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Biomedical subjects

H Chap

Publications and source records attributed to H Chap.

At least 73 records · Page 4Linked to original sources
PubMed1997

Protein tyrosine phosphatase SHP-1 fails to associate with cytoskeleton but is normally phosphorylated upon thrombin stimulation of thrombasthenic platelets.

SHP-1 is a cytoplasmic protein tyrosine phosphatase predominantly expressed in hematopoietic cells. Upon thrombin stimulation of human platelets, SHP-1 is rapidly phosphorylated on both serine and tyrosine residues, and becomes associated with the cytoskeleton, where it could participate in the formation of multiprotein signalling complexes. In order to discriminate between signalling events occurring downstream of G-protein-coupled thrombin receptor and those subsequent to integrin alpha IIb beta 3 engagement, SHP-1 behaviour was examined in platelets from two patients lacking integrin alpha IIb beta 3 (Glanzmann's thrombasthenia). Upon thrombin stimulation, phosphorylation of SHP-1 occurred normally in thrombasthenic platelets, whereas association with the cytoskeleton was abolished. Moreover, inhibition of normal platelet aggregation with the tetrapeptide arg-gly-asp-ser (RGDS) which impairs fibrinogen binding to integrin alpha IIb beta 3, did not alter significantly SHP-1 phosphorylation. It is concluded that SHP-1 phosphorylation is not a consequence of integrin signalling but might rather occur downstream of thrombin receptor and heterotrimeric G-proteins.

Blood Platelets↗
PubMed1996

A role for phosphoinositide 3-kinase in bacterial invasion.

Listeria monocytogenes is a bacterial pathogen that invades cultured nonphagocytic cells. Inhibitors and a dominant negative mutation were used to demonstrate that efficient entry requires the phosphoinositide (PI) 3-kinase p85alpha-p110. Infection with L. monocytogenes caused rapid increases in cellular amounts of PI(3, 4)P2 and PI(3,4,5)P3, indicating that invading bacteria stimulated PI 3-kinase activity. This stimulation required the bacterial protein InlB, host cell tyrosine phosphorylation, and association of p85alpha with one or more tyrosine-phosphorylated proteins. This role for PI 3-kinase in bacterial entry may have parallels in some endocytic events.

Androstadienes↗
PubMed1996

High-density lipoprotein 3 receptor-dependent endocytosis pathway in a human hepatoma cell line (HepG2).

The internalization of HLD3 into HepG2 cells at 37 degrees C was precisely measured, taking advantage of the previously observed rapid dissociation of HDL3 from its two binding sites [Barbaras, R., et al. (1994) Biochemistry 33, 2335-2340]. We observed a high level of HDL3 internalization (100 ng/mg of cell protein, corresponding to 45.5% of the total HDL3 associated to the cells at 37 degrees C) reaching a plateau at 15 min. Apolipoprotein A-I (the main HDL3 apolipoprotein) associated with dimyristoylphosphatidylcholine (DMPC) complexes was also internalized by HepG2 cells, at levels comparable to those obtained with HDL3 lipid-free apolipoprotein A-I, which can bind only to the HDL3 high-affinity binding site, and displayed a weak internalization (5 ng internalized/mg of cell protein compared to 250 ng/mg for apolipoprotein A-I complexed with DMPC). Clathrin-coated vesicle purification following HDL3 or LDL internalization at 37 degrees C showed radioactivity associated with these vesicles, and further content analysis evidenced the presence of radiolabeled apoA-I and apoB, respectively. Treatment of the cells either by saccharose hypertonic shock or by potassium depletion, in order to block clathrin-coated vesicle formation, completely inhibited HDL3 internalization, as also observed with LDL. Altogether, these observations clearly demonstrate that HDL3 internalization into HepG2 cells occurs through an endocytosis pathway involving an interaction between apolipoprotein A-I and a cell surface protein, leading to the formation of clathrin-coated vesicles.

