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Biomedical subjects

I Gaillard

Publications and source records attributed to I Gaillard.

15 recordsLinked to original sources
PubMed2004

Olfactory receptors.

Olfaction is an ancient sensory system allowing an organism to detect chemicals in its environment. The first step in odor transduction is mediated by binding odorants to olfactory receptors (ORs) which belong to the heptahelical G-protein-coupled receptor (GPCR) superfamily. Mammalian ORs are disposed in clusters on virtually all chromosomes. They are encoded by the largest multigene family (approximately 1000 members) in the genome of mammals and Caenorhabditis elegans, whereas Drosophila contains only 60 genes. Each OR specifically recognizes a set of odorous molecules that share common molecular features. In mammals, signal transduces through the G-protein-dependent signal pathway in the olfactory sensory neurons that synapse ultimately in the glomeruli of the olfactory bulb, and is finally processed in higher brain structures. The expression of a given OR conditions neuron and glomerulus choices. To date, the processes which monitor OR expression and axon wiring have emerged but are not completely elucidated.

Animals↗
PubMed2004

Efficiency of in vivo purging with rituximab prior to autologous peripheral blood progenitor cell transplantation in B-cell non-Hodgkin's lymphoma: a single institution study.

BACKGROUND: Rituximab induces clinical response in advanced B-cell lymphoma and is efficient in removing circulating B-cell from peripheral blood. We therefore postulated that rituximab might be a useful in vivo purging agent before high-dose therapy in this setting. PATIENTS AND METHODS: Fourteen patients with relapsed follicular, marginal zone and mantle cell lymphomas (11, two and one cases, respectively) and a PCR-detectable molecular marker were treated first with rituximab, then a mobilization chemotherapeutic regimen, followed by high-dose therapy with peripheral blood stem cell transplantation. PCR analyses were performed in peripheral blood before rituximab and during follow-up, and in harvest. RESULTS: Harvests were free of PCR-detectable molecular marker in nine of the 11 studied cases (82%). After high-dose therapy, clinical complete remission was obtained in 13 (93%) patients and molecular remission in 11 (79%). With a median follow-up of 3 years, the 14 transplanted patients were alive, 11 of them remaining in clinical complete remission and eight in molecular remission at last follow-up. CONCLUSION: Rituximab treatment followed by high dose therapy appears to be effective in achieving complete clinical and molecular response. In vivo harvest purging is predictive of prolonged clinical and molecular remission.

Adult↗
PubMed2004

A rapid method for determining the antimicrobial activity of novel natural molecules.

Preservatives used in the Agro-food industries may be of natural origin or obtained chemically. Because of the increasing interest of consumers in food products that contain only natural ingredients, studies on preservative molecules of natural origin, such as organic acids or peptides, have been reported in the past several years. Such studies, which require numerous assays, may be limited by the large amount of molecules required. Microscale assays provide an opportunity for testing natural components available in low quantity. This study examined a rapid method that used microplates for the evaluation of anti-microbial substances. The method was validated using five foodborne pathogens. It required a low amount of product and was convenient for the determination of correlations between the bacterial growth inhibition and concentration of the antimicrobial substance.

Anti-Bacterial Agents↗
PubMed2000

Adrenocorticotrophic hormone stimulates phosphotyrosine phosphatase SHP2 in bovine adrenocortical cells: phosphorylation and activation by cAMP-dependent protein kinase.

During activation of adrenocortical cells by adrenocorticotrophic hormone (ACTH), tyrosine dephosphorylation of paxillin is stimulated and this correlates with protrusion of filopodial structures and a decreased number of focal adhesions. These effects are inhibited by Na(3)VO(4), a phosphotyrosine phosphatase inhibitor [Vilgrain, Chinn, Gaillard, Chambaz and Feige (1998) Biochem. J. 332, 533-540]. However, the tyrosine phosphatases involved in these processes remain to be identified. In this study, we provide evidence that the Src homology domain (SH)2-containing phosphotyrosine phosphatase (SHP)2, but not SHP1, is expressed in adrenocortical cells and is phosphorylated upon ACTH challenge. ACTH (10(-8) M) treatment of (32)P-labelled adrenocortical cells resulted in an increase in phosphorylated SHP2. By probing SHP2-containing immunoprecipitates with an antibody to phosphoserine we found that SHP2 was phosphorylated on serine in ACTH-treated cells in a dose- and time-dependent manner. Furthermore, using an in vitro kinase assay, we showed that SHP2 was a target for cAMP-dependent protein kinase (PKA). Serine was identified as the only target amino acid phosphorylated in SHP2. Phosphorylation of SHP2 by PKA resulted in a dramatic stimulation of phosphatase activity measured either with insulin receptor substrate-1 or with the synthetic peptide [(32)P]poly(Glu/Tyr) as substrate. In an in-gel assay of SHP2-containing immunoprecipitates, phosphorylated in vitro by PKA or isolated from adrenocortical cells treated with 10 nM ACTH, a pronounced activation of SHP2 activity was shown. These observations clearly support the idea that a PKA-mediated signal transduction pathway contributes to SHP2 regulation in adrenocortical cells and point to SHP2 as a possible mediator of the effects of ACTH.

