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Biomedical subjects

M Renard

Publications and source records attributed to M Renard.

At least 19 recordsLinked to original sources
PubMed2001

Acyl-CoA elongase expression during seed development in Brassica napus.

The Bn-FAE1.1 and Bn-FAE1.2 genes encode the 3-ketoacyl-CoA synthase, a component of the elongation complex responsible for the synthesis of very long chain monounsaturated fatty acids (VLCMFA) in the seeds of Brassica napus. Bn-FAE1 gene expression was studied during seed development using two different cultivars: Gaspard, a high erucic acid rapeseed (HEAR), and ISLR4, a low erucic acid rapeseed (LEAR). The mRNA developmental profiles were similar for the two cultivars, the maximal expression levels being measured at 8 weeks after pollination (WAP) in HEAR and at 9 WAP in LEAR. Differential expression of Bn-FAE1.1 and Bn-FAE1.2 genes was also studied. In each cultivar the same expression profile was observed for both genes, but Bn-FAE1.2 was expressed at a lower level than Bn-FAE1.1. Secondly, VLCMFA synthesis was measured using particulate fractions prepared from maturating seeds harvested weekly after pollination. The oleoyl-CoA and ATP-dependent elongase activities increased from the 4th WAP in HEAR and reached the maximal level at 8 WAP, whereas both activities were absent in LEAR. In contrast, the 3-hydroxy dehydratase, a subunit of the elongase complex, had a similar activity in both cultivars and reached a maximum from 7 to 9 WAP. Finally, antibodies against the 3-ketoacyl-CoA synthase revealed a protein of 57 kDa present only in HEAR. Our results show: (i) that both genes are transcribed in HEAR and LEAR cultivars; (ii) that they are coordinately regulated; (iii) that Bn-FAE1.1 is quantitatively the major isoform expressed in seeds; (iv) that the Bn-FAE1 gene encodes a protein of 57 kDa responsible for the 3-ketoacyl-CoA synthase activity.

Acetyltransferases↗
PubMed2001

Mutations in the fatty acid elongation 1 gene are associated with a loss of beta-ketoacyl-CoA synthase activity in low erucic acid rapeseed.

Low erucic acid rapeseed (LEAR) is characterised by a near absence of very long chain fatty acids in the seed oil which has been correlated with a lack of acyl-CoA elongation activity. Here we show that the absence of acyl-CoA and ATP-dependent elongation activities in microsomes isolated from LEAR embryos is associated with an absence of beta-ketoacyl-CoA synthase activity encoded by the Bn-fatty acid elongation 1 (FAE1) genes. Size exclusion chromatography of solubilised microsomes revealed the presence of a high molecular mass acyl-CoA elongase complex in high erucic acid rapeseed which was absent in microsomes isolated from LEAR seeds. Although transcripts for the Bn-FAE1 genes were detected in LEAR embryos, immunoblots using antisera raised against the beta-ketoacyl-CoA synthase indicated an absence of this protein. Comparison of the deduced amino acid sequences of immature embryo cDNAs reveals that LEAR alleles of Bn-FAE1 encode variant beta-ketoacyl-CoA synthase proteins.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2001

Zirconium speciation in lactate solutions and polyacrylate gels.

Controlling gelation kinetics is an important objective for several applications (ceramic and thin film syntheses, reduction in permeability of porous rock, etc). There is a growing interest in studying the gelation of polymers by zirconium, a crosslinker of lower toxicity than the chromium which is still commonly used. XAS at the Zr K-edge was performed at the European Synchrotron Radiation Facility (ESRF) on the BM32 beamline. The fluorescence detection was used to carry out successful in situ speciation at concentrations as low as 36 ppm. The Zr speciation was determined both in ZrLa (where La stands for lactate) aqueous solutions and in gels of a terpolymer of acrylamide having 2% of zirconium reactive acrylate side groups and 2% of sulfonate groups introduced to prevent syneresis. XANES results show that Zr is always in a dodecahedral geometry. In ZrLa solutions. EXAFS results indicate that Zr species grow from a dimer Zr2(La)6 to a tetramer (Zr4(La)x) and then to larger polymers resulting from tetramer associations, as the Zr concentration decreases from 51840 ppm to 36ppm. In polymer gels, Zr species appear to be dimers at pH 6 while tetramers are found when gelation occurred at pH 7. Calculations taking into account multiple scattering effects as well as dynamic molecular calculation confirmed conclusions derived from conventional EXAFS analysis.