Apolipoprotein A-I↗
PubMed1996

Guinea pig intestinal phospholipase B: protein expression during enterocyte maturation and effects of N-oligosaccharide removal on enzymatic activities and protein stability.

Guinea pig phospholipase B (PLB) is an intestinal brush-border hydrolase displaying a broad substrate specificity towards various dietary lipids. PLB was detected by immunoblotting as a single 140-kDa polypeptide in all cell populations isolated from guinea pig intestinal mucosa, but increased in parallel to its activity from undifferentiated to mature cells, the specific activity of the enzyme remaining constant. Moreover, N-glycosylation, which contributed to 23% of the apparent molecular mass, was identical along the cell differentiation axis. In all cell fractions, N-linked sugar chains were of the complex type, since they were removed by N-glycosidase F, whereas PLB remained insensitive to endoglycosidase H. Moreover, lack of O-glycosylation was demonstrated by the insensitivity of PLB to O-glycosidase and by its failure to interact with Helix pomatia lectin after prior treatment with neuraminidase or alpha-fucosidase. Enzymatic removal of sugar chains reduced phospholipase A2, lysophospholipase and diacylglycerol lipase activities by 27-35%, kinetic analysis indicating a decrease in apparent Vmax values for the three enzymatic activities, whereas the Km remained unchanged. Finally, the carbohydrate-depleted form of PLB did not display gross changes in thermal stability, in contrast to PLB from microorganisms previously investigated. Our data indicate that the high level of PLB N-glycosylation is poorly related to its biological function. Whether carbohydrate chains are involved in proper targeting of the enzyme to the brush-border membrane remains to be established.

Animals↗
PubMed1996

G-protein beta gamma subunits mediate specific phosphorylation of the protein-tyrosine phosphatase SH-PTP1 induced by lysophosphatidic acid.

SH-PTP1 is a protein-tyrosine phosphatase preferentially expressed in hematopoietic cells and bearing two SH2 (src homology-2) domains. In the human megakaryocytic cell line Dami, lysophosphatidic acid (LPA) promoted a rapid increase in SH-PTP1 phosphorylation on both serine and tyrosine residues. Only tyrosine phosphorylation was significantly inhibited by pertussis toxin and by the protein kinase C inhibitor GF109203X. Moreover, SH-PTP1 was phosphorylated upon challenge with other agonists acting via G-protein-coupled receptors such as alpha-thrombin, epinephrine, and ADP, whereas the closely related protein-tyrosine phosphatase SH-PTP2 failed to share such a regulation in Dami cells. We developed an in vitro assay that reproduced LPA-dependent phosphorylation of SH-PTP1 in a cell-free system. The fusion protein glutathione S-transferase-beta-adrenergic receptor kinase 1-(495-689) or the transducin subunit Galphat-GDP, which act as specific antagonists of Gbetagamma, inhibited SH-PTP1 phosphorylation. Moreover, purified transducin Gbetagamma subunits mimicked the effect of LPA. Finally, stable expression of beta-adrenergic receptor kinase 1-(495-689) in Dami cells resulted in the inhibition of SH-PTP1 as a specific target of protein kinases linked to G-protein-coupled receptors via Gbetagamma subunits.

Cells, Cultured↗
PubMed1996

Opposite effects of tumor necrosis factor alpha on the sphingomyelin-ceramide pathway in two myeloid leukemia cell lines: role of transverse sphingomyelin distribution in the plasma membrane.