Adrenal Cortex↗
PubMed2000

ACTH-regulated expression of vascular endothelial growth factor in the adult bovine adrenal cortex: a possible role in the maintenance of the microvasculature.

Endothelial cells lining vessels of endocrine tissues are fenestrated. Interactions with the local environment via either soluble factors or cell-cell interactions appear to govern this terminal endothelial differentiation. Adrenocorticotropin (ACTH) has previously been reported to modulate endothelial fenestration in the rat adrenal cortex. Since vascular endothelial growth factor (VEGF) has been characterized as a potent inducer of endothelial fenestration, we aimed to characterize the status of VEGF expression in the bovine adult adrenal cortex and asked whether ACTH may regulate VEGF expression. By immunohistochemical analysis, we observed VEGF expression in steroidogenic cells from both zona glomerulosa and zona fasciculata of the bovine adrenal cortex. Double-labeling experiments performed on isolated cells in primary culture revealed VEGF immunoreactivity, essentially colocalized with the Golgi apparatus. The expression of two predominant VEGF isoforms, VEGF(121) and VEGF(165), was observed by RT-PCR analysis. ACTH (10 nM) was found to rapidly (within 2-4 h) increase the abundance of these VEGF transcripts, as assessed by both RT-PCR and Northern blot analysis. In parallel, ACTH significantly induced VEGF secretion into the medium of fasciculata cells in primary culture. Thus, our data are consistent with the involvement of ACTH, through its regulation of VEGF expression, in the maintenance of the adult adrenal cortex endothelium.

Adrenal Cortex↗
PubMed1999

[Interactions between steroidogenic cells and capillary endothelial cells in the adrenal gland].

Adrenocortical capillary endothelial (ACE) cells showed a two- to three-fold increase in number when they were cocultured with bovine adrenocortical (BAC) cells from the zona fasciculata-reticularis. This effect was detectable within 48 h, persisted throughout the seven-day coculture period, and occurred in the absence of addition of exogenous growth factors. It was similar to that obtained by addition of 1 ng/ml of FGF-2. Adrenal cortex tumor cells from humans (NCI) and mice (Y1) also stimulated ACE cell growth, whereas NIH 3T3 cells did not, suggesting an effect specific of steroid-producing cells. Addition of an FGF-2-neutralizing antibody failed to inhibit the BAC cell-induced ACE cell growth stimulation, indicating that FGF-2 was not involved. BAC cell-conditioned medium stimulated ACE cell growth, indicating a role for a diffusible factor. When BAC cells were cocultured with ACE cells in a 3D collagen gel, capillary-like tubes developed, consistent with secretion by BAC cells of angiogenic factors. Studies are under way to identify these factors.

3T3 Cells↗
PubMed1998

Hormonal regulation of focal adhesions in bovine adrenocortical cells: induction of paxillin dephosphorylation by adrenocorticotropic hormone.

A study of bovine adrenocortical cell shape on adrenocorticotropic hormone (ACTH) challenge showed that the cells round up and develop arborized processes. This effect was found to be (1) specific for ACTH because angiotensin II and basic fibroblast growth factor have no effect; (2) mediated by a cAMP-dependent pathway because forskolin reproduces the effect of the hormone; (3) inhibited by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, but unchanged by okadaic acid, a serine/threonine phosphatase inhibitor; and (4) correlated with a complete loss of focal adhesions. Biochemical studies of the focal-adhesion-associated proteins showed that pp125fak, vinculin (110 kDa) and paxillin (70 kDa) were detected in the Triton X-100-insoluble fraction from adrenocortical cells. During cell adhesion on fibronectin as substratum, two major phosphotyrosine-containing proteins of molecular masses 125 and 68 kDa were immunodetected in the same fraction. A dramatic decrease in the extent of tyrosine phosphorylation of these proteins was observed within 60 min after treatment with ACTH. No change in pp125fak tyrosine phosphorylation nor in Src activity was detected. In contrast, paxillin was found to be tyrosine-dephosphorylated in a time-dependent manner in ACTH-treated cells. Sodium orthovanadate completely prevented the effect of ACTH. These observations suggest a possible role for phosphotyrosine phosphatases in hormone-dependent cellular regulatory processes.

