosine phosphorylation of a panel of proteins observed in whole-cell lysates. In the human T cell line Jurkat, the activity of a 56-kDa tyrosine kinase was likewise activated by anti-Fas antibodies. Immunoprecipitation studies of anti-Fas-stimulated human Jurkat and murine 2B4.11 T cells revealed activation of the Src-family tyrosine kinases Lck and Fyn. Fas receptor-induced tyrosine phosphorylation of p120 c-cbl proto-oncogene product was observed in Jurkat T cells. Pharmacological experiments demonstrated that pretreatment of Jurkat cells with tyrphostins inhibited Fas-induced apoptosis; likewise, Lck activity was inhibited by tyrphostins in a dose-dependent fashion. Finally, Lck derived from unstimulated Jurkat T cells formed stable complexes with the intracellular domain of the Fas receptor. These data are consistent with the notion that expression and activation of members of the Src-family kinases is required for Fas-induced cell death in T lymphocytes and consistent with recent findings demonstrating decreased Fas-mediated thymocytic death in Fyn-knockout mice.

Animals↗
PubMed1996

Fas-induced programmed cell death is mediated by a Ras-regulated O2- synthesis.

Fas induces apoptosis in lymphocytes via a poorly defined intracellular signalling cascade. Previously, we have demonstrated the involvement and significance of a signalling cascade from the Fas receptor via sphingomyelinases and ceramide to Ras in Fas-induced apoptosis. Here we demonstrate rapid and transient synthesis of reactive oxygen intermediates (ROI) via activation of Ras after Fas. Genetic inhibition of Ras by transfection of transdominant inhibitory N17Ras blocked Fas-mediated ROI synthesis and programmed cell death. Likewise, the antioxidants N-acetyl-cysteine and N-t-butyl-phenylnitrone abolished Fas-induced cell death, pointing to an important role for Ras-triggered ROI synthesis in Fas-mediated programmed cell death.

Acetylcysteine↗
PubMed1995

Molecular analysis of Ras activation by tyrosine phosphorylated Vav.

Vav has been shown to activate Ras (1-3) and is regulated by tyrosine phosphorylation (1) or binding of diglycerides (3) to the cysteine rich domain. In the present study employing different Ras activation assay techniques using [3H]GDP release or [32P]alpha GTP-binding from membrane-bound or soluble recombinant Ras, we demonstrate that Ras activity can be increased by tyrosine phosphorylated Vav upon cellular stimulation via the IL-2 receptor or the TCR/CD3-complex. Increase of [32P]alpha GTP-binding to Ras catalyzed by phosphorylated Vav is similar to the activity of immunoprecipitated Sos. The activity of Vav measured by binding of [32P]alpha GTP to Ras was linear with respect to the concentration of Vav protein used. To study molecular characteristics of this Vav-Ras interaction, we used several Ras mutants and demonstrate that Vav activity towards Ras depends on the integrity of the same or similar domains as Ras activation by SDC 25 or CDC 25.

CD3 Complex↗
PubMed1995

Survival and death of prelymphomatous B-cells from N-myc/bcl-2 double transgenic mice correlates with the regulation of intracellular Ca2+ fluxes.

Coexpression of the proto-oncogenes c-myc and bcl-2 under the control of the immunoglobulin enhancer E mu provokes the rapid development of primitive lymphoid tumors in transgenic mice. In the present study we show that the myc family members N-myc and L-myc also cooperate with bcl-2 in oncogenesis and can provoke the development of more mature pre-B, B and T cell type lymphomas. The analysis of prelymphomatous B-cells from single E mu N-myc and bcl-2-Ig transgenic animals and from young, tumor free, double transgenic E mu N-myc/bcl-2-Ig mice revealed that E mu directed expression of N-myc leads to very rapid apoptosis after explantation and culturing compared to B-cells from normal mice. As expected, B-cells from bcl-2-Ig transgenics were protected to a certain degree from apoptosis. Strikingly however, B-cells from E mu N-myc/bcl-2-Ig double transgenic animals were found to be almost completely resistant towards a number of different apoptotic stimuli. Furthermore, after treatment with H2O2, which can trigger apoptosis, B-cells from E mu N-myc animals reach levels of intracellular free Ca2+ concentrations that are comparable to B-cells from normal mice, whereas B-cells from bcl-2-Ig or E mu N-myc/bcl-2-Ig double transgenic mice show no increase in intracellular Ca2+ concentrations after stimulation with H2O2. These findings suggest that the prevention of apoptosis conferred by bcl-2 correlates with the inhibition of intracellular Ca2+ fluxes whereas induction of apoptosis mediated by N-myc requires normal Ca2+ levels. We hypothesize therefore that the regulation of intracellular Ca2+ concentrations represent one important parameter in the oncogenic cooperation between bcl-2 and N-myc.

