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M Lecomte

Publications and source records attributed to M Lecomte.

At least 19 recordsLinked to original sources
PubMed1997

Advanced glycation end products induce specific glycoprotein alterations in retinal microvascular cells.

In order to investigate the mechanisms involved in diabetic retinopathy, we studied the effects of advanced glycosylation end products (AGE) on retinal microvascular cell glycoproteins. Bovine retinal pericytes (BRP) and endothelial cells (BREC) were incubated in the presence of AGE-modified albumin and cell glycoproteins analyzed by lectin affinoblotting and metabolic radiolabeling with sugar precursors. Selective modifications in the glycoprotein sugar chains were observed mainly in BREC and for a 210 kDa membrane glycoprotein. Indeed, a 40% decrease of alpha(2,3) sialic acid, beta(1,3) galactose or alpha(1,6) fucose content was observed without significant protein amount changes. These glycoprotein alterations were related to the concentration of AGE. Neither BRP nor BREC glycoproteins were modified when cells were incubated with high glucose or fructose concentrations. These results suggest a new diabetic pathogenic mechanism in which a protein post-translational modification, in this case glycation, could modify another post-translational process such as the enzymatic glycosylation.

Animals↗
PubMed1997

Growth modulation of retinal microvascular cells by early and advanced glycation products.

To investigate the possible implication of non-enzymatic glycosylation in the etiopathogenesis of the diabetic retinopathy, we studied the effect of early and advanced glycation products on the growth of retinal microvascular cells. Glucose modified products were obtained by incubating bovine serum albumin or fetal bovine serum with 0.5 M glucose for 10 (early glycation products: EG-BSA and EG-FBS, respectively) or 60 days (advanced glycation end products: AGE-BSA and AGE-FBS, respectively). Cell growth was assessed by cell counting and DNA content determination. EG-BSA or AGE-BSA significantly decreased pericyte proliferation after 8 days of culture (33 and 13% inhibition, respectively). Concerning endothelial cells, EG-BSA reduced proliferation to 40% whereas AGE-BSA increased it to 156% after 4 days of culture. The glucose-treated sera didn't exhibit the same growth effects, neither the EG-FBS nor the AGE-FBS significantly affected endothelial cell proliferation. Only the AGE-FBS showed a significant inhibitory effect on pericyte proliferation (40% inhibition). We conclude that retinal microvascular cell growth in vitro could be differently modulated by early and advanced glycation products. The inhibitory effect of AGEs observed on pericyte growth, suggests that glycoxidation could be implicated in the pericyte loss observed in diabetic retinopathy.

Animals↗
PubMed1997

Involvement of cell-cell interactions in the pathogenesis of diabetic retinopathy.

Retinopathy is a severely disabling complication of diabetes mellitus whose underlying mechanisms are still obscure. The key question is why retinal microvessels are so reactive to the diabetic environment, whereas other microvessels show no evidence of alteration. The answer could lie in the particular structure and location of retinal microvessels since they are composed of, and surrounded by, various types of cells, thereby favouring cell-cell interactions which occur between cells of the capillary wall itself but also with circulating blood cells and retinal neural cells. In the retinal capillary wall, pericytes are in close relation with underlying endothelial cells, and both cell types have close contacts with the capillary basement membrane. Adhesion molecules and cell surface glycoconjugates appear to be the main mediators of interactions between circulating blood cells and capillary endothelial cells, whereas growth factors seem to play a major role in interactions between glial and capillary wall cells in the retina. Biochemical dysfunctions observed in diabetes, such as glycation of proteins and enhanced oxidative stress, could modify these cell-cell and cell-matrix interactions, thereby disturbing the complex cellular organization in which retinal microvessels are embedded. The aim of this review was to provide an overall, nonexhaustive description of some types of cellular interactions that may underlie the pathogenic mechanisms involved in flow and growth changes leading to diabetic retinopathy.

Animals↗
PubMed1996

Involvement of arginine 120, glutamate 524, and tyrosine 355 in the binding of arachidonate and 2-phenylpropionic acid inhibitors to the cyclooxygenase active site of ovine prostaglandin endoperoxide H synthase-1.