Adrenal Cortex↗
PubMed1996

Purification and reconstitution of the glutamate carrier GltT of the thermophilic bacterium Bacillus stearothermophilus.

An affinity tag consisting of six adjacent histidine residues followed by an enterokinase cleavage site was genetically engineered at the N-terminus of the glutamate transport protein GltT of the thermophilic bacterium Bacillus stearothermophilus. The fusion protein was expressed in Escherichia coli and shown to transport glutamate. The highest levels of expression were observed in E. coli strain DH5 alpha grown on rich medium. The protein could be purified in a single step by Ni2+-NTA affinity chromatography after solubilization of the cytoplasmic membranes with the detergent Triton X100. Purified GltT was reconstituted in an active state in liposomes prepared from E. coli phospholipids. The protein was reconstituted in detergent-treated preformed liposomes, followed by removal of the detergent with polystyrene beads. Active reconstitution was realized with a wide range of Triton X100 concentrations. Neither the presence of glycerol, phospholipids, nor substrates of the transporter was necessary during the purification and reconstitution procedure to keep the enzyme in an active state. In B. stearothermophilus, GltT translocates glutamate in symport with protons or sodium ions. In membrane vesicles derived from E. coli cells expressing GltT, the Na+ ion dependency seems to be lost [Tolner, B., Ubbink-Kok, T., Poolman, B., & Konings, W. N. (1995) Mol. Microbiol. 18, 123-133], suggesting a role for the lipid environment in the cation specificity. In agreement with the last observation, glutamate transport catalyzed by purified GltT reconstituted in E. coli phospholipid is driven by an electrochemical gradient of H+ but not of Na+.

ATP-Binding Cassette Transporters↗
PubMed1994

Enhancement of reactive oxygen species production and cell surface markers expression due to haemodialysis.

Leukocyte activation during haemodialysis (HD) was evaluated by reactive oxygen species (ROS) production and cell surface markers expression (CD11b: C3bi receptor and CD25: IL2 receptor). Eight end-stage renal disease patients were exposed to three dialysis phases according to a A/B/A protocol study. During phase A polysulphone (PS) membranes were used and during phase B cuprophane (CU) membranes were used. Each phase lasted 3 weeks. Timed samples were collected during the last session of each phase at 0, 15, and 30 min of HD. Flow cytometry analysis was performed both on monocytes (MO), polymorphonuclears (PMN), and lymphocytes (Ly). Hydroethydine was used as a marker of cell-ROS production. Specific monoclonal antibodies were used to analyse the cell surface markers. CU increased ROS production in PMN and MO by 10- and 2.4-fold respectively and had no significant effect on Ly. CU enhanced 16-fold CD11b expression on PMN, and increased also CD11b and CD25 expression on MO by 7- and 40-fold respectively. On the contrary, PS did not affect either ROS production or cell surface markers expression in MO, PMN, Ly. We conclude that oxydative metabolism and cell surface markers expression of PMN and MO were significantly increased with cuprophane membranes and not with polysulphone membranes, suggesting that complex cell-cell interactions were involved in membrane-related bioincompatibility phenomena.

Aged↗
PubMed1989

Structure of human angiotensinogen gene.

A cDNA clone encoding human angiotensinogen was isolated from a cDNA library prepared from human liver mRNA and used to isolate the angiotensinogen gene. The complete exon sequence of this gene together with extensive intron and flanking sequences are reported. The human angiotensinogen gene contains five exons interrupted by four intervening sequences. We compared the intron-exon structure of this human gene with that of the rat gene or the genes coding for proteins such as alpha 1-antitrypsin and antithrombin III, whose primary amino acid sequences show similarities. The human angiotensinogen gene shows identical organization with the alpha 1-antitrypsin gene, but is different from the antithrombin III gene. The 5'-flanking sequence (-500 to -1 bp) of the human angiotensinogen gene was examined for hormone regulatory elements (HRE), which may be implicated in the interaction with the hormone receptor complexes.

