Biomedical subjects

Claude Villard

Publications and source records attributed to Claude Villard.

9 recordsLinked to original sources

PubMed2006

Proteomic characterization of lipid rafts markers from the rat intestinal brush border.

To assess intestinal lipid rafts functions through the characterization of their protein markers, we have isolated lipid rafts of rat mucosa either from the total membrane or purified brush-border membrane (BBM) by sucrose gradient fractionation after detergent treatment. In both membrane preparations, the floating fractions (4-5) were enriched in cholesterol, ganglioside GM1, and N aminopeptidase (NAP) known as intestinal lipid rafts markers. Based on MALDI-TOF/MS identification and simultaneous detection by immunoblotting, 12 proteins from BBM cleared from contaminants were selected as rafts markers. These proteins include several signaling/trafficking proteins belonging to the G protein family and the annexins as well as GPI-anchored proteins. Remarkably GP2, previously described as the pancreatic granule GPI-anchored protein, was found in intestinal lipid rafts. The proteomic strategy assayed on the intestine leads to the characterization of known (NAP, alkaline phosphatase, dipeptidyl aminopeptidase, annexin II, and galectin-4) and new (GP2, annexin IV, XIIIb, Galpha(q), Galpha(11), glutamate receptor, and GPCR 7) lipid rafts markers. Together our results indicate that some digestive enzymes, trafficking and signaling proteins may be functionally distributed in the intestine lipid rafts.

Animals↗

PubMed2005

A fast, sensitive method for the simultaneous determination of alpha-tocopherol and alpha-tocopheryl acetate in mixed micelles.

This report improves analytical procedures to investigate the behaviour of the two Vitamin E forms, alpha-tocopherol (Tol) and alpha-tocopheryl acetate (Tac), in model systems mimicking the intestinal medium. We describe how to prepare mixed micelles as vehicle for Tac and Tol and the HPLC method for their quantification in the micelles. Tac and Tol were extracted using ethanol-hexane-drying procedure, whereas the separation and detection were performed in methanol and by UV method, respectively. Both compounds were eluted in less than 4 min. In the range between 1.7 microM and 54 microM of Tac or Tol in the micelles, their recovery were 89% and 81%, respectively, with correlation coefficient over 0.99 and R.S.D. of less than 7.2% in all cases. Limits of detection and quantification for Tac and Tol in mixed micelles ranged between 1 microM and 2 microM and between 3 microM and 5 microM, respectively. The behaviours of Tac and Tol were quite different during the extraction procedure and both were influenced by the vitamin concentration and the relative volume of organic solvents.

Chromatography, High Pressure Liquid↗

PubMed2005

Ornithine decarboxylase activity is inhibited by the polyamine precursor amino acids at the protein stability level in Caco-2 cells.

High concentrations of certain amino acids are known to affect hormonal secretion, immune function, electrolyte balance or metabolic functions. However, there is a lack of knowledge regarding the molecular mechanisms responsible for these effects. We showed that, as well as spermidine transport, the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis, is decreased in human colon adenocarcinoma cells, Caco-2, following a 4-h supplementation with one of the two polyamine precursor amino acids, L-arginine or L-methionine. Dose-response assays indicated that the inhibitory effect of supplemental L-methionine was stronger than that of supplemental L-arginine. However, it was transient, being even replaced by ODC induction after 8 h, whereas the inhibitory effect of L-arginine lasted for at least 8 h. Unlike L-cysteine, neither L-methionine nor L-arginine could inhibit ODC activity in a crude acellular preparation of the enzyme. The inhibition of ODC activity in cells exposed to L-methionine or L-arginine was due to a decreased abundance of ODC protein without change at the mRNA level and each of these amino acids could counteract ODC induction by a glycine supplement. Contrary to the latter, supplemental L-methionine or L-arginine induced a marked decrease in ODC half-life, concomitantly with an increase in the activity of antizyme, an ODC inhibitory protein. Thus, depending on their nature, amino acids can up- or downregulate ODC activity at the protein stability level.

Arginine↗

PubMed2005

The eefABC multidrug efflux pump operon is repressed by H-NS in Enterobacter aerogenes.

The Enterobacter aerogenes eefABC locus, which encodes a tripartite efflux pump, was cloned by complementation of an Escherichia coli tolC mutant. E. aerogenes deltaacrA expressing EefABC became less susceptible to a wide range of antibiotics. Data from eef::lacZ fusions showed that eefABC was not transcribed in the various laboratory conditions tested. However, increased transcription from Peef was observed in an E. coli hns mutant. In addition, EefA was detected in E. aerogenes expressing a dominant negative E. coli hns allele.

Bacterial Outer Membrane Proteins↗

PubMed2004

The inhibition specificity of recombinant Penicillium funiculosum xylanase B towards wheat proteinaceous inhibitors.