Examination of the crystal structure of the ovine prostaglandin endoperoxide synthase-1 (PGHS-1)/S- flurbiprofen complex (Picot, D., Loll, P.J., and Garavito, R.M. (1994) Nature 367, 243-2491) suggests (a) that the carboxyl group of arachidonic acid interacts with the arginino group of Arg120; (b) that Arg120 forms an important salt bridge with Glu524; and (c) that Tyr355, which is in close proximity to Arg120, could determine the stereochemical specificity of PGHS-1 toward 2-phenylpropionic acid inhibitors. To test these concepts, we used site-directed mutagenesis to prepare ovine PGHS-1 mutants having modifications of Arg120 (R120K, R120Q, R120E), Glu524 (E524D, E524Q, E524K), and Tyr355 (Y355F) and examined the properties of the mutant enzymes expressed in COS-1 cells. All of the mutants retained at least part of their cyclooxygenase and peroxidase activities except the R120E mutant, which had no detectable activity. The Km values of the R120K and R120Q mutants with arachidonic acid were 87 and 3300 microM, respectively, versus 4 microM for native PGHS-1. The R120Q mutant failed to undergo suicide inactivation during catalysis or time-dependent inhibition by flurbiprofen. These results are consistent with Arg120 binding the carboxylate group of arachidonate and suggest that interaction of the carboxylate group of substrates and inhibitors with Arg120 is necessary for suicide inactivation and time-dependent inhibition, respectively. The Km values for the E524D, E524Q, and E524K mutants were not significantly different from values obtained for the native PGHS-1, suggesting that this residue is not importantly involved in catalysis or substrate binding. The effect of modifications of Arg120 and Tyr355 on the stereospecificity of inhibitor binding was determined. Ratios of IC50 values for cyclooxygenase inhibition by D- and L-ibuprofen, a competitive cyclooxygenase inhibitor, were 32, 67, and 7.1 for native PGHS-1, R120Q PGHS-1, and Y355F PGHS-1, respectively. The decreased stereochemical specificity observed with the Y355F PGHS-1 mutant suggests that Tyr355 is a determinant of the stereospecificity of PGHS-1 toward inhibitors of the 2-phenylpropionic acid class.

Amino Acid Sequence↗
PubMed1996

Description of an in vitro angiogenesis model designed to test antiangiogenic molecules.

Angiogenesis is involved in numerous pathologies. Studies with in vitro models allow the description and analysis of the different steps involved in this process under defined culture conditions. We describe a controllable and reproducible in vitro model. We assessed the usefulness of this model with two different cell lines: human umbilical vein endothelial cells and bovine retinal endothelial cells. These cells reorganize themselves and change their phenotypes within 24 h after seeding under our culture conditions (low human serum percentage, defined cell density, fibrin matrix) to form 'capillary-like structures' (CLS) in vitro. We showed that, depending on the cell line used, the fibrinolytic activity of the cells was a determining factor which could induce or prevent the formation of the CLS. Inhibitors of angiogenesis can be tested using such a model.

Angiogenesis Inducing Agents↗
PubMed1996

Docosahexaenoic acid is a major n-3 polyunsaturated fatty acid in bovine retinal microvessels.