Journal Article↗
PubMed2000

[Erythrocyte adhesion to vascular endothelium: clinical applications].

INTRODUCTION: The objectives of this paper are to review the environmental factors, the different erythrocyte ligands and the corresponding endothelial receptors involved in adhesion. CURRENT KNOWLEDGE AND KEY POINTS: Leukocyte adhesion to vascular endothelium is related to inflammation and has been widely studied. The adhesion of erythrocytes to vascular endothelium has been investigated more recently, mainly in the physiopathology of three diseases: diabetes mellitus, sickle cell disease and malaria. The three diseases are characterized by microvascular complications and are deleterious for the red blood cell membrane. They lead to abnormal erythrocyte adhesion to vascular endothelium. Thus better understanding of the mechanisms involved in red blood cell adhesion to the endothelium is important since it might lead to the development of new therapeutic targets. Progress in this field might contribute to therapeutic improvement in sickle cell disease and to the development of an antimalarial vaccine. FUTURE PROSPECTS AND PROJECTS: However, additional studies focusing on in vivo endothelium heterogeneity, the different subpopulations of red blood cells and the diversity of Plasmodium falciparum strains are required. The consequences of such erythrocytes/endothelium interactions on the endothelial functions remain to be established.

Anemia, Sickle Cell↗
PubMed2000

Evidence for the existence of two soluble NAD(+) kinase isoenzymes in Euglena gracilis Z.

Two soluble NAD(+) kinase isoenzymes (isoenzymes 1 and 2) from Euglena gracilis were separated by preparative electrophoresis and characterized. They display several similar properties: both have an identical apparent molecular weight of 68 kDa and their activities are independent on calmodulin, insensitive to 2-mercaptoethanol but inhibited by p-chloromercurybenzoate, 5, 5'-dithiobis(2-nitrobenzoate) and, surprisingly, by low dithiothreitol concentrations, the inhibition by dithiothreitol being irreversible for isoenzyme 1 but reversible for isoenzyme 2. Nevertheless, the two isoenzymes mainly differ by their specificities towards triphosphate nucleotides and their catalytic mechanisms. Isoenzyme 1 is as active in the presence of ATP as of GTP and acts by a ping-pong mechanism with a k(M) for NAD(+) of 0.26 mM and a k(M) for low MgATP(2-)concentrations of 0.03 mM. Isoenzyme 2 is three-fold more active in the presence of GTP than of ATP and operates by a sequential mechanism with k(M)s for NAD(+) and MgGTP(2-) of 1.03 and 0.20 mM, respectively. This study shows the evidence for the existence of two structurally similar but catalytically different NAD(+) kinase isoenzymes in E. gracilis. One resembles the enzyme previously described in bacteria. The other displays a catalytic mechanism identical to that of NAD(+) kinase from other organisms but remains unique among all the NAD(+) kinases studied to-date regarding its specificity towards GTP.

Adenosine Triphosphate↗
PubMed2000

Homocysteine-thiolactone induces caspase-independent vascular endothelial cell death with apoptotic features.

OBJECTIVE: Cell death is generally classified into two large categories: apoptosis, which represents active, physiological programmed cell death, and necrosis, which represents passive cell death without underlying regulatory mechanisms. Apoptosis plays an important role in tissue homeostasis and its role in endothelium integrity can be influenced by the functional status of endothelial cells. Homocysteine, a sulfated amino-acid product of methionine demethylation, is an independent risk factor for vascular disease (arterial and venous thombosis). Our goal was to investigate the thiol-derivatives effect on the endothelial cell apoptosis. METHODS: Three parameters were measured: mitochondrial membrane potential using DiOC6(3) as the probe, DEVDase activation, and phosphatidylserine exposure on the cell surface with fluorosceinated annexin V labeling which allows apoptosis to be distinguished from necrosis. RESULTS: Homocysteine-thiolactone induced endothelial cell apoptosis in a concentration-dependent manner (range: 50-200 microM), independently of the caspase pathway. Only homocysteine-thiolactone, among the thiol derivatives tested, induced apoptosis. Apoptosis was not influenced by the serum concentration in culture medium, suggesting that the observed apoptotic process could occur in vivo. None of the inhibitors used (e.g., leupeptin, fumosinin B1, catalase, or z-VAD-fmk) was able to prevent homocysteine-induced apoptosis of vascular endothelial cells. CONCLUSION: The apoptosis of vascular endothelial cells induced by high concentration of homocysteine-thiolactone might be one step atherosclerotic cardiovascular disease, and contribute to its complication.

