Biomedical subjects

J P Breittmayer

Publications and source records attributed to J P Breittmayer.

At least 19 recordsLinked to original sources

PubMed2001

Inhibition of phosphatidylserine synthesis in Jurkat T cells by hydrogen peroxide.

Incubation of Jurkat cells in the presence of H2O2 either directly added to the culture medium or generated with glucose oxidase, menadione or the couple xanthine/xanthine oxidase induced a marked decrease of phosphatidylserine synthesis in the absence of changes in the synthesis of phosphatidylcholine and phosphatidylethanolamine. Concentration dependent response curves indicated that H2O2 induced inhibition of phosphatidylserine synthesis with an IC(50)=5 microM while both induction of tyrosine phosphorylation of proteins and Ca(2+) signals were obtained with an EC(50)=300 microM. The tyrosine kinase and Ca(2+) independent mechanism was confirmed by comparing the H2O2-induced and the CD3-induced inhibition of phosphatidylserine synthesis using several Jurkat clones differing in the expression of cell surface receptors such as CD3/TCR and CD45 and protein tyrosine kinase such as p72syk, ZAP-70 and p56lck. While CD3-induced inhibition of phosphatidylserine synthesis necessitates protein tyrosine phosphorylation and Ca(2+) signals, H2O2 provoked its effect in all the clones studied independently of the presence or absence of the proteins previously shown to be key elements in T cell signal transduction. Conversely, the antioxidant molecule, butylated hydroxanisole, generates an increased PtdSer synthesis, suggesting that the synthesis of this phospholipid is regulated by the redox status of the cells.

Antioxidants↗

PubMed2001

Persistent viral load in patients on HAART is associated with a higher level of activation-induced apoptosis of CD4(+) T cells.

Apoptosis is one of the mechanisms involved in the persistent CD4(+) T cell lymphopenia occurring in human immunodeficiency virus (HIV)-infected patients treated with highly active antiretroviral therapy (HAART). The aim of this study was to look at the relationship between the level of T cell apoptosis in patients on HAART and their HIV viral load measurement. Forty-five patients receiving HAART for a median time of 5 months were included: 13 had an undetectable viral load and 32 had a viral load > or = 200 copies/mL. No relationship was observed between the level of spontaneous T cell apoptosis and the viral load measurement. Patients with a viral load > or = 200 copies/mL exhibited a significantly higher level of CD4-induced apoptosis of CD4(+) T cells, compared with patients with undetectable viraemia (38 versus 15%, respectively, P = 0.03). Activation-induced apoptosis may be involved in the paucity of the immune reconstitution observed in HIV-infected patients treated with HAART.

Adult↗

PubMed2000

Priming by microbial antigens from the intestinal flora determines the ability of CD4+ T cells to rapidly secrete IL-4 in BALB/c mice infected with Leishmania major.

Infection of BALB/c mice with Leishmania major results in the rapid accumulation of IL-4 transcripts within CD4(+) T cells that react to the parasite Leishmania homologue of mammalian RACK1 (LACK) Ag. Because memory/effector cells secrete IL-4 more rapidly than naive cells, we sought to analyze the phenotype of these lymphocytes before infection. Indeed, a fraction of LACK-specific CD4(+) T cells expressed a typical CD62 ligand(low)CD44(high)CD45RB(low) phenotype in uninfected mice. LACK-specific T cells were primed in gut-associated lymphoid tissues by cross-reactive microbial Ags as demonstrated by their reactivity with bacterial extracts and by the ability of APCs from the mesenteric LN of BALB/c mice to induce their proliferation. Also, mice in which the digestive tract has been decontaminated exhibited a reduced proportion of LACK-specific T cells expressing a memory/effector phenotype and did not exhibit the early accumulation of IL-4 transcripts induced by L. major. Thus, LACK-specific T cells represent a subset of CD4(+) T cells which have acquired the ability to rapidly secrete IL-4 as the result of their priming by cross-reactive microbial Ags. Tracking the fate of these cells may provide information about the regulation of cell-mediated immune responses to gut Ags in physiological and pathological situations.

ATP-Binding Cassette Transporters↗

PubMed2000

Regulation of phosphatidylserine exposure at the cell surface by the serine--base exchange enzyme system during CD95-induced apoptosis.

