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Biomedical subjects

M Gaudry

Publications and source records attributed to M Gaudry.

At least 37 records · Page 2Linked to original sources
PubMed1991

Vitamin K dependent carboxylation: determination of the stereochemical course using 4-fluoroglutamyl-containing substrate.

The stereochemical course of the vitamin K dependent carboxylation has been elucidated using a (4S)-4-fluoroglutamyl-containing pentapeptide as a substrate. The absolute configuration of the [13C]-4-carboxy-4-fluoroglutamate obtained when the carboxylation was carried out with 13C-labeled sodium bicarbonate, was determined after reduction of the [13C]-4-carboxy-4-fluoroglutamyl residue into 4-fluoro-5,5'-dihydroxyleucine, hydrolysis, lactonization, and peracetylation. The absolute configuration at C-4 was determined to be S by locating the 13C label in the lactone ring of the trans isomeric lactone and in the hydroxymethyl group of the cis isomer following HPLC separation of both isomers and analysis by GC/MS/MS techniques. It follows that the vitamin K dependent carboxylation occurs with inversion of configuration.

Amino Acid Sequence↗
PubMed1991

Priming of calcium mobilization in human neutrophils by granulocyte-macrophage colony-stimulating factor: evidence for an involvement of phospholipase D-derived phosphatidic acid.

Human neutrophils pre-incubated with granulocyte-macrophage-colony-stimulating factor (GM-CSF) exhibit an enhanced mobilization of calcium in response to secondary stimuli such as chemotactic factors. The mechanisms underlying this priming effect of GM-CSF were examined. It was first demonstrated that the additional calcium mobilized by chemotactic factors in GM-CSF-treated cells was derived from intracellular stores and was associated neither with an increased permeability to calcium nor with production of inositol 1,4,5-trisphosphate. These results indicated that GM-CSF called upon a novel mechanism in order to enhance the mobilization of calcium in human neutrophils. The growth factor has recently been shown to prime phospholipase D leading to an enhanced activation by chemotactic factors and an augmented production of phosphatidic acid. Furthermore the ability of exogenous phosphatidic acid to mobilize calcium in cell types other than neutrophils has been previously demonstrated. Therefore, we examined the potential involvement of phospholipase D in the priming of the calcium response by GM-CSF in human neutrophils. Inhibition of the production of the fMet-Leu-Phe-stimulated production of phosphatidic acid by ethanol or wortmannin had only marginal effects on the concurrent mobilization of calcium. However, the priming of the mobilization of calcium by GM-CSF was greatly decreased in cells treated with either ethanol or wortmannin. These results provide strong support for the hypothesis that the production of phosphatidic acid, which is enhanced in GM-CSF-treated cells, is linked to an increased mobilization of intracellular calcium. These results may have relevance to the mechanism of action of GM-CSF in mature haematopoeitic cells as well to the mitogenic activity of other growth factors.

Adult↗
PubMed1991

Leukotriene B4 receptors on guinea pig alveolar eosinophils.

The existence of receptors for LTB4 on highly purified guinea pig alveolar eosinophils was investigated. Massive infiltration of eosinophils in alveolar spaces was induced in guinea pigs by i.v. injections of Sephadex beads G50 (16 mg/kg). Alveolar eosinophils (50 x 10(6) cells) were purified to approximately 98% by Percoll continuous density gradient centrifugation. The binding studies indicated that alveolar eosinophils bind LTB4 in a saturable, reversible and specific manner. Scatchard analysis indicated the existence of high-affinity binding sites (Kd1 = 1.00 +/- 0.22 nM; Bmax1 = 966 +/- 266 sites/cell) and low-affinity binding sites (Kd2 = 62.5 +/- 8.9 nM; Bmax2 = 5557 +/- 757 sites/cell). The metabolism of LTB4 by alveolar eosinophils in binding conditions was assessed by RP-HPLC and no significant degradation of [3H]LTB4 was observed. LTB4 dose-dependently stimulated eosinophil migration in both chemokinesis and chemotaxis assays with an EC50 value of 1.30 +/- 0.14 and 18.14 +/- 1.57 nM, respectively. LTB4 caused a dose-dependent increase in the production of superoxide anion with an apparent EC50 value of 50 X 10(-9) M in our experimental conditions. LTB4 also induced a dose-dependent increase in the generation of TxA2 with an EC50 value of 46.2 X 10(-9) M. Taken together, our results demonstrated that guinea pig alveolar eosinophils express two classes of specific receptors for LTB4. The high-affinity binding sites seem associated to chemokinesis and chemotaxis whereas the low-affinity binding sites seem associated to superoxide anion production and generation of TxA2. The existence of LTB4 receptors in eosinophils could explain the presence of these cells in hypersensitivity reactions.