The filamentous fungus Penicillium funiculosum produces a mixture of modular and non-modular xylanases belonging to different glycoside hydrolase (GH) families. In the present study, we heterologously expressed the cDNA encoding GH11 xylanase B (XYNB) and studied the enzymatic properties of the recombinant enzyme. Expression in Escherichia coli led to the partial purification of a glutathione fusion protein from the soluble fraction whereas the recombinant protein produced in Pichia pastoris was successfully purified using a one-step chromatography. Despite O-glycosylation heterogeneity, the purified enzyme efficiently degraded low viscosity xylan [K(m)=40+/-3 g l(-1), V(max)=16.1+/-0.8 micromol xylose min(-1) and k(cat)=5405+/-150 s(-1) at pH 4.2 and 45 degrees C] and medium viscosity xylan [K(m)=34.5+/-3.2 g l(-1), V(max)=14.9+/-1.0 micromol xylose min(-1)k(cat)=4966+/-333 s(-1) at pH 4.2 and 45 degrees C]. XYNB was further tested for its ability to interact with wheat xylanase inhibitors. The xylanase activity of XYNB produced in P. pastoris was strongly inhibited by both XIP-I and TAXI-I in a competitive manner, with a K(i) of 89.7+/-8.5 and 2.9+/-0.3 nM, respectively, whereas no inhibition was detected with TAXI-II. Physical interaction of both TAXI-I and XIP-I with XYNB was observed using titration curves across a pH range 3-9.

Cloning, Molecular↗

PubMed2003

Rat kidney acylase I: further characterisation and mutation studies on the involvement of Glu 147 in the catalytic process.

Rat kidney acylase I was characterised by performing site-directed mutagenesis and enzymatic analysis in the presence of various chemical inhibitors. Site-directed mutagenesis on E147 and overexpression of the protein in a bacterial system, revealed the importance of this residue in enzymatic activity, it corresponds to the putative catalytic E175 in carboxypeptidase G2 from Pseudomonas aeruginosa. The reactivity of histidine and cysteine residues of acylase I with diethylpyrocarbonate (DEPC) and mercuric chloride, respectively, showed that these two amino acids are required for the enzyme to be fully active. Interestingly, the effects of mercuric chloride on rat kidney acylase I were not as great as those on the porcine enzyme, in agreement with previously observed differences between the two enzymes. Moreover, N-[3-(2-furyl)-acryloyl-L-methionine] (FA-Met) a synthetic substrate of the porcine acylase I was found to be an inhibitor of the rat kidney enzyme. These results strongly suggest the existence of differences between the active site of rat and porcine kidney acylases I. Lastly, the rat kidney enzyme was as sensitive as its porcine counterpart to two metal chelating agents, 1,10-phenanthroline and ethylenediamine tetraacetate (EDTA).

Amidohydrolases↗

PubMed2003

Structural properties of porcine intestine acylpeptide hydrolase.

The acylpeptide hydrolase of porcine intestinal mucosa (pi-APH) is a serine peptidase belonging to the prolyl oligopeptidase family. The enzyme catalyzes the release of N-terminal acylamino acids, especially acetylamino acids, from acetylpeptides. pi-APH is an homotetramer of approximately 300 kDa. We report the loss of the native tetrameric structure of pi-APH upon citraconylation and the process was reversed at acidic pH, indicating that the subunits were noncovalently bound. Determination of free cysteines in combination with peptide mapping suggested the involvement of all cysteines in disulfide bridges. Two structural domains were identified based on the three-dimensional model of pi-APH monomer: a beta-propeller fold in the N-terminal sequence (113-455) and an alpha/beta hydrolase fold corresponding to the C-terminal catalytic domain (469-732). Preferential cleavage sites for limited proteolysis with trypsin occurred within the beta-propeller domain, in agreement with the three-dimensional model. The putative role of this domain in the specificity mechanism of APH enzymes is also discussed.

Amino Acid Sequence↗

PubMed2003

Development of the primary root and mobilisation of reserves in etiolated seedlings of Brassica napus grown on a slowly rotating clinostat.

The effect of the slow rotating clinostat (1 rpm) on the growth of the primary root was studied on Brassica napus seedlings. After 5 d in darkness, the primary root was longer and thinner in seedlings grown on the clinostat than in seedlings grown in the vertical position. However, the breakdown of lipid reserves, sucrose level and transport of 14C-labeled sucrose from the cotyledons to the primary root, were not altered by growth on the clinostat. Moreover, the activity of isocitrate lyase, one of the two enzymes necessary for the conversion of lipids into glucids also was also not modified in the cotyledons of clinorotated seedlings. Thus, there was clear evidence that clinorotation had a direct effect on the growth of the primary root that was independent of the mobilisation of lipid reserves in the cotyledons. As a sink, the primary root had the same strength on the clinostat as in the vertical position, but the reserves were used in a different way. The increase in root elongation on the clinostat could be due to the slight, but continuous, omnilateral gravitropic stimulation due to the rotation of the seedlings about a horizontal axis.

Brassica↗

PubMed2002

Cel9M, a new family 9 cellulase of the Clostridium cellulolyticum cellulosome.

A new cellulosomal protein from Clostridium cellulolyticum Cel9M was characterized. The protein contains a catalytic domain belonging to family 9 and a dockerin domain. Cel9M is active on carboxymethyl cellulose, and the hydrolysis of this substrate is accompanied by a decrease in viscosity. Cel9M has a slight, albeit significant, activity on both Avicel and bacterial microcrystalline cellulose, and the main soluble sugar released is cellotetraose. Saccharification of bacterial microcrystalline cellulose by Cel9M in association with two other family 9 enzymes from C. cellulolyticum, namely, Cel9E and Cel9G, was measured, and it was found that Cel9M acts synergistically with Cel9E. Complexation of Cel9M with the mini-CipC1 containing the cellulose binding domain, the X2 domain, and the first cohesin domain of the scaffoldin CipC of the bacterium did not significantly increase the hydrolysis of Avicel and bacterial microcrystalline cellulose.

Amino Acid Sequence↗