Early in the apoptotic process, CD95 induces a translocation of phosphatidylserine (PtdSer) from the inner to the outer leaflet of the cellular plasma membrane. In mammalian cells, PtdSer is only synthesized through a calcium-dependent exchange of the polar head group of pre-existing phospholipids, either phosphatidylcholine or phosphatidylethanolamine, by a serine. Using a pharmacological approach, we examined the influence of PtdSer synthesis on CD95-induced PtdSer exposure at the surface of Jurkat cells. We found that CD3/TCR triggering or thapsigargin treatment of Jurkat cells was accompanied both by a decreased PtdSer synthesis and by a strong reduction of CD95-induced PtdSer at the cell surface, as monitored by fluorescence-activated cell sorting (FACS) analysis of annexin V-fluorescein isothiocyanate (FITC)-labeled cells. PtdSer synthesis through the serine-base exchange enzyme system thus appeared as one of the mechanisms implicated in the recently discovered CD3/TCR-induced down-regulation of CD95-induced apoptosis. Conversely, increasing the activity of the serine-base exchange enzyme system with different drugs, either the K+ channel blocker quinine, the cationic amphiphil stearylamine, or three different calmodulin antagonists, chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), resulted in an increased appearance of PtdSer at the surface of CD95-treated cells. Both PtdSer synthesis and CD95-induced annexin V-FITC reactivity were abrogated in ATP-depleted cells. Also, modifying the membrane potential with valinomycin (hyperpolarization) or either gramicidin or KCl (depolarization) demonstrated a strong relationship between PtdSer synthesis and annexin V-FITC reactivity in CD95-treated cells. Together, our results indicate that CD95-induced exposure of PtdSer at the cell surface could be regulated by the activity of the serine-base exchange enzyme system.

Adenosine Triphosphate↗

PubMed2000

Responding to allegations of scientific misconduct: the procedure at the French National Medical and Health Research Institute.

Institutions in France are not yet well prepared to respond to allegations of scientific misconduct. Following a serious allegation in late 1997, INSERM, the primary organization for medical and health-related research in France, began to reflect on this subject, aided by scientists and jurists. The conclusions have resulted in establishing a procedure to be followed in cases of alleged misconduct, and also in reinforcing the application of good laboratory practices within each laboratory. Guidelines for authorship practices and scientific assessment must also be considered. Even though each institution must remain responsible for responding to allegations of scientific misconduct within its doors, INSERM would like to see national, European, and international co-ordination about the methods of such response.

Academies and Institutes↗

PubMed2000

Diadenosine polyphosphates as antagonists of the endogenous P2Y(1) receptor in rat brain capillary endothelial cells of the B7 and B10 clones.

1. Diadenosine polyphosphates (Ap(n)As, n=2 - 7) are considered as stress mediators in the cardiovascular system. They act both via identified P2 purinoceptors and via yet to be characterized receptors. This study analyses the actions of Ap(n)As in clones of rat brain capillary endothelial cells that express P2Y(1) receptors (B10 cells) or both P2Y(1) and P2Y(2) receptors (B7 cells). 2. B10 cells responded to Ap(3)A with rises in intracellular Ca(2+) concentration ([Ca(2+)](i)). This response was prevented by adenosine-3'-phosphate-5'-phosphate, an antagonist of P2Y(1) receptors. It was largely suppressed by a treatment with apyrase VII or with creatine phosphokinase/creatine phosphate to degrade contaminating ADP. 3. Ap(n)As inhibited ADP induced increases in [Ca(2+)](i) mediated by P2Y(1) receptors by shifting ADP concentration-response curves to larger concentrations. Apparent K(i) values were estimated to be 6 microM for Ap(4)A, 10 microM for Ap(5)A and 47 microM for Ap(6)A. Ap(2)A and Ap(3)A were much less active. 4. Ap(n)As were neither agonists nor antagonists of the endogenous P2Y(2) receptor in B7 cells. 5. Ap(n)As are neither agonists nor antagonists of the G(i)-coupled, ADP receptor in B10 cells. 6. The results suggest that most actions of Ap(n)As in B7 and B10 cells can be accounted for by endogenous P2Y(1) receptors. Ap(4)A, Ap(5)A and Ap(6)A are specific antagonists of endogenous Ca(2+)-coupled P2Y(1) receptors.