Amino Acid Sequence↗
PubMed1989

Regulation of angiotensinogen gene.

The development of recombinant DNA technology has introduced new directions for the study of the angiotensinogen molecule. The cloning and sequencing of the human and rat cDNAs demonstrate the similarity of angiotensinogen to various serine protease inhibitors produced by the liver and was the beginning of studies looking for new physiological roles of angiotensinogen, in addition to the substrate for renin. The determination of the nucleotide sequence of these cDNAs also allowed the identification of angiotensinogen mRNA in many tissues in addition to the liver that is the major site of synthesis. This multilocalization of angiotensinogen is one of the arguments for the presence and the function of local renin-angiotensin systems. Finally, the hepatic biosynthesis of angiotensinogen is regulated by many different hormonal factors including glucocorticoid, estrogen, thyroid hormone, insulin, and angiotensin II. The cloning of the angiotensinogen gene offers the opportunity to study this regulation at the transcriptional level. We present in this paper a review of the literature concerning the new aspects of angiotensinogen using molecular biological tools and its regulation together with the characterization of the human angiotensinogen gene.

Amino Acid Sequence↗
PubMed1989

Molecular characterization of antithrombin III (ATIII) variants using polymerase chain reaction. Identification of the ATIII Charleville as an Ala 384 Pro mutation.

The genes of seven structural mutants of antithrombin III (ATIII), presenting either defective serine protease reactivity or abnormal heparin binding, were analyzed. The polymerase chain reaction (PCR) was used to amplify the corresponding gene exon and the mutation was identified by either dot blot analysis using a battery of allele-specific oligonucleotide probes or sequencing. Variants Paris and Paris 2 were identified as Arg 47 Cys mutations, and Clichy, Clichy 2, and Franconville were found to be Pro 41 Leu mutations. All five are heparin binding-site variants. ATIII Avranches is an Arg 393 His mutation and ATIII Charleville is an Ala 384 Pro mutation. These two mutations impair the reactive site of the molecule. ATIII Charleville is a new mutation of the reactive center, as predicted by previous biochemical data. The position of this new mutation, together with the other previously described mutations of the reactive center, sheds light on the molecular function of this site in inhibiting thrombin. Finally, genomic amplification by PCR is a powerful technique for the fast identification of antithrombin III mutations and their homozygous/heterozygous status, and should be useful for predicting thrombotic risk.

Adult↗
PubMed1988

Expression and characterization of recombinant human angiotensinogen in a heterologous eukaryotic cell line.

Transfection of Chinese hamster ovary cells with an expression plasmid containing a full length human angiotensinogen cDNA has provided cell lines that secrete recombinant angiotensinogen in large quantities. This angiotensinogen is immunologically identical to plasma angiotensinogen and can be cleaved by human kidney renin (EC 3.4.23.15.). The peptide liberated by renin cleavage is immunologically identical to standard angiotensin I and shows a retention time on isocratic reversed-phase high-pressure liquid chromatography identical to that of standard angiotensin I. The heterogeneity of recombinant angiotensinogen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis differs from that of plasma angiotensinogen. Treatment with endoglycosidases demonstrated that this difference is restricted to that of N-glycans and that N-glycans correspond to the quasi-totality of the carbohydrate content of both recombinant and plasma angiotensinogens. The development of a system capable of expressing human angiotensinogen cDNA in mammalian cells and the ability to obtain the corresponding angiotensinogen in large quantities will allow new studies on structure-function relationships of this protein.

Angiotensinogen↗
PubMed1986

Analysis of inactive renin by renin profragment monoclonal antibodies.

Two peptides were synthesized, corresponding to the sequences (-19 to -7) and (-26 to -17) of the prorenin prosegment. Monoclonal antibodies were raised to these sequences and used to characterize human plasma inactive renin. Only anti (-19 to -7) reacted with inactive renin, as measured by direct assay or affinity chromatography. The data were used to evaluate two possible inactive renin stuctures: plasma inactive renin is a truncated prorenin lacking the prosegment N-terminal portion; its spatial conformation masks the N-terminal extremity, preventing interaction of this region with specific antibodies.

Antibodies, Monoclonal↗