Animals↗
PubMed1995

The role of p56lck in CD4-mediated suppression of CD3-induced early signaling events in T lymphocytes.

Crosslinking of the CD4 coreceptor can inhibit subsequent T-cell activation via the T-cell antigen receptor (TCR)/CD3 complex. The ability of the human immunodeficiency virus (HIV) envelope protein, gp 120, to cause similar inhibition has implicated this inhibitory signal in the induction of T-cell anergy and apoptosis observed in the acquired immunodeficiency syndrome (AIDS). In order to clarify the biochemical basis of this inhibition, we analyzed the effect of CD4 ligation on early signaling events induced by subsequent CD3xCD4 co-crosslinking. By comparison with CD3 crosslinking alone, CD3xCD4 co-crosslinking of a CD3+CD4+ human T-cell leukemia line (SupT1) resulted in an enhanced increase in free intracellular calcium concentration and tyrosine phosphorylation of several cellular substrates, including the prominent phosphorylation of an unidentified 120-kDa protein (p120). Prior CD4 ligation inhibited these responses. Similar results were obtained with A3.01, another CD3+CD4+ T leukemic line. However, P120 was only minor phosphorylated on tyrosine upon receptor crosslinking in A2.01/CD4(-cyt401), a derivative line expressing a truncated CD4 coreceptor lacking its cytoplasmic domain which binds the p56lck protein tyrosine kinase (PTK). Furthermore, prior CD4 ligation failed to inhibit in this line the increased tyrosine phosphorylation induced by subsequent CD3xCD4 co-crosslinking. Thus, prior CD4 crosslinking, or expression of truncated CD4, are both associated with reduced p120 phosphorylation. These results suggest that p120 is a p56lck substrate playing an important role during T-cell activation.

CD3 Complex↗
PubMed1995

Molecular basis of IsK protein regulation by oxidation or chelation.

Slowly activating IsK channels were expressed in Xenopus oocytes and exposed to oxidative agents. Oxidative treatment reduced the resulting current IsK, while no inhibition was observed for IsK protein mutants carrying a Ser mutation instead of a highly conserved Cys residue in the intracellular domain. In contrast, Hg2+, which may not only oxidize thiol groups but also form chelates with dibasic amino acids, caused a use-dependent, positive regulation of IsK. This effect was reversed in an IsK protein mutant with a deletion in the extracellular domain. These data suggest opposite effects of peroxides and Hg2+ on IsK, a peroxide-mediated IsK inhibition by intracellular oxidation and a Hg(2+)-mediated IsK increase, caused by extracellular Hg2+ chelation of the IsK protein.

Animals↗
PubMed1995

The nitroso-donor S-nitroso-cysteine regulates IsK expressed in Xenopus oocytes via a c-GMP independent mechanism.

In Xenopus oocytes expressing slowly activating IsK channels superfusion with the nitroso-donor S-Nitroso-Cysteine (SNOC) resulted in an increase of IsK, which was greatly enhanced when the amino acid-exchanger rBAT was coexpressed. The effects of SNOC on IsK could not be prevented by the guanylate cyclase inhibitor LY-83,583 and the cGMP kinase inhibitor H8, but was abolished in the presence of staurosporine. SNOC also increased the currents induced by the expression of protein mutants lacking intracellular sites, previously described to be involved in IsK regulation by oxidation and phosphorylation. These data suggest that the NO-donor SNOC regulates IsK indirectly via a cGMP independent, but staurosporine sensitive, pathway.

Alkaloids↗
PubMed1995

FAS-induced apoptosis is mediated via a ceramide-initiated RAS signaling pathway.

Fas receptor-induced apoptosis plays critical roles in immune homeostasis. However, most of the signal transduction events distal to Fas ligation have not been elucidated. Here, we show that Ras is activated following ligation of Fas on lymphoid lines. The activation of Ras is a critical component of this apoptotic pathway, since inhibition of Ras by neutralizing antibody or a dominant-negative Ras mutant interfered with Fas-induced apoptosis. Furthermore, ligation of Fas also resulted in stimulation of the sphingomyelin signalling pathway to produce ceramides, which, in turn, are capable of inducing both Ras activation and apoptosis. This suggests that ceramides acts as second messengers in Fas signaling via Ras. Thus, ligation of the Fas molecule on lymphocyte lines induces activation of Ras via the action of ceramide, and this activation is necessary, but not sufficient, for subsequent apoptosis.