Animals↗
PubMed1990

Selective inhibition of human neutrophil functional responsiveness by erbstatin, an inhibitor of tyrosine protein kinase.

The role of tyrosine kinases in the responses of human neutrophils to chemotactic factors was examined using the recently described inhibitor erbstatin. Pre-incubation with erbstatin decreased the amount of tyrosine phosphorylation induced by the formylated oligopeptide formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) without effecting the binding of [3H]-fMet-Leu-Phe. Erbstatin also dose-dependently inhibited the production of superoxide anion induced by fMet-Leu-Phe and platelet-activating factor, but did not affect the oxidative burst induced by either the calcium ionophore A23187 or the phorbol ester phorbol 12-myristate 13-acetate. Furthermore, erbstatin diminished the cytosolic acidification elicited by fMet-Leu-Phe, platelet-activating factor, and leukotriene B4. In contrast, erbstatin was without effect on the increase in the levels of cytoplasmic free calcium and polymerized actin elicited by fMet-Leu-Phe, C5a, leukotriene B4 and platelet-activating factor, whereas the increase in cytoplasmic free calcium elicited by platelet-derived growth factor was inhibited by erbstatin. In addition, erbstatin affected neither the release of elastase stimulated by these agonists nor the release of beta-glucosaminidase, lysozyme or vitamin B12-binding protein induced by fMet-Leu-Phe. These results indicate that tyrosine protein kinases are involved in the signaling pathways employed by chemotactic factors in the stimulation of selective functional responses (and superoxide production in particular) in human neutrophils.

Calcimycin↗
PubMed1990

Involvement of a phospholipase D in the mechanism of action of granulocyte-macrophage colony-stimulating factor (GM-CSF): priming of human neutrophils in vitro with GM-CSF is associated with accumulation of phosphatidic acid and diradylglycerol.

The generation of diradylglycerol (DRG) and phosphatidic acid (PdtOH) was investigated in neutrophils primed with granulocyte-macrophage colony-stimulating factor (GM-CSF). Mass accumulation of DRG and PdtOH was measured using reversed-phase high performance liquid chromatography and thin layer chromatography, respectively. GM-CSF had no direct effect on the levels of PdtOH and DRG, but it increased PdtOH generation and the late phase of DRG accumulation in human neutrophils stimulated with FMLP. The elevation of the mass of PdtOH peaked approximately 100 s and clearly preceded that of DRG, which peaked at 150 s. The diacylglycerol kinase inhibitor R59022 enhanced the sustained increase in DRG but did not produce a parallel inhibition in PdtOH production. GM-CSF was without effect on the level of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] and did not affect the liberation of Ins(1,4,5)P3 induced by FMLP. These findings exclude the involvement of the PtdIns(4,5)P2-specific phospholipase C/diacylglycerol pathway in the sustained phase of DRG accumulation. The early (30-s) appearance of PdtOH clearly suggests that GM-CSF enhanced FMLP receptor-linked phospholipase D (PLD) generation of PdtOH. PLD was assessed more directly by formation of labeled phosphatidylethanol (PEt) through PLD capacity of catalyzing a trans-phosphatidylation in presence of ethanol. The formation of PEt associated with a concomitant decrease in PdtOH directly demonstrated that the mechanism by which GM-CSF enhances PdtOH production is activation of a PLD active on phosphatidylcholine. This study provides evidence that the mechanism of action of GM-CSF involves upregulation of PLD activity leading to enhanced generation of PdtOH and DRG in FMLP-stimulated neutrophils. These findings may provide the basis for several of the priming effects of GM-CSF.