Tumor necrosis factor alpha (TNF alpha) mediates proliferation, functional activation, and apoptotic cell death depending on the target cell type. Although sphingomyelin (SPM) hydrolysis and ceramide generation may function as an important mediator in TNF alpha signaling, the molecular mechanisms of the signaling pathway(s) are still not well understood. The aim of the present study is to compare the effect of TNF alpha on SPM metabolism and cell growth in two myeloid leukemic cell lines (U937 and KG1a) that differ in their sensitivity to TNF alpha. Our results show that TNF alpha induced apoptosis in U937 but not in KG1a cells. TNF alpha triggered in KG1a cells neither SPM hydrolysis nor ceramide generation, but induced SPM synthesis and ceramide breakdown as well as dose-dependent cell proliferation. In contrast, TNF alpha induced in U937 SPM hydrolysis and ceramide generation as well as dose-dependent cell death. Synthetic cell permeant ceramide, as well as natural ceramide, generated by treatment with bacterial sphingomyelinase (SPMase), all induced apoptosis in both U937 and KG1a cells. These findings indicate that the SPM-ceramide pathway is altered in KG1a cells upstream of the ceramide generation. Analysis of the transverse distribution of SPM in the plasma membrane showed that the SPM pool involved in cell signaling (inner leaflet) was markedly reduced in KG1a cells; it is 7-fold lower than that found in the inner leaflet of U937 cells. Therefore, our study strongly suggests that the different responses induced by TNF alpha in myeloid cells are dependent on the SPM plasma membrane transverse asymmetry.

Animals↗
PubMed1996

alphaIIb beta 3-integrin mediated adhesion of human platelets to a fibrinogen matrix triggers phospholipase C activation and phosphatidylinositol 3',4'-biphosphate accumulation.

This study focused on the variations in phosphoinositide metabolism depending upon alphaIIbbeta3-integrin/fibrinogen interaction without previous activation of platelet agonist receptors. We found that adhesion of resting human platelets to immobilized fibrinogen stimulates phosphatidic acid production and a concomitant decrease in phosphatidylinositol 4',5'-bisphosphate. These results, and the absence of a transphosphatidylation reaction, argue in favor of the activation of a phospholipase C. Moreover, we observed the accumulation of phosphatidylinositol 3',4'-bisphosphate in adherent platelets as a consequence of the activation of a phosphatidylinositol 3-kinase. This effect was inhibited by ADP scavengers. Our results demonstrate that in adherent platelets, whereas phosphatidylinositol 3-kinase activation is controlled by both alphaIIbbeta-integrin engagement and released ADP, phospholipase C stimulation is triggered only by alphaIIbbeta-integrin/fibrinogen interaction.

Adenosine Diphosphate↗
PubMed1996

Expressed sequence tags identify human isologs of the ARF-dependent phospholipase D.

By searching into Expressed Sequence Tags databases (dbEST) using Blast X algorithm software and a plant phospholipase D as template, we have identified a cDNA from human brain (Z45777) which encodes for a protein similar to the amino acid region 743-929 of the human phospholipase D1 (PLD1), and a cDNA from human liver (R93485) which encodes for a protein similar to region 815-932 of PLD1. Sequence comparison between cloned phospholipases showed the presence of 3 conserved amino acid sequences: AFVGGIDLAYGRWD (box A), IIGSANINDRS (box B), and YIYIENQFFI (box C). Phylogenic analysis indicated that the cDNA from brain and liver encoded for human isologs of PLD1.

ADP-Ribosylation Factors↗
PubMed1996

A genetic defect in phosphatidylcholine biosynthesis triggers apoptosis in Chinese hamster ovary cells.

We have investigated the cell death of a Chinese hamster ovary mutant (MT-58) with a thermo-sensitive CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of the CDP-choline pathway for phosphatidylcholine biosynthesis (Esko, J. D., Wermuth, M. M., and Raetz, C. R. H. (1981) J. Biol. Chem. 256, 7388-7393). After MT-58 cells were shifted to the restrictive temperature of 40 degrees C, the cytidylyltransferase was inactivated immediately leading to a decrease in phosphatidylcholine biosynthesis and cell death. DNA content and number of cells in the S phase decreased significantly in the dying MT-58 cells according to flow cytometrical analyses. The fragmentation of genomic DNA was detected by DNA ladders in agarose gel and release of the prelabeled genomic DNA into cytosolic fractions 14 h after the temperature shift. The dying cells underwent a dramatic reduction of cellular volume while maintaining the membrane containment of cellular contents. These events indicated that the inactivation of cytidylyltransferase triggered apoptosis in Chinese hamster ovary cells. This is the first report that apoptosis was induced in cultured cells, not by an added agent, but by a mutation in phospholipid biosynthesis.

