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Biomedical subjects

C Breton

Publications and source records attributed to C Breton.

At least 19 recordsLinked to original sources

Ontogenesis and functional aspects of oxytocin and vasopressin gene expression in the thymus network.

Ontogenesis of oxytocin (OT) and vasopressin (VP) gene expression and function were investigated in murine thymus. OT and VP transcripts were detected in the thymus on embryonic days 13 and 15, respectively. Corresponding messenger RNAs were evidenced in thymic epithelial cells by in situ hybridization with a neurophysin probe. From all OT and VP receptors, only OTR was expressed by all T-cell subsets, while V1bR was found in double positive and single positive CD8 cells. In fetal thymic organ cultures, OTR antagonist d[D-Tyr(Et)2, Thr4]OVT increased early apoptosis of CD8 cells, while V1bR antagonist (Sanofi SSR149415) inhibited T-cell differentiation, and favored CD8 T-cell commitment.

Analysis of Variance↗

Leishmaniosis due to Leishmania infantum in a FIV and FelV positive cat with a squamous cell carcinoma diagnosed with histological, serological and isoenzymatic methods.

Leishmaniosis caused by Leishmania infantum is an endemic zoonosis present in the Mediterranean area. Canidae (dog and fox) constitute the main reservoir hosts for the parasite, whilst wild rodents or the cat can be carriers of the protozoan and are considered as secondary potential reservoirs. This paper describes a case of disseminated feline leishmaniosis with cutaneous (ulcerative), visceral (spleen and lymph nodes) and blood involvement in a FIV-FelV positive cat. The microscopic identification of the Leishmania infection was initially made on a skin biopsy of the temporal area, where a squamous cell carcinoma was diagnosed. The diagnosis of the disease was achieved by several serological techniques (ELISA, IFAT and Western-blot). The strain was obtained by blood culture, characterized by electrophoresis of isoenzymes and identified as Leishmania infantum zymodeme MON-1. Since the infection due to L. infantum is a zoonosis, the potential feline reservoir should be more investigated. Serological analysis by Western blot on domestic cats provides a useful tool. In veterinary practice, feline leishmaniosis should be systematically included in the differential diagnosis when compatible cutaneous lesions are present, especially in the endemic areas of canine leishmaniosis.

Animals↗

Cloning, expression and pharmacological characterization of a vasopressin-related receptor in an annelid, the leech Theromyzon tessulatum.

In annelids, it has been established that arginine-vasopressin (AVP)/oxytocin (OT) superfamily peptides are involved in the maintenance of water and electrolyte homeostasis as well as reproduction. At present, there is little information on their receptors. In this study, we report the characterization of a 1.7 kb cDNA for an AVP-related receptor from the leech Theromyzon tessulatum. The open reading frame encodes a 435-aminoacid transmembrane protein that displays seven segments of hydrophobic amino acids, typical of G-protein-coupled receptors. The overall predicted protein exhibits about 30% amino-acid identities to other invertebrate, as well as vertebrate, AVP/OT receptor family members, and displays conserved characteristic features belonging to the AVP/OT receptor superfamily. RT-PCR expression experiments showed that mRNA is expressed in the genital tract, the ovary and the brain. The receptor expression is stage specific, showing a weak expression after the two first blood meals, increasing dramatically after the last blood meal during the period of sexual maturation and disappearing after egg laying. Thus, the leech AVP-related receptor may mediate reproductive functions. When expressed in COS-7 cells, the receptor binds ligands with the following rank order of potency: AVP= Arg-vasotocin >Arg-conopressin >mesotocin = OT = Lys-conopressin=isotocin>annetocin. This shows an AVP-like pharmacological profile. The transfected receptor mediates AVP-induced accumulation of inositol phosphates, indicating that the leech AVP-related receptor is functional. This study describes the characterization of a novel AVP/OT superfamily receptor in annelids, which are considered the most distant group of coelomate metazoans possessing a functional AVP/OT-related endocrine system.