Chromatography, High Pressure Liquid↗
PubMed1990

Vitamin K-dependent carboxylation. Mechanistic studies with 3-fluoroglutamate-containing substrates.

The tripeptides t-butyloxycarbonyl-Xaa-Glu-[3H]Val, where Xaa is either (2R,3S)- or (2R,3R)-3-fluoroglutamate (respectively the erythro and the threo isomer), were synthesized and their behaviour during vitamin K-dependent carboxylation was studied. Neither peptide was carboxylated. The erythro compound gave rise to an HF-elimination product representing 1% of the starting material. This HF elimination did not occur during incubation of the threo compound. The formation of the dehydropeptide, probably by elimination of an F- anion from an intermediate carbanion, favours the ionic pathway for vitamin K-dependent carboxylation.

Amino Acid Sequence↗
PubMed1990

Dual effect of phorbol myristate acetate on chemoattractant-induced locomotion of human neutrophils.

Phorbol myristate acetate (PMA) is a potent activator of Ca2+/phospholipid-dependent protein kinase (PKC) and was used to study the involvement of this kinase in human polymorphonuclear neutrophil (PMN) locomotion. Preincubation of PMNs with low concentrations of PMA (4 to 64 x 10(-11) M) had the following effects: (1) fMet-Leu-Phe-induced migration under agarose was increased when the chemoattractant was used at the suboptimal concentration of 10(-8)M and not at the optimal concentration of 10(-7)M; (2) no effect on spontaneous or serum- or LTB4- induced migration at either suboptimal or optimal concentrations; (3) PMA enhanced fMet-Leu-Phe-induced migration, increasing the speed of locomotion but not affecting shape changes induced by fMet-Leu-Phe; (4) the number of fMet-Leu-Phe-specific receptors expressed on the PMN membrane was not altered. Intermediate concentrations of PMA (1.6 to 4.0 x 10(-9) M) had no effect on PMN migration, whereas higher concentrations (4.0 to 16 x 10(-9) M) reduced both spontaneous and fMet-Leu-Phe-, serum-, or LTB4-induced migration in a dose-dependent manner. Effects observed with low concentrations of PMA were not associated with a translocation of cytosolic PKC even when preincubation with PMA was followed by fMet-Leu-Phe stimulation. In contrast, effects observed with higher concentrations of PMA paralleled the decrease in cytosolic PKC activity.

Cells, Cultured↗
PubMed1988

Protective effect of indomethacin against chemotactic deactivation of human neutrophils induced by formylated peptide.

The effects of the nonsteroidal antiinflammatory drug indomethacin on the parameters relating to the migration and respiratory burst of human polymorphonuclear leukocytes (PMN) were studied in an attempt to clarify the mechanism of this drug's action on PMN. At various concentrations below 200 micrograms/ml, indomethacin partially inhibited the spontaneous migration of PMN but did not alter the directional migration induced by C5a-activated serum. In the presence of N-formyl-methionyl-leucyl-phenylalanine (FMLP) as chemoattractant, directed PMN migration was either inhibited or stimulated by indomethacin, depending on FMLP concentration. When PMN migration was induced by the optimal and suboptimal FMLP concentrations of 10(-7) and 10(-8) M, indomethacin inhibited this migration, but when the high FMLP concentration of 10(-6) M depressed this migration by chemotactic deactivation, indomethacin restored it to its maximum. Both the inhibitory and stimulatory effects of indomethacin on FMLP-induced PMN migration were due to changes in the migration speed. Indomethacin also inhibited FMLP-induced changes in the shape of floating PMN, and in respiratory burst, as well as specific FMLP binding to PMN. In contrast, indomethacin did not alter the PMN respiratory burst induced by phorbol myristate acetate or C5a-activated serum. These data show that indomethacin is able to prevent the loss of PMN chemokinetic activity induced by formylated peptides and suggest that it might be useful for investigating the mechanism of peptide-induced chemotactic deactivation.