Animals↗
PubMed1995

Heavy metal mediated inhibition of rBAT-induced amino acid transport.

rBAT, a protein which is located in the brush border membranes of intestine and renal proximal tubule cells, was recently shown to induce electrogenic countertransport of neutral and dibasic amino acids after its expression in Xenopus oocytes. Here, we studied the effects of heavy metals on rBAT induced amino acid transport in Xenopus oocytes to clarify a possible involvement of rBAT in heavy metal-induced aminoaciduria. The heavy metals Hg2+ and Pb2+ inhibited rBAT-induced amino acid transport with a different profile of action. The Pb2+ mediated inhibition occurred rapidly upon superfusion and was readily reversible upon washout. The maximal inhibition caused by Pb2+ was about 50% of the amino acid-induced currents at an apparent affinity (Km) of about 10 microM. In contrast, the Hg(2+)-mediated inhibition occurred rather slowly, depending on its concentration, and was not reversible during washout with control solution. However, the Hg(2+)-mediated amino acid transport inhibition could be reversed with Hg2+ chelating agents and reducing compounds. Other oxidative agents, such as the membrane permeable 2,2'-Dithio-bis(5-Nitropyridine) (DTNP), but not the membrane impermeable 5,5'-Dithio-bis (2-Nitrobenzoic acid) (DTNB), mimicked the effect of Hg2+, and their effect could similarly be reversed with 2,3-Dihydroxybutane-1,4-dithiol (DTE). In conclusion, Pb2+ and Hg2+ inhibit rBAT-induced amino acid transport in a noncompetitive, allosteric fashion. Blockade of rBAT-induced amino acid transport may be involved in aminoaciduria following mercury or lead intoxication.

Amino Acid Transport Systems, Basic↗
PubMed1994

Monocyte deactivation by interleukin 10 via inhibition of tyrosine kinase activity and the Ras signaling pathway.

Activation of monocytes by bacterial lipopolysaccharides (LPSs) is a central component in the pathogenesis of septic shock syndrome. Interleukin 10 (IL-10) is a potent monocyte-deactivating factor and transcriptionally inhibits LPS-induced expression of proinflammatory mediators. The intracellular signaling pathways of LPS have been only partially characterized and mechanisms of IL-10 signaling remain unknown. We show that LPS activates the protein tyrosine kinase (PTK) p56lyn and that this is associated with tyrosine phosphorylation of the protooncogene product Vav. These events are completely blocked by the tyrosine kinase inhibitor herbimycin A. LPS also increases Ras activation in monocytes. LPS-triggered phosphorylation of mitogen-activated protein kinase is a downstream activation event that is also reduced by herbimycin A. Analysis of the IL-10 effects shows that it completely inhibits the p56lyn tyrosine kinase activation and all other subsequent events in this pathway including Ras activation. The IL-10 effects are selective since it reduced PTK-dependent cytokine mRNA expression but not the PTK independent induction of c-jun and c-fos mRNA in LPS-activated monocytes. These results identify the Ras signaling pathway as a component of intracellular signaling in LPS-stimulated monocytes and define early events in this response as targets of monocyte deactivation by IL-10.

Base Sequence↗
PubMed1994

Tyrosine phosphorylation and activation of Vav GTP/GDP exchange activity in antigen receptor-triggered B cells.

Ag receptor triggering in B cells stimulates the activity of receptor-associated tyrosine protein kinases (TPK), leading to tyrosine phosphorylation of several cellular substrates, one of which is the Vav proto-oncogene product. We have recently determined that Vav is a TPK-regulated guanine nucleotide exchange factor for Ras in T cells. Here, we show that B cell extracts or Vav immunoprecipitates contain a Ras GDP/GTP exchange activity that is stimulated upon surface Ig (slg) triggering. The receptor-mediated stimulation of Vav exchange activity was blocked by the TPK antagonist, herbimycin A. Furthermore, immunodepletion of Vav from the B cell extracts removed approximately 80% of the Ras GDP/GTP exchange activity. These findings indicate, first, that B cell-derived Vav possesses GDP/GTP exchange activity for Ras; second, that the exchange activity of Vav is accelerated by a slg-triggered, herbimycin A-sensitive TPK and, third, that Vav accounts for most of the receptor-stimulated Ras GDP/GTP exchange activity. Thus, Vav may serve as a critical component in slg-mediated signal transduction pathways by coupling receptor-associated TPK to the activation of Ras proteins.

Animals↗
PubMed1994

Identification of a protein, SPY75, with repetitive helix-turn-helix motifs and an SH3 domain as a major substrate for protein tyrosine kinase(s) activated by Fc epsilon RI cross-linking.