Amino Acid Sequence↗

Neurohypophysial receptor gene expression by thymic T cell subsets and thymic T cell lymphoma cell lines.

Neurohypophysial oxytocin (OT) and vasopressin (VP) genes are transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

Animals↗

The use of molecular markers for germplasm management in a French olive collection.

With more than 100 accessions, the CBNMP olive collection includes a major part of the French germplasm. We used molecular markers to characterise all accessions and to study genetic relationships between cultivars. Firstly, 497 olive trees were genotyped using 32 RAPD markers. We identified 114 RAPD profiles and detected several cases of mislabelling, synonymy and homonymy. Secondly, for each RAPD profile, one tree was analysed using mtDNA RFLPs to determine the cytoplasmic lineage of each cultivar and using five nuclear SSR loci. French germplasm displayed ME1, MOM and MCK mitotypes with ME1 prevailing (84%). Based on SSR markers, we revealed a slight differentiation between French cultivars growing in the West and the East side of the Rhône Valley. This study allowed us to construct a molecular data-base for the reference collection and to analyse genetic diversity for further prospecting, and for introducing new olive accessions.

DNA Fingerprinting↗

Laparoscopic repair of incisional hernia: a retrospective study of 159 patients.

BACKGROUND: In this long-term retrospective study, a laparoscopic technique was used for incisional hernia repair. METHODS: Over a 6-year period, we performed laparoscopic repairs with prosthetic mesh in 159 patients suffering from incisional hernia. Morbidity factors were noted and operative data were collected. In addition, early and long-term complications and recurrences were analyzed. RESULTS: There were no deaths as a result of the procedure. In 21 patients (13.8%), the operation was converted to an open procedure. Small bowel perforation occurred in three patients (1.9%). Mean hospital stay was 3.5 days. Early complications occurred in 61 patients (44%). The mean follow-up time was 49 months. There were no infections of the prosthetic mesh. Residual abdominal pain was reported in 31 patients (26%). Bowel obstructions requiring resection were found in two patients (1%), and hernia recurrence was observed in 19 patients (15.7%). CONCLUSIONS: Laparoscopic herniorraphy is a promising technique with all the advantages of minimal-invasive surgery. Nevertheless, close attention needs to be paid to the choice of the hernia and mesh size and to the fixing of the mesh.

Adult↗

Biochemical characterization of the beta-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C. 31461.

Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequential transfer of sugars, from the appropriate sugar donor to an activated lipid carrier, by committed glycosyltransferases (GTs). Few studies on the mechanism of action for this type of GT are available. Sphingomonas paucimobilis A.T.C.C. 31461 produces the industrially important polysaccharide gellan gum. We have cloned the gelK gene from S. paucimobilis A.T.C.C. 31461. GelK belongs to family 1 of the GT classification [Campbell, Davies, Bulone, Henrissat (1997) Biochem. J. 326, 929-939]. Sequence similarity studies suggest that GelK consists of two protein modules corresponding to the -NH(2) and -CO(2)H halves, the latter possibly harbouring the GT activity. The gelK gene and the open reading frames coding for the -NH(2) (GelK(NH2)) and -CO(2)H (GelK(COOH)) halves were overexpressed in Escherichia coli. GelK and GelK(NH2) were present in both the soluble and membrane fractions of E. coli, whereas GelK(COOH) was only present in the soluble fraction. GelK catalysed the transfer of [(14)C]glucuronic acid from UDP-[(14)C]glucuronic acid into a glycolipid extracted from S. paucimobilis or E. coli, even in the presence of EDTA, and the radioactive sugar was released from the glycolipid by beta-1,4-glucuronidase. GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PP(i)-lipid moiety is needed for enzymic activity. Recombinant GelK(NH2) and GelK(COOH) did not show detectable activity. Based on the biochemical characteristics of GelK and on sequence similarities with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily.

Amino Acid Sequence↗

Design, synthesis and pharmacological characterization of a potent radioiodinated and photoactivatable peptidic oxytocin antagonist.