Adenosine Diphosphate↗

PubMed2000

Inhibition of phosphatidylserine synthesis during Jurkat T cell activation. The phosphatase inhibitor, sodium ortho-vanadate bypasses the CD3/T cell receptor-induced second messenger signaling pathway.

Sodium ortho-vanadate (Na3VO4), an inhibitor of protein tyrosine phosphatase, induces a rapid (15 min) and strong inhibition of phosphatidylserine synthesis with an IC50 = 100 microM. The mode of action of Na3VO4 was compared to that of CD3 mAbs. It was found that Na3VO4 bypasses the major CD3-induced T cell activation signals including protein tyrosine phosphorylation, p56lck activation and the generation of second messengers including inositol phosphates and its subsequent Ca2+ mobilization as well as diacylglycerol production. These facts were confirmed by using a panel of Jurkat clones that differs by the expression of either tyrosine kinases involved in the CD3-induced T cell activation pathway such as p56lck, p72syk and ZAP-70 or some cell surface receptors such as the CD3/TCR complex or the CD45 phosphatase.

Antibodies, Monoclonal↗

PubMed2000

The p42/p44 MAP kinase pathway prevents apoptosis induced by anchorage and serum removal.

Anchorage removal like growth factor removal induces apoptosis. In the present study we have characterized signaling pathways that can prevent this cell death using a highly growth factor- and anchorage-dependent line of lung fibroblasts (CCL39). After anchorage removal from exponentially growing cells, annexin V-FITC labeling can be detected after 8 h. Apoptosis was confirmed by analysis of sub-G1 DNA content and Western blotting of the caspase substrate poly (ADP-ribose) polymerase. Growth factor withdrawal accelerates and potentiates suspension-induced cell death. Activation of Raf-1 kinase in suspension cultures of CCL39 or Madin-Darby canine kidney cells stably expressing an estrogen-inducible activated-Raf-1 construct (DeltaRaf-1:ER) suppresses apoptosis induced by growth factor and/or anchorage removal. This protective effect appears to be mediated by the Raf, mitogen- or extracellular signal-regulated kinase kinase (MEK), and mitogen-activated protein kinase module because it is sensitive to pharmacological inhibition of MEK-1 and it can be mimicked by expression of constitutively active MEK-1 in CCL39 cells. Finally, apoptosis induced by disruption of the actin cytoskeleton with the Rho-directed toxin B (Clostridium difficile) is prevented by activation of the DeltaRaf-1:ER chimeric construct. These findings highlight the ability of p42/p44 mitogen-activated protein kinase to generate survival signals that counteract cell death induced by loss of matrix contact, cytoskeletal integrity, and extracellular mitogenic factors.

Animals↗

PubMed2000

Aspirin and sodium salicylate inhibit endothelin ETA receptors by an allosteric type of mechanism.

Aspirin is a commonly used drug with a wide pharmacological spectrum including antiplatelet, anti-inflammatory, and neuroprotective actions. This study shows that aspirin and sodium salicylate, its major blood metabolite, reverse contractile actions of endothelin-1 (ET-1) in isolated rat aorta and human mammary arteries. They also prevent the intracellular Ca(2+) mobilizing action of ET-1 in cultured endothelial cells but not those of neuromedin B or UTP. Inhibition of the actions of ET-1 by salicylates is apparently competitive. Salicylates inhibit (125)I-ET-1 binding to recombinant rat ETA receptors. Salicylic acid promotes dissociation of (125)I-ET-1 ETA receptor complexes both in the absence and the presence of unlabeled ET-1. It has no influence on the rate of association of (125)I-ET-1 to ETA receptors. Salicylates do not promote dissociation of (125)I-ET-1 ETB receptor complexes. Salicylates potentiate relaxing actions of receptor antagonists such as bosentan. It is concluded that salicylates are allosteric inhibitors of ETA receptors. The results also suggest that: 1) irreversible ET-1 binding probably limits actions of receptor antagonists in vivo, and 2) an association of salicylates and ETA receptor antagonists should be used to evaluate the physiopathological role of ET-1 and may be of therapeutic interest in the treatment of ischemic heart disease.

