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Biomedical subjects

F Gautier

Publications and source records attributed to F Gautier.

At least 19 recordsLinked to original sources
PubMed2005

Effect of tepoxalin on renal function in healthy dogs receiving an angiotensin-converting enzyme inhibitor.

The objective of this study was to investigate renal function in clinically normal dogs receiving tepoxalin, a nonsteroidal inflammatory drug, either in association with or without an angiotensin-converting enzyme inhibitor (ACEI). Ten adult female Beagle dogs were used in the three phases of the study. The dogs were administered the drugs once daily for 7 days (experiment 1: placebo/tepoxalin/tepoxalin and benazepril; experiment 2: enalapril/tepoxalin and enalapril) or for 28 days (experiment 3: tepoxalin and benazepril together). Renal function was assessed by measurement of glomerular filtration rate (GFR) by renal scintigraphy [(renal uptake of 99mTc-diethylenetriaminepentacetic acid (DTPA)] and plasma clearance of 99mTc-DTPA. Compared with the placebo group, renal uptake and plasma clearance of 99mTc-DTPA were not significantly modified after a 7-day period of treatment with tepoxalin or enalapril alone, tepoxalin and benazepril or tepoxalin and enalapril together. No significant change was obtained in GFR after a 28-day period of dosing with tepoxalin and benazepril together. Therefore, it was concluded that tepoxalin did not alter renal function in healthy Beagle dogs receiving ACEI.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2000

Identification of an apoptotic cleavage product of BARD1 as an autoantigen: a potential factor in the antitumoral response mediated by apoptotic bodies.

We have shown previously that rats can be cured from induced peritoneal colon carcinomatosis by injections of apoptotic bodies derived from tumor cells and interleukin 2. This curative treatment generated a tumor-specific cytotoxic T-cell response associated with a humoral response. Autoantibodies from sera of cured rats strongly recognized a Mr 67,000 protein from apoptotic bodies and weakly reacted with a protein of Mr approximately 97,000 in PROb parental cells. We now show that these autoantibodies are directed against BARD1, originally identified as a protein interacting with the product of the breast cancer gene 1, BRCA1. We demonstrate that the Mr 67,000 antigen is a cleaved form of BARD1 present in apoptotic bodies derived from rat and human colon and mammary carcinoma cell lines. Moreover, we show that the cleavage site of BARD1 is located NH2 terminally but downstream of the RING domain essential for BARD1 and BRCA1 protein interaction. In vitro studies using [35S]methionine-labeled human BARD1 and apoptotic cellular extracts derived from SW48 carcinoma cells indicate that BARD1 proteolysis occurs at an early stage of apoptosis and in a cell cycle-dependent manner. This hydrolysis is inhibited by EGTA, and the calpain inhibitor I, N-acetyl-leu-leu-norleucinal, but not by several caspases inhibitors, suggesting that BARD1 is hydrolyzed by the calcium-dependent cysteine proteases, calpains. Thus, the highly immunogenic form of cleaved BARD1 could contribute to the antitumoral response mediated by apoptotic bodies.

Amino Acid Sequence↗
PubMed1999

Production and characterisation of a monoclonal antibody specific for apoptotic bodies derived from several tumour cell lines.

We have recently shown that apoptotic bodies (apobodies) derived from rat colon carcinoma cell lines (PROb) after sodium butyrate (NaB) treatment were able to cure rats with induced peritoneal carcinomatosis ( [BOISTEAU] ). A specific immune response was assumed to be involved since the serum of cured rats contained antibodies specific for apobodies. In the present study, a mAb (clone 6E8) produced by immunisation of rats with apobodies strongly recognized apobodies but had little reactivity with parental tumour cell lines, as demonstrated by enzyme-linked immunosorbent assay (ELISA), immunostaining and flow cytometry. Immunoelectron microscopy showed that 6E8 mAb mainly stained the hyaloplasm or cytosol of apobodies. A protein was detected at 67 kDa by immunoprecipitation of apobodies with mAb, followed by immunoblotting, using serum of rats immunised with apobodies. The 6E8 mAb recognized apobodies derived from several rat or human colon cancer cell lines and a rat glioma cell line, regardless of the apoptosis stimulus used (NaB, staurosporine or UV). Our results clearly show that 6E8 mAb defines an epitope specifically generated during apoptosis, which suggests that the protein recognized may be involved in the molecular cascade of apoptotic cell death.