The aim of this study was to purify microvessels from bovine retina and also to cultivate bovine retinal endothelial cells (BRECs) or intramural pericytes, to determine their fatty acid composition. Microvessels were obtained after Dounce homogenization of the retina followed by centrifugation on albumin cushion and finally microvessels in the pellet were trapped on a 100-microns nylon filter. Contamination of microvessel preparations by neuronal tissue, assessed after both microscopic examination and western blotting with a monoclonal antibody raised against rhodopsin, was minor. In the entire bovine retina, docosahexaenoic acid (DHA) represented 23.3% of the total fatty acids and there was about three times less arachidonic acid (AA) (8.2%) than DHA. In contrast, DHA and AA levels were almost equivalent in the retinal microvessels with approximately 10% of total fatty acids. When compared with intact microvessels, the DHA proportion of confluent monolayers of both BRECs or pericytes in primary cultures dropped to approximately 2% of the total fatty acids, whereas AA was unchanged. Culture medium supplementation with unesterified DHA (10 microM) restored the DHA proportion of BRECs close to the microvascular value at the expense of linoleic acid without affecting AA very much. In contrast, DHA supplementation in pericytes increased the DHA proportion of these cells at the expense of AA. In conclusion, DHA of intact microvessels represented 10% of the total fatty acids, which was close to the AA proportion. Mild DHA supplementation of BRECs or pericytes in primary cultures restored their DHA proportion to the original microvessel value. This high percentage of polyunsaturated fatty acids in retinal microvessels should allow us to test the hypothesis that oxidation products derived from these fatty acids may be involved in the pathogenic process leading to diabetic retinopathy.

Animals↗
PubMed1996

Immunohistochemical study of 100 pancreatic tumors in 28 patients with multiple endocrine neoplasia, type I.

One hundred pancreatic tumors ranging in size from 0.3 to 7 cm were studied in 28 patients (17 male and 11 female patients; mean age 35 years) with multiple endocrine neoplasia, type I. An immunohistochemical study was performed on deparaffinized sections using the following antibodies: neuron-specific enolase, chromogranin A or synaptophysin, insulin, glucagon, somatostatin, pancreatic polypeptide (PP), vasoactive intestinal peptide (VIP), gastrin, adrenocorticotropic hormone, alpha-subunit of human chorionic gonadotropin, gonadotropin-releasing factor, serotonin, and calcitonin. Among the 100 tumors (all multiple), seven were unclassified, 10 were plurihormonal, and 83 produced a predominant hormonal secretion (with 50-90% of the same cell type), including 37 "A-cell tumors" (glucagon), 27 "B-cell tumors" (insulin), 11 PP-cell tumors, one G-cell tumor (gastrin) and one vasoactive intestinal peptide (VIP)-cell tumor. These multiple tumors had a different predominant hormonal secretion in the same patient in 23 of the 28 cases. There was a preferential association of A-cell tumor and B-cell tumor. Hyperplasia of the islets of Langerhans was not detected in adjacent pancreas. Nesidioblastosis was observed in 30% of cases.

Adolescent↗
PubMed1995

Selective inhibition of prostaglandin endoperoxide synthase-1 (cyclooxygenase-1) by valerylsalicylic acid.

Aspirin causes a time-dependent inhibition of prostaglandin endoperoxide H synthases (PGHS)-1 and -2 by acetylating active site serines present in both isozymes. In the case of PGHS-1, aspirin acetylation blocks cyclooxygenase activity, apparently by preventing arachidonate binding to the cyclooxygenase active site. With PGHS-2, acetylation does not block substrate binding but rather alters the enzyme in such a way that the acetylated form of PGHS-2 produces 15R-hydroxyeicosatetraenoic acid (15R-HETE) instead of the usual prostaglandin endoperoxide product. Based on these differences between PGHS-1 and PGHS-2, we reasoned that a salicylate ester containing an acyl group somewhat larger than the acetyl group of aspirin might be a selective inhibitor of PGHS-2. Accordingly, we prepared and tested eight different acyl salicylates as inhibitors of human (h) PGHS-1 and -2 expressed transiently in cos-1 cells. Valeryl(pentanoyl)salicylate (VSA) was the only compound in this series which showed isozyme selectivity, and, surprisingly, VSA inhibited hPGHS-1 much more effectively than hPGHS-2. Inhibition of hPGHS-1 by VSA was time-dependent. VSA also inhibited ovine PGHS-1 but did not inhibit the S530A mutant of ovine PGHS-1. This latter mutant, which lacks the active site serine hydroxyl group, is also refractory to inhibition by acetylsalicylate. Thus, we conclude that VSA acylates the active site serine of PGHS-1. VSA inhibited prostanoid synthesis by serum-starved murine NIH 3T3 cells which express only PGHS-1; in contrast, VSA caused only partial inhibition of prostanoid synthesis by serum-stimulated 3T3 cells which express both PGHS isozymes. Our results establish that VSA can be used as a reasonably selective inhibitor of PGHS-1.