Chemotaxis, Leukocyte↗
PubMed1988

Negative effect of a protein kinase C inhibitor (H-7) on human polymorphonuclear neutrophil locomotion.

The effects of 1-(5-isoquinoline sulphonyl)-2-methyl piperazine (H-7), a recently described inhibitor of Ca2+/phospholipid-dependent protein kinase (protein kinase C), were studied during under-agarose migration of human polymorphonuclear neutrophils (PMN) stimulated by various chemoattractants, in order to determine whether protein kinase C is involved in PMN locomotion. The effect of H-7 on the oxidative burst induced by phorbol 12-myristate 13-acetate or N-formyl-methionyl-leucyl-phenylalanine (FMLP) was also measured. Pre-incubation of PMN with H-7 concentrations ranging from 50 to 400 microM inhibited: (i) spontaneous PMN migration under agarose; (ii) the directed migration induced by activated serum, leukotriene B4 or FMLP; and (iii) the speed of the migration induced by FMLP. The inhibition by H-7 of FMLP-induced directed migration was less when FMLP was used at high concentrations which, in the absence of H-7, inhibit locomotion. H-7 depressed the oxidative burst induced by phorbol myristate acetate (PMA) but not that induced by FMLP. All the effects of H-7 on the oxidative burst and migration were reversed by washing PMN after H-7 treatment. These findings indicate that protein kinase C, inhibitable by H-7, is involved in a mechanism controlling the speed of PMN locomotion.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1985

Interaction of L-threo and L-erythro isomers of 3-fluoroglutamate with glutamate decarboxylase from Escherichia coli.

L-threo-3-Fluoroglutamate and L-erythro-3-fluoroglutamate were tested with glutamate decarboxylase from Escherichia coli. Both isomers were substrates: the threo isomer was decarboxylated into optically active 4-amino-3-fluorobutyrate, whereas the erythro isomer lost the fluorine atom during the reaction, yielding succinic semialdehyde after hydrolysis of the unstable intermediate enamine. The difference between the two isomers demonstrates that the glutamic acid-pyridoxal phosphate Schiff base is present at the active site under a rigid conformation. Furthermore, although the erythro isomer lost the fluorine atom, yielding a reactive aminoacrylic acid in the active site, no irreversible inactivation of E. coli glutamate decarboxylase was observed.

4-Aminobutyrate Transaminase↗
PubMed1985

Biotin uptake: influx, efflux and countertransport in Escherichia coli K12.

Biotin uptake by Escherichia coli K12 has been reinvestigated. The vitamin uptake is an active process depending on energy and inhibited by uncouplers. The kinetic parameters (Km = 0.27 microM, Vmax = 6.8 pmol/min per mg dry cells) are close to those previously determined for a biotin-dependent strain E. coli C162 (Piffeteau, A., Zamboni, M. and Gaudry, M. (1982) Biochim. Biophys. Acta 688, 29-36). By use of biotin p-nitrophenyl ester, an affinity label of the biotin transport system, it was shown, under conditions of steady state, that the efflux of biotin is not energy dependent and is mainly mediated by a diffusion mechanism. Reexamination of the regulation of the biotin transport by biotin, revealed that only 50% of the biotin uptake system is under control by the vitamin.

Affinity Labels↗
PubMed1984

Vitamin K dependent carboxylation: synthesis and biological properties of tetrazolyl analogues of pentapeptidic substrates.

The pentapeptides Phe-Leu-X-Glu-Val and Phe-Leu-X-X-Val, where X = 4-(5H-tetrazolyl)-2-aminobutyric acid (tetrazolyl analogue of glutamic acid), were synthesized by addition of tri-n-butyltin azide to the corresponding nitrile-containing peptides. These tetrazolyl peptides and a dinitrile precursor were tested as possible substrates and inhibitors of the vitamin K dependent carboxylation. Phe-Leu-X-Glu-Val was carboxylated (40% of the reference peptide Phe-Leu-Glu-Glu-Val, Km = 20 mM) exclusively on the glutamyl residue, whereas the dinitrile precursor and Phe-Leu-X-X-Val were not carboxylated. The latter was a competitive inhibitor with an affinity (Ki = 3 mM) close to that of the reference peptide (Km = 3 mM).

