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Biomedical subjects

A Renard

Publications and source records attributed to A Renard.

At least 37 records · Page 2Linked to original sources
PubMed1990

Expression in Escherichia coli of two mutated genes encoding the cholera toxin B subunit.

To allow subsequent genetically mediated fusion of foreign antigens to cholera toxin B subunit (CTB), two mutated CTB encoding genes (ctxB) were constructed and overexpressed in Escherichia coli. The signal peptide coding sequence was deleted and restriction sites were created at both ends of the modified sequence. Both synthesized CTBs contain additional amino acid(s) at the N terminus (one and three). They were purified as insoluble products and refolded into the natural pentameric CTB structure by a denaturation-renaturation cycle. After renaturation, both recombinant proteins recovered CTB antigenicity and the ability to bind to GM1 gangliosides, as shown by in vitro analysis. Preliminary data indicated that both properties were unaltered by fusion of a foreign peptide to the mutated CTBs.

Base Sequence↗
PubMed1990

The coding region for the 54-kDa protein of several pestiviruses lacks host insertions but reveals a "zinc finger-like" domain.

We sequenced cDNAs, amplified by the polymerase chain reaction (PCR), which correspond to the carboxy-terminal portion of the 54-kDa protein of various cytopathic (cp) or noncytopathic (ncp) pestiviral strains. Except for the previously described insertions in two cp strains of the bovine viral diarrhea virus (BVDV), we did not find comparable insertions in this gene in eight pestiviral strains. The predicted amino acid sequences of this 54-kDa protein portion contain a conserved cysteine-rich stretch remarkably similar to a "zinc finger-type" binding domain found in many gene-regulatory proteins. Thus, this protein may be involved in the binding to viral RNA.

Amino Acid Sequence↗
PubMed1990

Molecular cloning of the mRNA coding for the G protein of the viral haemorrhagic septicaemia (VHS) of salmonids.

Viral haemorrhagic septicaemia virus (VHSV), a rhabdovirus, is a major threat for continental European trout fish farming. The development of a recombinant subunit vaccine could solve that problem. The neutralizing epitopes are located on the glycoprotein or G protein, the surface antigen. The G protein has a molecular weight of 65 kDa, reduced to 55 kDa by deglycosylation. cDNA was synthetized from mRNA of VHS virus infected cells, and cloned in E. coli. The viral cDNA was recognized by positive hybridization with a labelled probe made from infected cell RNA, and negative hybridization with labelled cDNA made from cellular RNA. The Northern blot hybridization with different clones on VHS infected cell RNA revealed two VHS mRNA whose lengths, 2.0 and 1.5 kb, were compatible with the mRNA length for G and N proteins respectively. This mRNA must contain about 400 bp of untranslated sequence.

Animals↗
PubMed1990

Synthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the alpha/beta-barrel proteins.

We have attempted to construct an artificial polypeptide that folds like the eight-stranded parallel beta-barrel structures. Our approach consists of repeating eight times a unit peptide designed to adopt a 'beta-strand/alpha-helix' pattern. A first 'test' sequence for this structural unit was deduced from a series of parameters defined after an analysis of three natural alpha/beta-barrel proteins and including principally the lengths of the secondary structure elements, the alpha/beta packing and the fitting on average Garnier profiles. The gene encoding this structural unit was synthesized, cloned and expressed in Escherichia coli either as a monomer or as direct repeats of 2-12 units. Preliminary structural characterization of the 7-, 8- and 9-fold unit polypeptides by circular dichroism measurements indicates the presence of the predicted amount of alpha-helix in the three proteins. Further analysis by urea-gradient gel electrophoresis demonstrates that, in the conditions tested, only the 8-fold unit polypeptide forms a compact structure through a cooperative and rapid two-state folding transition involving long-range molecular interactions.

Amino Acid Sequence↗
PubMed1988

The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R61 DD-peptidase family.

Homology searches and amino acid alignments, using the Streptomyces R61 DD-peptidase/penicillin-binding protein as reference, have been applied to the beta-lactamases of classes A and C, the Oxa-2 beta-lactamase (considered as the first known member of an additional class D), the low-Mr DD-peptidases/penicillin-binding proteins (protein no. 5 of Escherichia coli and Bacillus subtilis) and penicillin-binding domains of the high-Mr penicillin-binding proteins (PBP1A, PBP1B, PBP2 and PBP3 of E. coli). Though the evolutionary distance may vary considerably, all these penicillin-interactive proteins and domains appear to be members of a single superfamily of active-site-serine enzymes distinct from the classical trypsin or subtilisin families. The amino acid alignments reveal several conserved boxes that consist of strict identities or homologous amino acids. The significance of these boxes is highlighted by the known results of X-ray crystallography, chemical derivatization and site-directed-mutagenesis experiments.