Animals↗
PubMed1996

Differential labelings suggest two specific phospholipid subclass hydrolysis promoted by PDGF-BB in vascular smooth muscle cells.

We have used differential phospholipid subclass labelings performed with [3H]lyso PAF and [3H] myristic acid into vascular smooth muscle cells (vSMC) to characterize the subclasses of phospholipid substrates upon different stimulation times with platelet-derived growth factor (PDGF-BB). In cells labeled with [3H]lyso PAF, PDGF-BB induced a sustained hydrolysis of alkyl-PE. In contrast, in [3H]myristic acid-labeled cells, PDGF-BB promoted a rapid and transitory hydrolysis of diacyl-PC. This hydrolysis was concomitant with an synthesis of diglyceride (DG) and phosphatidic acid (PA). Thus, both diacyl-PC and alkyl-PE appear to be major targets in PDGF-BB stimulation of SMC. These results suggest that agonists could induce the hydrolysis of precise phospholipid subclasses leading to a new specificity into the signal transduction cascade.

Animals↗
PubMed1996

Biochemical characterization of pre-beta 1 high-density lipoprotein from human ovarian follicular fluid: evidence for the presence of a lipid core.

In order to isolate pre-beta 1 HDL, we have focused our interest on a particular model, namely, human preovulatory follicular fluid, which contains only HDL as a lipoprotein class as well as a high proportion of pre-beta 1 HDL relative to total HDL (1.5 times more than in homologous plasma) as evidenced by double-dimension gel electrophoresis. Apo A-I in pre-beta 1 HDL represented 17.6% of total apo A-I. Stokes' radii corresponded to 3.42 nm in follicular fluid pre-beta 1 HDL and 3.48 nm in homologous plasma counterparts. After electroelution from agarose, pre-beta 1 HDL were isolated in amounts sufficient to allow characterization by size-exclusion chromatography using HPLC. The estimated apparent molecular mass of these particles is 61.6 kDa. Lipid composition of pre-beta 1 HDL evidenced a low lipid content compared to follicular fluid HDL isolated by ultracentrifugation. Phospholipid composition showed a dramatic decrease in phosphatidylcholines (40.5% of total phospholipids), and the presence of lysophosphatidylcholines and of acidic phospholipids such as phosphatidylserine and phosphatidylinositol (13.6 and 13.7%, respectively). Furthermore, cholesteryl ester and triacylglycerol molecules were quantified by gas-liquid chromatography and represented 8-9% of the pre-beta 1 HDL total weight. Thus, a lipid core is present in pre-beta 1 HDL, which would be compatible with a spherical shape. The follicular fluid appears to be a good model to a better understanding of HDL metabolism.

Apolipoprotein A-I↗
PubMed1996

Involvement of vicinal dithiols in differential regulation of fMLP and phorbol ester-activated phospholipase D in stimulated human neutrophils.

In the human neutrophil, the fMLP-activated phospholipase D (PLD) was entirely calcium and tyrosine kinase dependent, but protein kinase C independent. An opposite regulation was observed with phorbol ester PMA, since the phospholipase D activity was mostly calcium insensitive, tyrosine kinase independent, but protein kinase C dependent. The arsenical compound, phenylarsine oxide (PAO), which reacts with vicinal sulhydryl groups, activated twofold at one minute the PMA stimulated-PLD activity, whereas it inhibited half of the fMLP-activated PLD after a time lag of 30 sec. Our data indicate that PAO acted on a mechanism regulating the balance between fMLP-activated and PMA-activated phospholipase D activity. Noteworthy, PAO similarly inhibited fMLP and PMA-induced O2(-)-production.

Arsenicals↗
PubMed1996

Transacylase-mediated alkylacyl-GPC synthesis and its hydrolysis by phospholipase D occur in separate cell compartments in the human neutrophil.