Animals↗
PubMed1999

Expression of collagens type I and IV, osteonectin and transforming growth factor beta-1 (TGFbeta1) in biliary atresia and paucity of intrahepatic bile ducts during infancy.

BACKGROUND/AIMS: Biliary atresia and paucity of intrahepatic bile ducts are the main causes of neonatal cholestasis leading to hepatic fibrosis. Fibrotic evolution is slow in paucity of bile ducts as compared to the rapid progression to biliary cirrhosis in biliary atresia when cholestasis persists despite hepatoportoenterostomy. Our aim was to compare the expression of collagens type I and IV, alpha-smooth muscle actin, osteonectin and transforming growth factor beta1 in biliary atresia and paucity of bile ducts. METHODS: Liver biopsies were obtained in 12 children with biliary atresia and in five with paucity of bile ducts. Collagens type I and IV, alpha-smooth muscle actin were detected with immunostaining. Collagens type I and IV, osteonectin and transforming growth factor beta1 mRNAs were detected by in situ hybridization. RESULTS: Expression of mRNA and proteins was roughly parallel. In ductular proliferation areas of biliary atresia: (1) the expression of collagens type I and IV and osteonectin was increased, and was localized to periductular myofibroblasts; (2) transforming growth factor beta1 was expressed around biliary ductules, probably in inflammatory cells, and also in biliary cells. Osteonectin expression was also increased in the lobules. In paucity of bile ducts, there was no overexpression of collagens type I and IV and transforming growth factor beta1, except in the only child with marked fibrosis. However, osteonectin expression was enhanced at the periphery of the lobules, even when fibrosis was mild or absent. CONCLUSIONS: These findings suggest that in biliary atresia ductular proliferation areas are the site of a marked production of extracellular matrix proteins in periductular myofibroblasts, probably secondary to transforming growth factor beta1 production by inflammatory cells and by biliary cells. The weak expression of transforming growth factor beta1 could explain the slow progression of fibrosis in paucity of bile ducts.

Actins↗
PubMed1998

High-level secretion of a wheat lipid transfer protein in Pichia pastoris.

Plant nonspecific lipid transfer proteins are small basic proteins with eight cysteine residues, all engaged in disulfide bonds. The sequence encoding the wheat 9-kDa LTP was cloned into the secretion vector pYAM7SP8 giving rise to pYTdltp4.90. Production in shake-flasks and a fermentor led to the synthesis of two major species of LTP: a larger than expected species of 14 kDa and a species of 10 kDa, close to the expected size of wheat LTP. When production was carried out in a fermentor with regulation of pH, oxygen level, and feed rate of carbon source, the 10-kDa species was the main protein at the end-point of culture. The recombinant wheat LTP (rLTP), secreted at a level of 720 mg/liter into the culture medium, is soluble. The rLTP was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography, with a recovery yield of 36%. However, the molecular mass of rLTP, determined by mass spectrometry, is 9996 Da, while its naturally occurring counterpart has a molecular mass of 9607 Da. This discrepancy in size corresponds to a protein carrying three extra amino acids (DKR) at its N-terminal end, and this was confirmed by sequencing. In vitro lipid transfer activity showed that rLTP behaves in a similar way to the naturally occurring protein. These data indicate that Pichia pastoris is an efficient system for production of large quantities of soluble and biologically active rLTP for structure/function analysis.

Amino Acid Sequence↗
PubMed1997

Apoptosis induced by sodium butyrate treatment increases immunogenicity of a rat colon tumor cell line.

We have recently demonstrated that a treatment combining the cell differentiating agent sodium butyrate (NaBut) and interleukin-2 (IL2) resulted in a remission of established peritoneal colorectal carcinomatosis in rats. NaBut or IL2 treatment alone, never cured these tumour-bearing rats. In the present investigation, we report that NaBut-treatments induce apoptosis in the colonic cancer cells both in vitro and in vivo. We postulated that the significant therapeutic effect of NaBut/IL2 treatment can be mainly attributed to a NaBut-induced apoptosis of the tumoural cells increasing their immunogenicity. Indeed, treatment which combined apoptotic bodies (apobodies) as cell vaccine, plus IL2 immunotherapy significantly increased tumour remission and survival rate of the vaccinated rats, whereas IL2 treatment alone did not. We observed that the cured rats presented long-term protection against subsequent challenge with the parental tumour cells. This latter result suggests that these treatments generate an immune protection. This was confirmed by the presence, in the sera of the cured rats, of anti-tumoural antibodies directed against both the apobodies and the tumour cells, but not against normal colonocytes. In addition, we show that injections of apobodies before administration of the parental tumour cells results in a partial protection. We provide the first evidence that apobodies, derived from cancer cells after NaBut-treatment, induce a specific immune response against parental tumours cells. These data suggest that the distinctive immunologic properties of apobodies could provide a valuable tool in colorectal cancer immunotherapy.