Using a segment strategy, we have synthesized four iodinated photoactivatable cyclic peptidic ligands of oxytocin, bearing a beta-mercapto-betabeta-cyclopentamethylene propionic group (Pmp) on their N-terminus. All the syntheses were RP-HPLC monitored, and the compounds were HPLC purified. They were characterized by 1H NMR, MALDI-TOF, or FAB mass spectrometries. The affinities of Pmp-Tyr(Me)-Ile-Thr-Asn-Cys-Gly-Orn-Phe(3I,4N3)-NH2 (20), Pmp-Tyr-Ile-Thr-Asn-Cys-Gly-Orn-Phe(3I,4N3)-NH2 (21), Pmp-Tyr(Me)-Ile-Thr-Asn-Cys-Pro-Orn-Phe(3I,4N3)-NH2 (22), and Pmp-Tyr-Ile-Thr-Asn-Cys-Pro-Orn-Phe(3I,4N3)-NH2 (23) were evaluated as inhibition constants (K(i), in nM) for the human oxytocin receptor expressed in Chinese hamster ovary cells by displacement of a radioiodinated disulfide-cyclized antagonist (Elands et al. Eur. J. Pharmacol. 1987, 147, 197-207). The most potent of them, compound 22, was synthesized by another method in order to allow its radiolabeling by 125I. Its dissociation constant (K(d)) for the human oxytocin receptor, directly measured in saturation studies, was 0.25 +/- 0.04 nM, and its antagonist properties were determined by inactivation of phospholipase C, thus obtaining an inactivation constant (K(inact)) of 0.18 +/- 0.02 nM, evaluated by inositol phosphate accumulation. This compound is a very good tool for the mapping of peptidic antagonist binding sites in the human oxytocin receptor.

Animals↗

Direct identification of human oxytocin receptor-binding domains using a photoactivatable cyclic peptide antagonist: comparison with the human V1a vasopressin receptor.

Understanding of the molecular determinants responsible for antagonist binding to the oxytocin receptor should provide important insights that facilitate rational design of potential therapeutic agents for the treatment of preterm labor. To study ligand/receptor interactions, we used a novel photosensitive radioiodinated antagonist of the human oxytocin receptor, d(CH(2))(5) [Tyr(Me)(2),Thr(4),Orn(8),Phe(3(125)I,4N(3))-NH(2)9]vasotocin. This ligand had an equivalent high affinity for human oxytocin and V(1a) vasopressin receptors expressed in Chinese hamster ovary cells. Taking advantage of this dual specificity, we conducted photoaffinity labeling experiments on both receptors. Photolabeled oxytocin and V(1a) receptors appeared as a unique protein band at 70-75 kDa and two labeled protein bands at 85-90 and 46 kDa, respectively. To identify contact sites between the antagonist and the receptors, the labeled 70-75- and the 46-kDa proteins were cleaved with CNBr and digested with Lys-C and Arg-C endoproteinases. The fragmentation patterns allowed the identification of a covalently labeled region in the oxytocin receptor transmembrane domain III consisting of the residues Leu(114)-Val(115)-Lys(116). Analysis of contact sites in the V(1a) receptor led to the identification of the homologous region consisting of the residues Val(126)-Val(127)-Lys(128). Binding domains were confirmed by mutation of several CNBr cleavage sites in the oxytocin receptor and of one Lys-C cleavage site in the V(1a) receptor. The results are in agreement with previous experimental data and three-dimensional models of agonist and antagonist binding to members of the oxytocin/vasopressin receptor family.

Amino Acid Sequence↗

Evidence for an endocannabinoid system in the central nervous system of the leech Hirudo medicinalis.