Subcellular localizations of CoA-independent transacylase and phospholipase D enzymes have been investigated in human neutrophils performing a two-step gradient system to separate plasma membranes from internal membranes and from the bulk of granules. The internal membranes were constituted by endoplasmic reticulum and by a subpopulation of specific and tertiary granules. The enzymes activities were assayed in vitro on gradient fractions using exogenous substrates. Following cell prelabelling with [3H]alkyllyso-GPC, we also analyzed the in situ localization of labelled products involving the action of both enzymes. The CoA-independent transacylase activity, together with the CoA-dependent transacylase and acyltransferase activities were only located in the internal membranes. Following 15 min cell labelling, part of the [3H]alkylacyl-GPC was recovered in plasma membranes indicating a rapid redistribution of the acylated compound. Very high contents in arachidonate containing [3H]alkylacyl-GPC were recovered both in plasma membranes and internal membranes. Phospholipase D activity being assayed in the presence of cytosol, GTP gamma S and gradient fractions, only the plasma membrane fractions from resting or stimulated cells allowed the enzyme to be active. The [3H]alkylacyl-GP and [3H]alkylacyl-GPethanol, phospholipase D breakdown products from [3H]alkylacyl-GPC, obtained after neutrophil prelabelling and activation by phorbol myristate acetate, were exclusively present in the plasma membranes. In contrast, the secondary generated [3H]alkylacylglycerols were equally distributed between plasma and internal membranes. No labelled product was recovered on azurophil granules. These data demonstrate that internal membranes are the site of action of the CoA-independent transacylase and plasma membranes are the site of action of the phospholipase D. This topographical separation between CoA-independent transacylase which generated substrate and phospholipase D which degraded it, suggested that subcellular localisation and traffic of substrates within the cell can be important to regulate the enzymes.

Amidohydrolases↗
PubMed1996

Distinct signal transduction pathways for activation of rabbit alveolar macrophages in vitro by cotton bract tannin.

These experiments were designed to study signal transduction pathways in alveolar macrophages stimulated by condensed tannin or zymosan. Condensed tannins, present in cotton mill dust, alter the host-defense function of alveolar macrophages and may contribute to the pathogenesis of byssinosis. We tried to determine the early steps in signal transduction mechanisms of cell activation by tannin. With the quantification of 51Cr release, we determined that tannin was cytotoxic for the cells after 30 min activation with 130 micrograms for 2 x 10(6) cells. 51Cr release was similar for control cells and zymosan- or 30 micrograms tannin-activated cells. Using the luciferine luciferase reaction, we showed that tannin markedly depleted ATP cell content. In inositol-labeled cells, tannin increased inositolphosphate release in a dose-dependent manner. In lysoPAF-labeled cells, tannin induced synthesis of phosphatidic acid and diglycerides. In the presence of ethanol, the level of tannin-induced phosphatidic acid was slightly reduced, and phosphatidylethanol was synthesized. No phosphatidylethanol was found in alveolar macrophages stimulated by zymosan in the presence of ethanol. GF 109203X, a specific inhibitor of protein kinase C decreased only tannin-induced phosphatidylethanol synthesis. In conclusion, tannin (at 30 or 130 micrograms/ml) activated an inositol phospholipase C in alveolar membranes. Phosphatidylcholine phospholipases C and D were found only at the higher concentration of tannin.

Adenosine Triphosphate↗
PubMed1996

Increased content of annexin II (p36) and p11 in human placenta brush-border membrane vesicles during syncytiotrophoblast maturation and differentiation.