3T3 Cells↗
PubMed1994

Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin.

Aspirin (acetylsalicylate) treatment of human (h) prostaglandin endoperoxide H synthase (PGHS)-1 expressed in cos-1 cells caused a time-dependent inactivation of oxygenase activity. Aspirin treatment of hPGHS-2 produced an enzyme which retained oxygenase activity but formed exclusively 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) instead of PGH2. The 15-HETE was exclusively of the 15R configuration. The Km values for arachidonate of native and aspirin-treated hPGHS-2 were about the same suggesting that arachidonate binds to both aspirin-treated and native hPGHS-2 in a similar manner. If, as expected, the formation of 15R-HETE proceeds through abstraction of the 13proS hydrogen from arachidonate, O2 insertion must occur from the same side as the hydrogen abstraction; with all other lipoxygenases and cyclooxygenases, O2 addition is antarafacial. When microsomal hPGHS-2 was incubated with [acetyl-14C]aspirin, the enzyme was acetylated. An S516A mutant of hPGHS-2, which retains enzyme activity, was not acetylated. This indicates that Ser-516 is the site of aspirin acetylation of hPGHS-2; this residue is homologous to the "active site" serine of PGHS-1. An S516N mutant of hPGHS-2 was catalytically active; in contrast, an S516Q mutant lacked cyclooxygenase but retained peroxidase activity. Because in the case of PGHS-1 a smaller asparagine substitution is sufficient to eliminate cyclooxygenase activity, we conclude that the active site of PGHS-2 is slightly larger than that of PGHS-1. An S516M mutant of hPGHS-2 was obtained which resembled aspirin-acetylated hPGHS-2 in that this mutant made 15R-HETE as its major product; however, unlike the aspirin-acetylated hPGHS-2, the Km value of the S516M mutant for arachidonate was 100 times that of native hPGHS-2.

Acetylation↗
PubMed1994

Role of the cyclic adenosine 3',5'-monophosphate and the phosphatidylinositol-Ca2+ cascades in mediating the effects of thyrotropin and iodide on hormone synthesis and secretion in human thyroid slices.

There are two major known regulatory pathways in human thyrocytes: the phosphatidylinositol-Ca2+ cascade (PiP2 cascade) and the cAMP cascade. We study here the regulation of the PiP2 cascade by TSH, ATP, NaF, and bradykinin. Our data show that protein iodination and, thus, the synthesis of thyroid hormones in human thyroid is under the control of both the PiP2 cascade and the cAMP cascade. Activation of the PiP2 cascade by TSH (10 mU/mL), NaF, bradykinin, ionomycin, and 12-O-tetradecanoylphorbol-13-acetate stimulates iodide organification. Conversely, activation of the cAMP cascade by forskolin, TSH (0.3 mU/mL), and dibutyryl cAMP inhibits iodide organification. These metabolic effects are correlated to activations and inhibitions of the H2O2-generating system, showing that H2O2 is a limiting factor for protein iodination in these cells. The cascades also regulate in parallel the activity of the pentose phosphate pathway. The effects of various concentrations of TSH on H2O2 generation and [1-14C]glucose oxidation were tested, showing a dual effect with an inhibition of these metabolisms for low concentrations of TSH (that stimulate the cAMP cascade) and an activation for high concentrations of TSH (that stimulate the PiP2 cascade). The control of thyroid secretion differs from that of protein iodination, in that the cAMP cascade greatly enhances secretion, whereas the PiP2 cascade has no effect on basal secretion and even an inhibitory effect on TSH-stimulated secretion (1 mU/mL). We also demonstrate here the presence of an inhibitory effect of iodide on its own organification in human thyroid (Wolff-Chaikoff effect). This effect is probably mediated through an inhibition of the inositol trisphosphate response to TSH and of the H2O2 response to Ca2+.