Allosteric Regulation↗

PubMed2000

Allosteric inhibition of endothelin ETA receptors by 3, 5-dibromosalicylic acid.

Derivatives of salicylic acid (SA) and benzoic acid prevent endothelin-1 (ET-1) binding to ETA receptors. This study analyzed actions of 30 derivatives of benzoic acid and salicylic acid on (125)I-ET-1 binding to recombinant rat ETA receptors. The most active compounds were 3,5-dibromosalicylic acid (Br2SA, K(i) = 0.5 mM) and 3,5-diiodosalicylic acid (K(i) = 0.3 mM). They were about 50 times more potent than SA and aspirin. Br2SA inhibited equilibrium (125)I-ET-1 binding in an apparently competitive manner. It accelerated 8-fold the dissociation of (125)I-ET-1 receptor complexes and did not modify the second order rate constant of association of (125)I-ET-1 to its receptors. Br2SA also decreased the affinity of ETA receptors for receptor antagonists BQ-123 and bosentan. Br2SA accelerated dissociation of (125)I-ET-1-solubilized ETA receptor complexes and decreased the apparent molecular size of solubilized receptors. Br2SA and 3,5-diiodosalicylic acid inhibited two cellular actions of ET-1: the mobilization of intracellular Ca(2+) stores in isolated cells and contractions of rat aortic rings. They accelerated the relaxing action of BQ-123 and bosentan in ET-1-treated aortic rings. The results suggest the existence of an allosteric modifier site on ETA receptors that recognizes selected derivatives of SA. SA derivatives might be of therapeutic interest to relieve tight ET-1 binding and to favor actions of receptor antagonists.

Allosteric Regulation↗

PubMed1999

Regulation of phosphatidylserine synthesis in Jurkat T cell clones: caffeine bypasses CD3/TCR-induced protein tyrosine kinases and calcium signals.

Phosphatidylserine synthesis as measured by the incorporation of [(3)H]serine into phosphatidylserine (PtdSer) through the serine-base exchange enzyme system (serine-BEES) is markedly inhibited in Jurkat cells treated with caffeine. The caffeine-induced inhibition was compared to that observed in cells treated with either CD3 mAb or thapsigargin. While CD3- and thapsigargin-induced inhibition was related to the release of Ca(2+) from the endoplasmic reticulum (ER), a process that deprives the serine-BEES of its major cofactor, caffeine modified PtdSer synthesis in the absence of decreased Ca(2+) content of ER. Using Jurkat clones differing by the expression of cell surface markers or protein tyrosine kinases implicated in the CD3/TCR signal transmission pathway, we have shown that CD3 mAb-induced inhibition of PtdSer synthesis necessitates the expression of both the CD3/TCR and the protein tyrosine phosphatase CD45 at the cell surface as well as the presence of p56(lck) and ZAP-70 protein tyrosine kinases. By contrast, thapsigargin, a blocker of the Ca(2+)-ATPase of the ER, known for its Ca(2+) releasing properties, inhibited PtdSer synthesis in all the Jurkat clones tested, indicating that this compound bypasses the CD3/TCR-induced signals. Despite its lack of effect on Ca(2+) release from ER and on protein tyrosine phosphorylations, caffeine inhibited PtdSer synthesis in all the Jurkat clones. The use of several cAMP-inducing drugs and of others xanthine derivatives indicated that caffeine modify PtdSer synthesis either by a direct action on the serine-BEES or by a modification of the structure of the phospholipids used as substrate by the enzyme.

Antibodies, Monoclonal↗

PubMed1999

Cleavage and relocation of the tyrosine kinase P59FYN during Fas-mediated apoptosis in T lymphocytes.

Ligation of Fas with its natural ligand or with anti-Fas antibodies induces an apoptotic program in Fas sensitive cells. We report here the identification of the tyrosine kinase p59Fyn as a substrate for CPP32-like proteinases and more particularly caspase 3 during Fas-mediated apoptosis in Jurkat T cells. Inhibition of CPP32-like proteinases by Ac-Asp-Glu-Val-Asp-aldehyde but not by Ac-Tyr-Val-Ala-Asp-aldehyde prevents CPP32, PARP and p59Fyn cleavage indicating that CPP32 or CPP32-like proteinases are responsible for the cleavage of p59Fyn. Cleavage occurs in the N-terminal domain of p59Fyn between Asp19 and Gly20 and is accompanied by relocation of an active p57Fyn kinase to cytoplasm of Fas-stimulated Jurkat cells as judged by both biochemical and confocal microscopy experiments. Thus, p59Fyn relocation and activity may play an important role during Fas-mediated cell death in human T lymphocytes.