Apoptosis↗
PubMed2000

Comparative methodologic study of NFkappaB activation in cultured endothelial cells.

The transcriptional regulatory protein nuclear factor kappaB (NFkappaB) participates in the control of gene expression of many modulators of the inflammatory and immune responses. Various activators trigger NFkappaB release and nuclear translocation after phosphorylation and proteolytic degradation of IkappaB. This study evaluated the abilities of fluorescence and confocal microscopies, laser scanning cytometry (LSC), electrophoretic mobility-shift assay (EMSA), and Western blotting to detect NFkappaB activation in endothelial cells (ECs) and to investigate the role of homocysteine (Hcy) in NFkappaB activation. ECs were treated with interleukin-1B (10 ng/mL) or Hcy thiolactone (1 and 5 mmol/L) as NFkappaB activators. Hcy, a thiol-containing amino acid, has been shown to directly damage ECs in vitro. Experimental evidence suggests that the atherogenic propensity associated with hyperhomocysteinemia results from EC dysfunction. When ECs were pretreated with an inhibitor (pyrrolidine dithiocarbamate, 100 micromol/L) or with staurosporine (5 microL/mL), no NFkappaB activation was observed. NFkappaB activation in ECs could be detected with all five techniques, clearly showing NFkappaB translocation from the cytoplasm to the nuclei. Confocal microscopy was more sensitive and less subjective than immunofluorescence microscopy. LSC was even more sensitive, specific, and reproducible. EMSA, the reference method, has the disadvantages of being radioactive, expensive, and time consuming. Western blot analysis detected the NFkappaB p50 subunit implicated in NFkappaB activation. The techniques usually used to detect NFkappaB activation in ECs are immunofluorescence microscopy and confocal microscopy, LSC, EMSA, and Western blot analysis, but none of them is ready for routine use.

Cells, Cultured↗
PubMed2000

[Interest in variations in soluble ICAM-1 plasma levels. From physiology to clinical applications].

Intracellular adhesion molecule 1 (ICAM-1) is an adhesion-related molecule belonging to the immunoglobulin superfamily. This molecule is found on the cell membrane of endothelial cells. When activated ICAM-1 allows stable leukocyte adhesion to the endothelial surface. ICAM-1 is found not only in the membrane form but also circulating in serum. ICAM-1 (extracellular part of ICAM-1). This enables ICAM-1s to bind leukocyte integrin receptors such as LFA-1 (CDI1a/CD18) and Mac-1 (CDI1b/CD18) and therefore provide adaptive changes in the adhesion process between circulating cells and the endothelium.

Cell Adhesion↗
PubMed1999

Functional characterization of the somatic hypermutation process leading to antibody D1.3, a high affinity antibody directed against lysozyme.

The impact of somatic hypermutation on the affinity of Abs directed against protein Ags remains poorly understood. We chose as a model the secondary response Ab D1.3 directed against hen egg lysozyme. During the maturation process leading to this Ab, five replacement somatic mutations occurred. After reconstituting the germline Ab from which D1.3 originated, we assessed the energetic and kinetic importance of each of the somatic mutations, individually or combined, using the BIAcore apparatus. We found that the mutations induced an overall 60-fold improvement of affinity, principally due to a decrease in the kinetic rate of dissociation. We showed that their effects were additive and context independent; therefore, in the case of D1.3, the order in which somatic mutations were introduced and selected is unimportant. Interestingly, most of the affinity improvement was due to a single somatic mutation (Asn50-->Tyr in VL), involving a residue that belongs to the functional interface between Ab D1.3 and lysozyme. This replacement could either establish new Van der Waals contacts between the Ab and the Ag or help stabilize the conformation of a closely situated crucial residue of the Ab paratope. The four other mutations played only a marginal part in affinity maturation; potential reasons for which these mutations were nevertheless selected are discussed.