Amino Acid Sequence↗
PubMed1987

A new natural hGH variant--17.5 kd--produced by alternative splicing. An additional consensus sequence which might play a role in branchpoint selection.

From a human pituitary cDNA library, we have cloned 3 distinct human growth hormone (hGH) cDNAs, coding respectively for the 22 K hGH, the 20 K variant, and a yet unknown 17.5 K variant. S1 mapping analysis using human pituitary RNA confirms the existence of at least four distinct hGH mRNAs originating from alternative acceptor sites at the second intron of the primary transcript. We have analysed the hGH gene sequence to explain the high frequency of alternative splicings which occur only at this location. In this study we propose CTTGNNPyPyPy as an additional consensus sequence guiding the selection of the branched nucleotide.

Base Sequence↗
PubMed1987

Molecular cloning of the bovine viral diarrhea virus genomic RNA.

The genomic RNA from Bovine Viral Diarrhea Virus (BVD) was cloned in E. coli. The complete sequence has been obtained and the genomic organization has been deduced. Some of the BVD specific cDNA have been expressed in bacteria, eucaryotic cells and in vitro. We suggest that BVDV belongs to a new family different from Togaviridae and Flaviviridae.

Cloning, Molecular↗
PubMed1985

Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2).

The nucleotide sequence of molecular clones of DNA from a retrovirus, ARV-2, associated with the acquired immune deficiency syndrome (AIDS) was determined. Proviral DNA of ARV-2 (9737 base pairs) has long terminal repeat structures (636 base pairs) and long open reading frames encoding gag (506 codons), pol (1003 codons), and env (863 codons) genes. Two additional open reading frames were identified. Significant amino acid homology with several other retroviruses was noted in the predicted product of gag and pol, but ARV-2 was as closely related to murine and avian retroviruses as it was to human T-cell leukemia viruses (HTLV-I and HTLV-II). By means of an SV-40 vector in transfected simian cells, the cloned gag and env genes of ARV-2 were shown to express viral proteins.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Primary structure and gene organization of human hepatitis A virus.

The RNA genome of human hepatitis A virus (HAV) was molecularly cloned. Recombinant DNA clones representing the entire HAV RNA were used to determine the primary structure of the viral genome. The length of the viral genome is 7478 nucleotides. An open reading frame starting at nucleotide 734 and terminating at nucleotide 7415 encodes a polyprotein of Mr 251,940. Comparison of the HAV nucleotide sequence with that of other picornaviruses has failed to reveal detectable areas of homology. However, a computer analysis of the putative amino acid sequence of HAV and poliovirus demonstrated the existence of short areas of homology in virion protein 3 (VP3) and throughout the carboxyl-terminal portion of the polyproteins. In addition, extensive protein structural homologies with poliovirus were detected.

Amino Acid Sequence↗
PubMed1985

Molecular cloning of bovine viral diarrhea viral sequences.

Bovine viral diarrhea virus (BVDV) genomic RNA was identified as a 12.5-kb single-stranded RNA molecule in both infected bovine embryonic kidney cells (BEK-1) and partially purified virions. BVD virion RNA was partially purified and used as a template for cDNA synthesis. BVDV-specific cDNA sequences were molecularly cloned and shown to hybridize to infected cell RNA but not to uninfected cell RNA or DNA. A single RNA species of 12.5 kb, representing the viral RNA genome, was detected in infected cells. A preliminary map of the BVDV specific cDNA clones was constructed and five major, nonoverlapping families were observed, accounting for approximately one-half of the viral genome.

Animals↗
PubMed1984

Isolation and characterization of the human prolactin gene.