Cross-linking of the high affinity receptor for IgE (Fc epsilon RI) initiates various biochemical and morphologic changes leading to degranulation and synthesis and release of cytokines and lipid mediators. Tyrosine phosphorylation of several cellular proteins was previously reported as the earliest signaling event for the Fc epsilon RI signal transduction pathway. By amino acid sequence determination and cDNA cloning analysis, a 75-kDa protein, termed SPY75, was identified as a major tyrosine-phosphorylated protein in activated mouse mast cells. SPY75, barely tyrosine phosphorylated in resting cells, was rapidly and transiently tyrosine phosphorylated on Fc epsilon RI cross-linking in an Ag concentration-dependent manner. Similar SPY75 tyrosine phosphorylation was observed when Ag receptors on B and T lymphocytes were cross-linked by appropriate antibodies. However, IL-3, granulocyte macrophage-CSF, or stem cell factor did not induce tyrosine phosphorylation of SPY75 in PT-18 mast cells, despite their responsiveness to these cytokines. SPY75 was not physically associated with the receptor or other known signaling molecules. This protein, the mouse homologue of the human HS1 gene product, has putative repetitive helix-turn-helix motifs found in many DNA-binding proteins and a putative nuclear transport signal. It also has a Src homology 3 domain, which is found in many signaling molecules and cytoskeletal proteins. These structural features and the rapid tyrosine phosphorylation on Fc epsilon RI cross-linking suggest that the signal generated by Fc epsilon RI cross-linking is transmitted through tyrosine phosphorylation of SPY75.

Amino Acid Sequence↗
PubMed1994

Effects of systemic complement activation on renal circulation of rats.

In a variety of immunopathological diseases activation of the complement cascade occurs either systemically or localized in the kidney. To elucidate the functional impact of complement activation upon the renal microcirculation, we administered cobra venom factor of Naja naja kaouthia (CVF) i.v. into thiobarbital anaesthetized female rats. CVF is a potent activator of the alternative pathway of complement by forming the C3-convertase CVF, Bb which cannot be downregulated by the natural inhibitor factors H and I and thereby leads to generation of the anaphylatoxins C3a and C5a and formation of the membrane attack complex (MAC). We utilized creatinine clearance and flowmeter measurements in the normal kidney and intravital microscopy of the split hydronephrotic rat kidney model to observe the microvascular changes. Bolus injection of CVF (100 U kg-1) resulted in an immediate reduction of RBF (-68% after 10 min), which remained decreased during the entire experiment (90 min). Systemic blood pressure was significantly reduced only in the early period (-23% of control: 126 mmHg after 10 min). After an initial anuric phase of 30 min duration, the glomerular filtration rate was significantly diminished by 47%. White cell count was decreased by about 50% after the experiments. Application of the competitive thromboxane A2-antagonist, BM 13505, reversed all renal and systemic CVF-effects. Continuous infusion of the competitive leukotriene D4-antagonist, ICI 198615, attenuated the late renal CVF-effects (i.e. 30 min after injection of CVF). Depletion of polymorphonuclear cells (PMN) attenuated the CVF-effects similar to BM 13505. Intravenous administration of CVF in the hydronephrotic kidney model resulted in a massive constriction of the interlobar and arcuate artery, with a fall in glomerular blood flow comparable to the reduction of RBF in the normal kidney. Diameters of the afferent arterioles--most sensitive to many vasoconstricting agents--were not significantly altered. Our results suggest that injection of CVF and the liberation of high amounts of the anaphylatoxins, C3a and C5a, induces the release of TXA2, which contributes to the early renal effects and the formation of cysteinyl-leukotrienes which play an important role in the late phase of systemic complement activation. Utilizing the split hydronephrotic kidney model we demonstrated the predominant action of complement activation on the large preglomerular vessels for the first time. PMN are seemingly involved in the liberation of secondary mediators which appear to reduce renal blood flow and glomerular filtration.

Animals↗
PubMed1994

Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein.

We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatically activated by lymphocyte antigen receptor-coupled protein tyrosine kinases or independently by diglycerides. To further evaluate the physiological role of Vav, we assessed its GDP-GTP exchange activity against several Ras-related proteins in vitro and determined whether Vav activation in transfected NIH 3T3 fibroblasts correlates with the activity status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) or phosphorylation with recombinant p56lck displayed GEF activity against Ras but not against recombinant RacI, RacII, Ral, or RhoA proteins. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a approximately 10-fold increase in basal or PMA-stimulated Ras exchange activity, respectively, in total-cell lysates and Vav immunoprecipitates. Elevated GEF activity was paralleled in each case by a significant increase in the proportion of active, GTP-bound Ras. PMA had a minimal effect on the low Ras. GTP level in untransfected control fibroblasts but increased it from 20 to 37% in proto-vav-transfected cells. vav-transfected cells displayed a constitutively elevated Ras. GTP level (35%), which was not increased further by PMA treatment. MAP kinases, known downstream intermediates in Ras-dependent signaling pathways, similarly exhibited increased basal or PMA-stimulated activity in Vav-expressing cells by comparison with normal NIH 3T3 cells. These results demonstrate a physiologic interaction between Vav and its target, Ras, leading to MAP kinase activation.

3T3 Cells↗