Apoptosis↗

PubMed1999

Benzoyl ATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation.

The effects of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) on intracellular Ca2+ mobilization and cyclic AMP accumulation were investigated using rat brain capillary endothelial cells which express an endogenous P2Y1 receptor, human platelets which are known to express a P2Y1 receptor, and Jurkat cells stably transfected with the human P2Y1 receptor. In endothelial cells, BzATP was a competitive inhibitor of 2-methylthio ADP (2-MeSADP) and ADP induced [Ca2+]i responses (Ki = 4.7 microM) and reversed the inhibition by ADP of adenylyl cyclase (Ki = 13 microM). In human platelets, BzATP inhibited ADP-induced aggregation (Ki = 5 microM), mobilization of intracellular Ca2+ stores (Ki = 6.3 microM), and inhibition of adenylyl cyclase. In P2Y1-Jurkat cells, BzATP inhibited ADP and 2-MeSADP-induced [Ca2+]i responses (Ki = 2.5 microM). It was concluded that BzATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation. In contrast to other P2Y1 receptor antagonists (A2P5P and A3P5P) which inhibit only ADP-induced Ca2+ mobilization, BzATP inhibits both the Ca2+- and the cAMP-dependent intracellular signaling pathways of ADP. These results provide further evidence that P2Y1 receptors contribute to platelet ADP responses.

Adenosine Diphosphate↗

PubMed1999

Highly active anti-retroviral therapy (HAART) is associated with a lower level of CD4+ T cell apoptosis in HIV-infected patients.

HAART may increase CD4+ T cell counts despite a persistently detectable HIV load. The impact of HAART on apoptosis, which may play a role in the disease process in HIV-infected patients, has not been extensively studied. We performed a study to compare the level of spontaneous T cell apoptosis and anti-retroviral treatments in a cohort of HIV-1-infected patients. Data were obtained from a computerized medical record. Quantification of apoptotic cells was by cytofluorometric technique. From November 1995 to December 1997 we studied T cell apoptosis in 112 HIV-infected patients. Forty patients were classified A, 36 B and 36 C. Thirty patients were naive and 82 received an anti-retroviral treatment, 49 including a protease inhibitor (PI). The median plasma viraemia determined in 63 patients was 3.6 (range 1.3-5.6) log10. The median apoptotic cell count was 22% (range 2-73%) and 12% (range 2-60%) for CD4+ and CD8+ T cells, respectively. We did not observe any correlation between the HIV viraemia and the level of apoptosis of T cell subsets. Patients with HAART showed a lower percentage of apoptotic CD4+ T cells only: 16% (range 2-61%) versus 25% (range 5-73%) for patients receiving two nucleoside analogues (P = 0.02). This effect was significant in stage A patients and remained observable during the whole course of HIV disease. In conclusion, HAART, without any relation to plasma viraemia, is able to reduce apoptosis of CD4+ T cells.

Adult↗

PubMed1999

Overexpression of Fas/CD95 and Fas-induced apoptosis in a patient with idiopathic CD4+ T lymphocytopenia.

The mechanisms of apoptosis have become better understood, in part with the discovery of Fas/CD95. We report the case of a patient characterized by a decreased CD4+ T cell count and an overexpression of Fas/CD95 resulting in apoptosis. A 54-year-old man presented with disseminated Mycobacterium xenopi infection. Analysis showed CD4+ T lymphopenia. Tests for human immunodeficiency virus (HIV) types 1 and 2 were negative. We compared the patient with eight healthy controls and five HIV-infected patients in terms of the expression of Fas/CD95 and Fas-mediated apoptosis of peripheral T lymphocytes. The percent of CD95+ cells in lymphocytes was 98% for the patient, and the mean percent of CD95+ cells in lymphocytes +/- SD for HIV-infected patients and healthy controls was 75% +/- 16% and 36% +/- 26%, respectively. The patient had a high level of spontaneous apoptosis, and apoptotic cells were all identified as being CD4+ T cells. Monoclonal antibodies to CD95 dramatically increased apoptosis of CD4+ T cells exclusively. CD4+ T lymphopenia observed in our patient correlated with an overexpression of Fas together with spontaneous and Fas-induced apoptosis.