In invertebrates, like Hydra and sea urchins, evidence for a functional cannabinoid system was described. The partial characterization of a putative CB1 cannabinoid receptor in the leech Hirudo medicinalis led us to investigate the presence of a complete endogenous cannabinoid system in this organism. By using gas chromatography-mass spectrometry, we demonstrate the presence of the endocannabinoids anandamide (N-arachidonoylethanolamine, 21.5+/-0.7 pmol/g) and 2-arachidonoyl-glycerol (147.4+/-42.7 pmol/g), and of the biosynthetic precursor of anandamide, N-arachidonylphosphatidyl-ethanolamine (16.5+/-3.3 pmol/g), in the leech central nervous system (CNS). Anandamide-related molecules such as N-palmitoylethanolamine (32.4+/-1.6 pmol/g) and N-linolenoylethanolamine (5.8 pmol/g) were also detected. We also found an anandamide amidase activity in the leech CNS cytosolic fraction with a maximal activity at pH 7 and little sensitivity to typical fatty acid amide hydrolase (FAAH) inhibitors. Using an antiserum directed against the amidase signature sequence, we focused on the identification and the localization of the leech amidase. Firstly, leech nervous system protein extract was subjected to Western blot analysis, which showed three immunoreactive bands at ca. approximately 42, approximately 46 and approximately 66 kDa. The former and latter bands were very faint and were also detected in whole homogenates from the coelenterate Hydra vulgaris, where the presence of CB1-like receptors, endocannabinoids and a FAAH-like activity was reported previously. Secondly, amidase immunocytochemical detection revealed numerous immunoreactive neurons in the CNS of three species of leeches. In addition, we observed that leech amidase-like immunoreactivity matches to a certain extent with CB1-like immunoreactivity. Finally, we also found that stimulation by anandamide of this receptor leads, as in mammals, to inhibition of cAMP formation, although this effect appeared to be occurring through the previously described anandamide-induced and CB1-mediated activation of nitric oxide release. Taken together, these results suggest the existence of a complete and functional cannabinoid system in leeches.

Adenylyl Cyclases↗

Exploring the acceptor substrate recognition of the human beta-galactoside alpha 2,6-sialyltransferase.

Human beta1,4-galactoside alpha2,6-sialyltransferase I (ST6GalI) recognition of glycoprotein acceptors has been investigated using various soluble forms of the enzyme deleted to a variable extent in the N-terminal half of the polypeptide. Full-length and truncated forms of the enzyme have been investigated with respect to their specificity for a variety of desialylated glycoproteins of known complex glycans as well as related proteins with different carbohydrate chains. Differences in transfer efficiency have been observed between membrane and soluble enzymatic forms, indicating that deletion of the transmembrane fragment induces loss of acceptor preference. No difference in substrate recognition could be observed when soluble enzymes of similar peptide sequence were produced in yeast or mammalian cells, confirming that removal of the membrane anchor and heterologous expression do not alter enzyme folding and activity. When tested on free oligosaccharides, soluble ST6GalI displayed full ability to sialylate free N-glycans as well as various N-acetyllactosaminyl substrates. Progressive truncation of the N terminus demonstrated that the catalytic domain can proceed with sialic acid transfer with increased efficiency until 80 amino acids are deleted. Fusion of the ST6GalI catalytic domain to the N-terminal half of an unrelated transferase (core 2 beta1,6-N-acetylglucosaminyltransferase) further showed that a chimeric form of broad acceptor specificity and high activity could also be engineered in vivo. These findings therefore delineate a peptide region of approximately 50 amino acids within the ST6GalI stem region that governs both the preference for glycoprotein acceptors and catalytic activity, thereby suggesting that it may exert a steric control on the catalytic domain.

Animals↗

The angiotensin system elements in invertebrates.

In this review, the different components of the renin-angiotensin system (RAS) in invertebrates are discussed. This system is implicated in osmoregulation, reproduction, memory processes and immune system regulation. As the elements of this hormone-enzymatic system also exist in invertebrates, it appears that the RAS originated very early in evolution.

Angiotensins↗

Phorbol ester differentially regulates oxytocin receptor binding activity in hypothalamic cultured neurons and astrocytes.