1-Carboxyglutamic Acid↗
PubMed1984

Vitamin-K-dependent carboxylation. Synthesis and biological properties of diastereoisomeric gamma-substituted glutamic acid containing peptidic substrates.

Pentapeptides Phe-Leu-X-Glu-Val where X is successively L-threo-gamma-fluoro-glutamyl, L-erythro-gamma-fluoro-glutamyl, L-threo-gamma-methyl-glutamyl or L-erythro-gamma-methyl-glutamyl have been synthesized and tested as substrates for the vitamin K-dependent carboxylation. L-threo- or L-erythro-gamma-methyl-glutamyl are not carboxylated but both corresponding peptides are inhibitors of the reaction. The L-threo-gamma-methyl-glutamyl containing peptide has the highest affinity described so far for the active site of the carboxylase (80 microM). In the gamma-fluoro-glutamyl series, only the L-erythro-gamma-fluoro-glutamyl residue is carboxylated, showing that the enzymatic hydrogen abstraction is stereospecific and corresponds to the elimination of the pro S hydrogen of glutamic acid. The lack of in vitro dicarboxylation of model peptides in contrast with the in vivo polycarboxylation of endogenous precursors is discussed along with the regiospecificity of the reaction with the different substrates.

Amino Acid Sequence↗
PubMed1983

Vitamin K-dependent carboxylation. Study of the hydrogen abstraction stereochemistry with gamma-fluoroglutamic acid-containing peptides.

Two pentapeptides Phe-Leu-X-Glu-Val where X is either the L-threo-gamma-fluoroglutamic acid or the L-erythro-isomer have been synthesized and tested as substrates in the vitamin K-dependent carboxylation. Both peptides are carboxylated, but the reaction occurs exclusively on the glutamic acid of the L-threo-gamma-fluoroglutamate-containing peptide, whereas both glutamic and fluoroglutamic residues of the L-erythro-gamma-fluoroglutamate-containing peptide are carboxylated. These results reveal that the enzymatic hydrogen abstraction step is stereospecific and corresponds, in the gamma-fluoroglutamate case, to the elimination of the hydrogen equivalent to the pro-S hydrogen of glutamic acid.

Chemical Phenomena↗
PubMed1983

Vitamin K dependent carboxylation: study of diastereoisomeric gamma-methylglutamic acid containing peptidic substrates.

Two pentapeptides Phe-Leu-X-Glu-Val where X is either the L-threo-gamma-methylglutamic acid or the L-erythro isomer have been synthesized and tested as substrates in the vitamin K dependent carboxylation. The gamma-methylglutamic residue is not carboxylated and both peptides are inhibitors of the carboxylation of the reference peptide Phe-Leu-Glu-Glu-Val. The threo containing isomer has a much better affinity than the reference and is the best inhibitor of this reaction described so far.

Animals↗
PubMed1983

Biotin dependent carboxylase activities in normal human and multicarboxylase deficient patient fibroblasts: relationship to the biotin content of the culture medium.

Three mitochondrial carboxylase activities can be depleted in skin fibroblasts from patients with the neonatal form of multiple carboxylase deficiency when the biotin content of the culture medium is lowered to 25 nmol/l. On the other hand this depletion can be achieved in control fibroblasts or in fibroblasts from patients with the late onset form of the deficiency when avidin (50 U/l) is added to the culture medium. The kinetics of the carboxylase activity decrease are nevertheless identical for control and for both types of fibroblasts. After depletion, control and late onset form fibroblasts recover their carboxylase activities at the same rate, whereas fibroblasts with the neonatal form of deficiency need longer times or higher concentrations of biotin to restore their carboxylase activities. These results are consistent with previous hypotheses concerning the origin of both forms of the deficiency. In addition, this technique provides a convenient access to human apocarboxylases, i.e. substrates for in vitro holocarboxylase synthetase investigation.

Biotin↗