Apoptosis↗

PubMed1998

CD95 (Fas/APO-1) induces an increased phosphatidylserine synthesis that precedes its externalization during programmed cell death.

CD95 (Fas, APO-1)-induced programmed cell death (apoptosis) in T cell lines is accompanied by a rapid flip-flop of phosphatidylserine (PtdSer). Externalization of this phospholipid has been previously recognized as one of the early detectable events of cells undergoing apoptosis. We show here that CD95 induces a rapid (detectable at time < 15 min), strong (2.5-fold) but transitory neosynthesis of PtdSer in the Jurkat cell line that precedes its externalization. PtdSer decarboxylation, a mitochondrial specific process, was strongly inhibited by CD95 suggesting that changes in mitochondrial activity take place in the early events of Fas-induced apoptosis and participate in the increased PtdSer synthesis observed. In cells undergoing apoptosis, newly synthesized PtdSer first exposed at the cell surface was in part shed with CD95-induced plasma membrane vesicles, a process that likely explains the transitory effect observed.

Antibodies, Monoclonal↗

PubMed1998

PPADS inhibits P2Y1 purinoceptors in rat brain capillary endothelial cells and in rat ileal myocytes by an indirect mechanism.

P2Y1 receptor-like responses were analyzed in rat ileal myocytes and in rat brain capillary endothelial cells. In endothelial cells, pyridoxal phosphate-6-azophenyl-2',4'disulfonic acid (PPADS) inhibits ADP induced intracellular Ca2+ transients with a half maximum effect at 3 microM. PPADS shifts ADP dose response curves to larger concentrations. Yet PPADS is inactive when added at the same time as ADP. A preequilibration of the cells with PPADS is necessary to observe its inhibitory action. Similarly in ileal myocytes, PPADS has no action on ADP responses when it is applied at the same time as ADP. Actions of PPADS require a preequilibration with the cells and are fully reversible. These results suggest that PPADS is not a competitive antagonist of P2Y1 receptors and caution about its usefulness to distinguish subtypes of P2Y1 receptors.

Adenosine Diphosphate↗

PubMed1998

Regulation of the serine-base exchange enzyme system by CD4: effects of monoclonal antibodies, jacalin, interleukin 16 and the HIV membrane protein gp120.

Phosphatidylserine (PtdSer) is synthesized by an exchange of the polar head group of phospholipids for a serine residue. The enzyme responsible for this reaction, the serine-base exchange enzyme system (serine-BEES) is inhibited during lymphocyte activation. We show here that triggering the CD4 cell surface molecule in several CD4+ T-cell lines regulates the serine-BEES activity, thus resulting in marked changes in PtdSer synthesis. CD4 ligands able to generate an activating signal in T-cells such as the lectin jacalin, down-regulate the synthesis of PtdSer. In contrast, monoclonal antibodies (mAbs) directed against the CD4 molecule, such as IOT4 and IOT4a, which have previously been described as generating an inhibitory signal to T-cells, induced an up-regulation of the serine-BEES and impaired CD3-induced inhibition of PtdSer synthesis. Similarly, the HIV-gp120 envelope glycoprotein, in both soluble and cross-linked forms, induces an increase in PtdSer synthesis. The protein tyrosine kinase p56lck participates in the regulation of serine-BEES activity because the effect of CD4 mAbs was additive to that of amino-hydroxyflavone, an inhibitor of p56lck. Also, CD4 mAbs were inactive in J Cam 1.6 cells or when the CD3 signals were bypassed by using thapsigargin. These results demonstrate that the CD4 surface molecule can transmit both activating and inhibiting intracellular signals depending on the CD4 ligand used. We suggest that PtdSer synthesis would be one of the intracellular signals that could explain the opposite effects of different CD4 ligands on T-cells.

Antibodies, Monoclonal↗