Adenosine Triphosphate↗
PubMed1991

Treatment of diversion colitis by short-chain fatty acids. Prospective and double-blind study.

Diminished production of short-chain fatty acids (SCFA) by altered flora has been suggested in the pathogenesis of diversion colitis (DC). We evaluated prospectively the effectiveness of SCFA irrigation in 13 patients with excluded colon (eight males, five females; mean age, 48 years). The causes of diversion were inflammatory bowel disease (n = 4), colonic cancer (n = 2), sigmoid diverticulitis with perforation (n = 3), ischiorectal abscess (n = 2), and miscellaneous (n = 2). Patients were given, twice a day for 14 days in a double-blind manner, a 60-ml enema containing either SCFA (acetate: 60 mmol/liter; propionate: 30 mmol/liter; and N-butyrate: 40 mmol/liter) (Group 1; n = 7) or isotonic NaCl (Group 2; n = 6). Endoscopy with biopsies was performed before starting the trial (D1) and 14 days later (D14). On D1 all patients had endoscopic and histologic findings suggestive of DC. No endoscopic or histologic changes were observed on D14 in either group. We conclude that endoscopic and histologic lesions of DC were not improved by SCFA irrigation during the 14 days.

Adult↗
PubMed1990

Covalent binding of arachidonic acid metabolites to human platelet proteins. Identification of prostaglandin H synthase as one of the modified substrates.

The covalent modification of proteins by metabolites of arachidonic acid (AA) was investigated in human platelets. Following incubation of washed human platelets with radiolabeled AA, ethanol precipitation of the proteins, and lipid extraction by organic solvents, a small fraction of the radioactivity added (0.3%) was tightly bound to the protein pellet. A dozen labeled protein bands were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Exhaustive hydrolysis of platelet proteins by proteases released an amphipathic radiolabeled material which had a chromatographic behavior similar to that of a known peptidolipid, leukotriene C4. These findings suggest a covalent nature for the observed binding. This binding was specific for AA since palmitate, myristate, or linoleate did not bind to a significant extent. It involved products of both cyclooxygenase and lipoxygenase pathways: it was indeed inhibited to a greater extent by eicosatetraynoic acid than by indomethacin. The protein-associated radioactivity was increased by the thromboxane synthase inhibitor dazoxiben. Indomethacin completely abolished this increase in binding, which could not be reproduced by exogenous prostaglandin (PG) E2, F2 alpha, or D2, and might thus involve PGG2 and/or PGH2. Diamide, an agent known to inhibit the reduction of 12-hydroperoxyeicosatetraenoic acid in platelets, produced an increase of the covalent binding, which was abolished by eicosatetraynoic acid but not by indomethacin: this suggests that the lipoxygenase product bound was 12-hydroperoxyeicosatetraenoic acid or a by-product. Dazoxiben and diamide produced distinct patterns of protein labeling after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One labeled band had a Mr of 70,000 as the PGH synthase monomer. Addition of AA at 17 microM enhanced the labeling of this band, while 100 microM was inhibitory. Labeling of this band was also induced by thrombin in prelabeled platelets. Two monoclonal antibodies against PGH synthase caused immune precipitation of a 70-kDa labeled protein in homogenates of [3H]AA-labeled platelets. PGH synthase, purified from ram seminal vesicles, was covalently modified after incubation with [3H]AA: this labeling was almost completely abolished by indomethacin. As much as 40% of platelet PGH synthase was covalently modified after incubation with 17 microM AA. It can be concluded that in intact platelets PGH synthase is covalently modified by an eicosanoid following incubation with exogenous AA or after AA mobilization from phospholipids by thrombin.

Animals↗
PubMed1990

Amiloride analogs induce the phosphorylation of elongation factor-2 in vascular endothelial cells.