Annexins are a group of proteins abundant in placental membranes where they may play diverse functional roles. Annexins are expressed in high levels in mature placenta but little is known about their presence at very early stages of gestation and later. We used the model of brush-border membrane vesicles (BBMV) at different stages of gestation to assess precise localization of some of these proteins in syncytiotrophoblast apical membrane and to determine their appearance along the maturation process of placenta. Here we describe annexins type I, II, IV, V and VI which are present all along gestation in BBMV. Annexin II (p36) is present with the S100 like calcium-binding protein p11 in BBMV, where they can constitute heterotetrameric forms of annexin II linked to cytoskeleton structures. No variation of annexins I, IV and VI content was observed in BBMV along pregnancy. Annexin V undergoes significant decrease after 12th week, which could be related to local anticoagulant activity. Levels of annexin II and p11 increased progressively during gestation suggesting that heterotetrameric forms of annexin II play a role in the differentiation process of placenta and in function of the mature microvilli.

Annexin A2↗
PubMed1996

Identification and quantification of diacylglycerols in HDL and accessibility to lipase.

We have investigated the presence of diacylglycerols in lipoproteins and especially in HDL. Lipoprotein diacylglycerols are very difficult to isolate and to quantify using classical enzymatic techniques, as they are measured in the presence of triacylglycerols and monoacylglycerols. Using a rapid and very sensitive method of gas-liquid chromatography, developed for neutral lipid analysis on an Ultra 1 Hewlett-Packard fused silica capillary column, diacylglycerols (DG) were identified in HDL and classified into five groups: DG 14-16, DG 16-16, DG 16-18, DG 18-18, and DG 18-20. However, their quantitation was difficult due to only partial resolution of molecular species. HDL lipids were submitted to preparative gas-liquid chromatography and diacylglycerols were then silylated using trimethylsilyl reagents. The trimethylsilyl ethers were analyzed by gas-liquid chromatography on a Restek 50 capillary column and were resolved on the basis of carbon number, degree of unsaturation, and double bond positions. The amount of HDL diacylglycerols was twice that of triacylglycerols. The major molecular species of diacylglycerols consisted of 16:0-18:2n-6, 18:0-18:2n-6, and 16:0-18:1n-9 as the major molecular species (33.4, 22.2, and 16.1 mol % of total diacylglycerols, respectively). Using guinea pig cationic pancreatic lipase in order to test the accessibility of diacylglycerols at the surface of HDL, we measured 59% of diacylglycerol hydrolysis, whereas no triacylglycerol hydrolysis was obtained. In addition, most of diacylglycerols having long chain fatty acids, such as 18-20, were completely hydrolyzed, whereas 18-18 and 16-18 were only partially hydrolyzed (64 and 46% respectively). This reflects a different partition of diacylglycerol molecular species between the particle's surface and the lipid core in HDL. This is the first analysis of diacylglycerol molecular species and their distribution in native lipoprotein particles.

Chromatography, Gas↗
PubMed1995

Increase in receptor-like protein tyrosine phosphatase activity and expression level on density-dependent growth arrest of endothelial cells.

Protein tyrosine phosphatase (PTPase) activity was examined in two cell lines: human umbilical vein endothelial (HUVE) cells, which display contact inhibition of cell growth, and A427 human adenocarcinoma cells, which have lost this ability. HUVE cells harvested at high density displayed a 10-fold increase in membrane-associated PTPase activity. A427 cells exhibited no such phenomenon. Moreover, modification of HUVE cell growth rate by a stimulating agent such as basic fibroblast growth factor or by blocking compounds such as thymidine or suramin resulted in no change in PTPase activity, suggesting that the observed increase in membrane-associated activity at confluency was specific for cell-cell-contact-directed growth arrest. The expression of various PTPase mRNAs was examined in HUVE and A427 cells. Of the receptor-like PTPases tested, two were exclusively expressed in HUVE cells (PTP gamma and HPTP beta). Only HPTP beta, which is structurally similar in its extracellular region to cell-adhesion receptors of the immunoglobulin superfamily, displayed a pattern of expression related to the increase in PTPase activity. Competitive PCR was used to quantify its expression during cell culture. A 12-fold increase in HPTP beta mRNA expression was detected and it parallelled the time course of PTPase activity. This observation strongly implicates receptor-like PTPases in density-dependent growth arrest.

Adenocarcinoma↗