Hypothalamic cultured neurons and astrocytes were used to investigate the cellular mechanisms underlying the oxytocin receptor-mediated downregulation through a possible involvement of protein kinase C (PKC). For this purpose, the effects of PKC activators, inhibitor and of OT on OT receptor binding activity were compared in both cultures. In neurons, phorbol-myristate-acetate (PMA), a potent PKC activator, increased the binding of an OT receptor antagonist whereas in astrocytes, a decrease was observed. Pre-treatment of the cells with bisindolylmaleimide (10(-4) M), a PKC inhibitor, prevented the PMA-induced up- and downregulation. In contrast, receptor downregulation resulting from treatment of both cells with OT (10(-9) M) was not affected by the PKC inhibitor. On the other hand, when PMA (10(-7) M) was tested along with OT (10(-9) M), a subsequent decrease in ligand binding was observed in astrocytes. In neurons, PMA attenuated the OT-induced downregulation. Structural analysis of neuron and astrocyte OT receptor mRNA by RT-PCR, subcloning and sequencing, demonstrated identical sequence to rat uterine receptor. In conclusion, these data suggest that activation of PKC has opposite effect on OT receptor binding activity in neurons and astrocytes but they do not support the involvement of PKC in the OT-induced downregulation.

Animals↗

Structural and functional features of glycosyltransferases.

Most of the glycosylation reactions that generate the great diversity of oligosaccharide structures of eukaryotic cells occur in the Golgi apparatus. This review deals with the most recent data that provide insight into the functional organization of Golgi-resident glycosyltransferases. We also focus on the recent successes in X-ray crystal structure determination of glycosyltransferases. These new structures begin to shed light on the molecular bases accounting for donor and acceptor substrate specificities as well as catalysis.

Amino Acid Sequence↗

Identification of essential amino acids in the Azorhizobium caulinodans fucosyltransferase NodZ.

The nodZ gene, which is present in various rhizobial species, is involved in the addition of a fucose residue in an alpha 1-6 linkage to the reducing N-acetylglucosamine residue of lipo-chitin oligosaccharide signal molecules, the so-called Nod factors. Fucosylation of Nod factors is known to affect nodulation efficiency and host specificity. Despite a lack of overall sequence identity, NodZ proteins share conserved peptide motifs with mammalian and plant fucosyltransferases that participate in the biosynthesis of complex glycans and polysaccharides. These peptide motifs are thought to play important roles in catalysis. NodZ was expressed as an active and soluble form in Escherichia coli and was subjected to site-directed mutagenesis to investigate the role of the most conserved residues. Enzyme assays demonstrate that the replacement of the invariant Arg-182 by either alanine, lysine, or aspartate results in products with no detectable activity. A similar result is obtained with the replacement of the conserved acidic position (Asp-275) into its corresponding amide form. The residues His-183 and Asn-185 appear to fulfill functions that are more specific to the NodZ subfamily. Secondary structure predictions and threading analyses suggest the presence of a "Rossmann-type" nucleotide binding domain in the half C-terminal part of the catalytic domain of fucosyltransferases. Site-directed mutagenesis combined with theoretical approaches have shed light on the possible nucleotide donor recognition mode for NodZ and related fucosyltransferases.

Amino Acid Sequence↗

Oxytocin receptor gene expression in rat mammary gland: structural characterization and regulation.