5-(N-Ethyl-N-isopropyl)amiloride (EIPA), a potent inhibitor of Na+/H+ antiport, reduced [35S]methionine incorporation in proteins and induced the phosphorylation of a Mr 95,000 protein in bovine aortic endothelial cells. This protein was previously shown to become phosphorylated in response to ATP, bradykinin, and A23187 (1) and was identified as elongation factor-2 (2). The action of EIPA was independent of changes in cytosolic pH, because it was neither mimicked by sodium acetate nor inhibited by ammonium chloride, and it was reproduced by 2',4'-dimethylbenzamil, an analog of amiloride that is inactive on the Na+/H+ antiport. Furthermore, EIPA enhanced the Ca2(+)-dependent phosphorylation of a similar Mr 95,000 protein in a cell-free system, rabbit reticulocyte lysate, where an inhibitory effect of amiloride on protein synthesis has already been described (3). Because phosphorylation decreases the activity of elongation factor-2, our observation might explain why amiloride analogs inhibit protein synthesis.

Amiloride↗
PubMed1988

Pattern of protein phosphorylation in aortic endothelial cells. Modulation by adenine nucleotides and bradykinin.

In bovine aortic endothelial cells, ATP (10-100 microM) and bradykinin (0.1-1.0 microM) enhanced the phosphorylation of two major protein substrates with apparent molecular masses of 95 and 28 kDa. The action of ATP involved P2y purinoceptors. The kinetics were distinct for the two phosphopeptides. The phosphorylation of the 95-kDa protein was rapid (within 30 s) but transient (maintained for only 2 min). This time course agrees with that observed for the increase of the cytosolic Ca2+ level induced by ATP in these cells. Ionophore A23187 (greater than or equal to 100 nM) induced this phosphorylation for a longer period (5-10 min), whereas phorbol 12-myristate 13-acetate (PMA) was completely inactive. The enhancement of the 28-kDa protein phosphorylation was detectable after a 5-min lag and was maintained for at least 20 min. PMA (50 nM) stimulated weakly the phosphorylation of the 28-kDa protein, whereas A23187 (100-300 nM) was even more effective than ATP and bradykinin. The 95-kDa phosphoprotein seems to be related to a 100-kDa substrate of calmodulin-dependent protein kinase III recently identified as elongation factor-2. The 28-kDa protein, which was resolved as three variants in bidimensional gel electrophoresis, appears very similar to a slightly heavier phosphoprotein from thrombin-stimulated human platelets. In addition, bidimensional electrophoresis allowed the detection of at least 10 substrates (from 18 to 46 kDa) whose phosphorylation was enhanced equally well by ATP, bradykinin, and A23187 and only partially by PMA. In conclusion, protein phosphorylation induced by ATP and bradykinin in aortic endothelial cells seems to be catalyzed mostly by Ca2+-dependent kinases, distinct from protein kinase C.

Adenosine Triphosphate↗
PubMed1986

Covalent binding of eicosanoids to platelet proteins.

Following an incubation of washed human platelets with 14C-arachidonic acid, a small fraction of the radioactivity became tightly bound to the protein pellet. Three criteria suggested that it was actually a covalent binding: it was not removed by exhaustive extractions with solvents of various polarities, it was not dialysable against SDS-buffer and it corresponded to the labeling of several protein bands after SDS-polyacrylamide gel electrophoresis. The use of several pharmacological agents (indomethacin, eicosatetraynoic acid, dazoxiben, diamide) has allowed us to divide this binding into three components: the first one, independent from both cyclooxygenase and lipoxygenase, the second one dependent on cyclooxygenase products and finally the third one, dependent on lipoxygenase products.

Arachidonic Acid↗
PubMed1986

[Induced prematurity].

Voluntary induction of a premature delivery, which goes against our constant battle to reduce prematurity, represents, at this time, a paradoxical situation which is not exceptional in vasculorenal syndromes and premature rupture of the membranes. Advances in ressuscitation and the care of premature newborn babies, on the one hand, and techniques of artificial induction of labor, on the other hand, account for this obvious paradox. Even advances in artificial induction of labor explain also the decrease in the cesarean sections indications, under such circumstances.

Female↗