Animals↗
PubMed1999

Endothelial dysfunction during acute methionine load in hyperhomocysteinaemic patients.

Hyperhomocysteinaemia has been associated with arterial and venous thrombosis possibly by causing damage to the endothelium. We hypothesised that an oral load in methionine, that increases plasma homocysteine, would also result in an increase in biological markers of endothelial or platelet dysfunction. Then we investigated two groups of patients with arterial or venous occlusive disease: 17 with hyperhomocysteinemia and 12 without hyperhomocysteinemia. We measured in both groups plasma soluble thrombomodulin, von Willebrand factor, P-selectin and tissue factor plasma inhibitor before and 6 hours after a load with 100 mg/kg oral methionine. Methionine load resulted in a significant increase in von Willebrand factor in both groups (P<0.02), suggesting that endothelial dysfunction occurs during the load.

Administration, Oral↗
PubMed1999

Three months supplementation of hyperhomocysteinaemic patients with folic acid and vitamin B6 improves biological markers of endothelial dysfunction.

Hyperhomocysteinaemia is a risk factor for premature atherosclerosis and venous thromboembolic disease. Supplementation with folic acid and vitamin B6 has been shown to decrease plasma homocysteine but data fail to assess an effect on the progression of vascular disease. We measured plasma homocysteine and two markers of endothelial injury (plasma soluble thrombomodulin and von Willebrand factor) at baseline and after 3 months of treatment with folic acid and vitamin B6. After this treatment there was a significant decrease in fasting soluble thrombomodulin (-15 ng/ml, 95%CI 5-22.2). Von Willebrand factor was significantly raised after methionine load at baseline but did not significantly rise after supplementation.

Adult↗
PubMed1999

A plastidial lysophosphatidic acid acyltransferase from oilseed rape.

The biosynthesis of phosphatidic acid, a key intermediate in the biosynthesis of lipids, is controlled by lysophosphatidic acid (LPA, or 1-acyl-glycerol-3-P) acyltransferase (LPAAT, EC 2.3.1.51). We have isolated a cDNA encoding a novel LPAAT by functional complementation of the Escherichia coli mutant plsC with an immature embryo cDNA library of oilseed rape (Brassica napus). Transformation of the acyltransferase-deficient E. coli strain JC201 with the cDNA sequence BAT2 alleviated the temperature-sensitive phenotype of the plsC mutant and conferred a palmitoyl-coenzyme A-preferring acyltransferase activity to membrane fractions. The BAT2 cDNA encoded a protein of 351 amino acids with a predicted molecular mass of 38 kD and an isoelectric point of 9.7. Chloroplast-import experiments showed processing of a BAT2 precursor protein to a mature protein of approximately 32 kD, which was localized in the membrane fraction. BAT2 is encoded by a minimum of two genes that may be expressed ubiquitously. These data are consistent with the identity of BAT2 as the plastidial enzyme of the prokaryotic glycerol-3-P pathway that uses a palmitoyl-ACP to produce phosphatidic acid with a prokaryotic-type acyl composition. The homologies between the deduced protein sequence of BAT2 with prokaryotic and eukaryotic microsomal LAP acytransferases suggest that seed microsomal forms may have evolved from the plastidial enzyme.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗
PubMed1999

Ventricular fibrillation secondary to ergotamine in a healthy young woman.

A 34-year-old woman collapsed secondary to ventricular fibrillation 3 hours following the ingestion of ergotamine tartrate for migraine. She underwent defibrillation and recovered rapidly without any subsequent consequences. The mechanism of action and the side effects of ergotamine and other antimigraine drugs are discussed. We hypothesize that a coronary spasm induced by ergotamine could be the aetiologic factor leading to ischaemic ventricular fibrillation.

Adult↗