Journal Article↗
PubMed1996

Altered glycosylation of alpha(s)beta 1 integrins from rat colon carcinoma cells decreases their interaction with fibronectin.

Malignant cell transformation is generally accompanied by changes in their interactions with environing matrix proteins in a way to facilitate their migration and generate invasion. Our results show the binding of rat colon adenocarcinoma PROb cells to fibronectin strongly reduced when compared to normal rat intestine epithelial cells. This decrease was not due to the level of alpha(s)beta 1 integrins expressed at the surface of the cell line. However, beta 1- and alpha(s)-associated subunits appeared to be structurally altered as shown by immunoprecipitation followed by electrophoresis. Pulse chase experiments using 35S methionine evidenced differences in the biosynthesis of beta 1- and alpha(s)associated integrins: normal epithelial IEC18 cells required 16 h for maximal biosynthesis of the completely mature beta 1 subunit, while PROb cells did it within 4-6 h. Studies using endoglycosidases O, H, D, and N glycanase confirmed that the molecular weight alterations were due to abnormal glycosylation and suggested that alpha(s)beta 1 integrins of PROb cells could bear both mature complex and immature high mannose types while IEC18 cells borne only mature complex type oligosaccharidic chains. Treatment of both cell types with castanospermine, an inhibitor of N-glycosylation, reduced the differences observed in their adhesion to the fibronectin without significantly affecting beta 1 receptors expression at the cell surface. These results strongly suggest a role of the glycosylation of beta 1 receptors in the adhesion of rat colon adenocarcinoma PROb cells to fibronectin substrata.

Animals↗
PubMed1980

The use of plasmid R1162 and derivatives for gene cloning in the methanol-utilizing Pseudomonas AM1.

A physical map for plasmid R1162 (Sm, Su, IncP4) was constructed. Neither EcoRI, PstI nor EcaI cut within a region essential for replication, molbilization or streptomycin resistence. Plasmid R1162 can replicate in E. coli as well as in Pseudomonas species and shows a strong dependence for DNA polymerase I in E. coli. By RP4 induced mobilization, R1162 can be transferred from E. coli to Pseudomonas AM1. A hybrid plasmid pFG7 (MW=8.4 x 10(6), Sm, Su, Ap, Tc) was constructed between pBR322 and R1162, which allows the selection of hybrid plasmids by insertional inactivation with the restriction enzymes HindIII, BamHI, SalI, ClaI. Transformation of E. coli SK1592 with Ecal-cut and ligated R1162-DNA and Pseudomonas AMI-DNA and subsequent mobilization of the hybrid plasmids into Pseudomonas AM1/M15a (methanol dehydrogenase-) led to the isolation of Pseudomonas AM1/M15a colonies, which could grow on methanol again. Back-conjugation into E. coli SK1592, subsequent mobilization studies and plasmid analysis suggests that the gene for Pseudomonas methanol dehydrogenase has been cloned in this vector.

Cloning, Molecular↗
PubMed1978

The expression of tetracycline resistance after insertion of foreign DNA fragments between the EcoRI and HindIII sites of the plasmid cloning vector pBR 322.

In vitro recombination techniques were used to construct hybrid plasmids between pBR 322 and different DNA fragments derived from pML 21 and the E. coli chromosome. Some of the resulting hybrid plasmids express the tetracycline resistance gene of pBR 322, depending on the DNA fragment which has been ligated into the HindIII-site of pBR 322. From these studies we could conclude the direction of transcription of the kanamycin resistance gene in plasmid pML 21 with respect to the SalI, SmaI, HincII, KpnI, EcoRI and HindIII restriction sites. The replacement of the small HindIII-EcoRI-fragment in pBR 322 by other DNA fragments from the E. coli chromosome and selection for tcr-phenotypes showed that this system may be very useful for screening and analysis of promotor-containing DNA fragments.

DNA, Bacterial↗