The differential, tissue-specific regulation of oxytocin (OT) binding sites allows the neurohypophysial nonapeptide OT to fulfill a dual role: to induce uterine contractions at parturition and to mediate milk ejection during lactation. Whereas uterine OT binding sites are up-regulated prior to parturition and are rapidly down-regulated thereafter, mammary gland OT binding sites gradually increase throughout gestation and remain up-regulated during the ensuing lactation period. Here, we structurally characterized OT receptor (OTR) mRNA in mammary gland and analyzed its expression during gestation and lactation and in response to steroid treatment. In mammary gland tissues, we found a 6.7 and a 5.4 kb OTR mRNA species, and both species were further analyzed by RACE (rapid amplification of cDNA ends). The 6.7 kb mRNA was found to be common to mammary gland and uterus and to extend 618 nucleotides beyond the published sequence of the rat OTR gene. The 5.4 kb mRNA species is unique to the mammary gland and terminates at a mammary gland-specific polyadenylation site that is not preceded by a classical polyadenylation signal. RT-PCR analysis did not provide any evidence for differences in the coding regions, suggesting that both uterine and mammary gland OTR mRNAs encode the same receptor protein. Furthermore, primer extension experiments showed that no differences exist in the specific transcriptional initiation sites of the OTR gene in the two tissues. During pregnancy, OTR mRNA per mammary gland increased approximately 150-fold and remained high during lactation, consistent with the previously identified regulation of OT binding sites and the role of OT during lactation. Whereas estrogen administration strongly induced the uterine OTR mRNA levels (>5-fold), mammary gland remained unaffected by steroid treatment. Moreover, tamoxifen had no effect on the mammary gland OTR mRNA level. In summary, our data demonstrate a differential control of OTR expression in uterus versus mammary gland and a mammary gland-specific OTR mRNA polyadenylation site. However, this differential control apparently does not involve the expression of different receptor genes nor the utilization of tissue-specific transcriptional initiation sites.

Animals↗

[Evaluation of continuing medical training in private sector French cardiologists in 1999].

The authors present the results of a retrospective national enquiry which took place in 1999 and was mailed and faxed to the 3,800 cardiologists practising in the private sector in order to assess the different types of continuous, individual and collective postgraduate training which they had benefited from in the preceding 12 months. The data was analysed by comparison with that obtained from an individualized representative sample in a panel of private sector cardiologists. The results were then compared with the criteria of a yardstick proposed by the National Committee of Continuous Medical Education of 1997, according to the April 25th 1996 decree. The meeting of these criteria would require carrying out 114,000 to 76,000 hour-equivalents of continuous education whereas the present offer is about 100,000 hour-equivalents. The different forms of individual or collective training were compared in the 327 questionnaires which were exploitable following adhesion to the French Society of Cardiology, to the Cardiologists' Union, to local cardiological societies, by age, gender and type of practice. The average number of annual hours of collective education was 52.2 +/- 60.1 hours (25% quartile = 25 hours, 75% = 60 hours). The average value of hours of individual education was 89.7 +/- 89.3 hours (25% quartile = 25 hours; 75% = 120 hours). This evaluation indicates that about 15% of cardiologists practising in the private sector have inadequate continuous medical education and that 68% would satisfy the criteria laid down in 1997. Moreover, the present offer would seem to be adequate providing the criteria of accreditation have been met.

Adult↗

Control of Mung bean pectinmethylesterase isoform activities. Influence of pH and carboxyl group distribution along the pectic chains.

Well-characterized pectin samples with a wide range of degrees of esterification (39-74%) were incubated with the solubilized pure alpha and gamma isoforms of pectinmethylesterase, from mung bean hypocotyl (Vigna radiata). Enzyme activity was determined at regular intervals along the deesterification pathway at pH 5.6 and pH 7.6. It has been demonstrated that the distribution of the carboxyl units along the pectin backbone controls the activity of the cell wall pectinmethylesterases to a much greater extent than the methylation degree, with a random distribution leading to the strongest activity. Polygalacturonic acid was shown to be a competitive inhibitor of the alpha isoform activity at pH 5.6 and to inhibit the gamma isoform activity at both pH 5.6 and pH 7.6. Under these conditions, the drop in enzyme activity was shown to be correlated to the formation of deesterified blocks of 19 +/- 1 galacturonic acid residues through simulations of the enzymatic digestion according to the mechanisms established previously (Catoire, L., Pierron, M., Morvan, C., Herve du Penhoat, C., and Goldberg, R. (1998) J. Biol. Chem. 273, 33150-33156). However, even in the absence of inhibition by the reaction product, activity dropped to negligible levels long before the substrate had been totally deesterified. Comparison of alpha and gamma isoform cDNAs suggests that the N-terminal region of catalytic domains might explain their subtle differences in activity revealed in this study. The role of pectinmethylesterase in the cell wall stiffening process along the growth gradient is discussed.

Amino Acid Sequence↗