Prolactin (PRL) and growth hormone (GH) genes derive from a common ancestor and still share some sequence homologies. Their expression in the pituitary gland is regulated in opposite directions by most of the many hormones acting on them. This provides an interesting system to study sequences involved in gene expression. Using a human PRL cDNA clone as a probe, we screened a human genomic DNA library in lambda phage and isolated a single recombinant comprising the whole hPRL gene. It was characterized by restriction endonuclease mapping and cDNA hybridization, by DNA heteroduplex analysis and by nucleotide sequencing. The hPRL gene is present as a single copy per haploid genome, is approximately 10 kb long and contains four introns, three of which interrupt the coding sequence at the same locations as in the known GH and PRL genes. The origin of transcription was determined by S1 mapping on prolactinoma mRNAs. The search for direct and inverted repeats, as well as dyad symmetries was carried out in the 900-bp sequenced in the 5'-flanking region. Sequence homologies between hPRL, hGH and rPRL were derived from computer drawn matrices for these upstream regions.

Animals↗
PubMed1984

[Efficacy of a new antisecretory agent (40 749 RP) on human gastric secretion induced by a meal (intragastric titration)].

An antisecretory drug of a new series, 40 749 RP, without anticholinergic or H2 receptor antagonist activities, was tested on meal-induced gastric acid secretion in 6 healthy volunteers. Gastric acid secretion and emptying of liquid were measured using intragastric titration. Oral dosages tested were 1, 2 and 4 mg/kg versus placebo. Inhibitions obtained were dose-related and expressed in percentage of the placebo values: 36 +/- 10 p. 100 for 1 mg/kg; 51 +/- 13 p. 100 for 2 mg/kg and 83 +/- 5 p. 100 for 4 mg/kg. Statistically significant correlation (p less than 0.001) was observed between maximal blood concentration of 40 749 RP and the percentage of secretory inhibition during the 90 min of the test. No change in gastrin response or in gastric emptying was observed whatever the dose.

Adult↗
PubMed1983

Repair of O6-ethylguanine DNA lesions in isolated cell nuclei. Presence of the activity in the chromatin proteins.

Cell nuclei prepared from rat liver were alkylated in vitro with ethylnitrosourea; the nuclear DNA was found to lose O6-ethylguanine and 7-ethylguanine during a subsequent incubation at 37 degrees C. The rate of O6-ethylguanine loss is comparable to that observed in vivo, indicating that no cytoplasmic component is needed for the repair; no free O6-ethylguanine was found in the incubation medium of the ethylated nuclei. The rate of 7-ethylguanine loss is higher than the spontaneous depurination in vitro and an amount of free 7-ethylguanine equivalent to that lost by the nuclear DNA was found in the incubation medium; these results suggest that this DNA lesion is excised by a DNA glycosylase. The proteins of the chromatin prepared from the isolated nuclei induced the disappearance of O6-ethylguanine from an added ethylated DNA. No free O6-ethylguanine was released indicating that the repair is not catalyzed by a DNA glycosylase; no oligonucleotides enriched in O6-ethylguanine were released either, indicating that the disappearance of O6-ethylguanine from DNA is not the result of the cooperative action of a specific endonuclease and an exonuclease. Activities capable of removing O6-ethylguanine from DNA were found in other cell compartments; most of it, however, is in the nucleus where the main location is chromatin. A pretreatment of the rats with daily low doses of diethylnitrosamine during 3 or 4 weeks increased 2-3-times the repair activity of the chromatin proteins.

Animals↗
PubMed1983

Properties of the chromatin repair activity against O6-ethylguanine lesions in DNA. Mechanism of the reaction.

Chromatin proteins from rat liver contain a repair activity that removes O6-ethylguanine from ethylnitrosourea-treated DNA. This activity does not depend on divalent cations and works in the presence of EDTA, but does depend on the presence of free thiol groups. Thus, it is destroyed by N-ethylmaleimide and is protected by dithiothreitol. The repair activity on single-stranded DNA is only 20% of what it is on double-stranded DNA; its half-life at 35 degrees C is 55 min, but DNA, ethylated or not, affords some protection. The repair reaction is a transethylation from O6-ethylguanine in DNA onto two different cysteine residues contained in acceptor proteins. The reaction can be followed by monitoring the appearance of ethylated proteins or by disappearance of O6-ethylguanine from DNA.

Animals↗
PubMed1983

Lineweaver-Burk, Hanes, Eadie-Hofstee and Dixon plots in non-steady-state situations.

Lineweaver-Burk, Hanes, Eadie-Hofstee and Dixon plots can only be used when a true initial rate is measured. Despite the fact that this point has often been stressed, it is far too often ignored in favour of restricting the assay time to one where low amounts of substrate are used. When one or several irreversible and slow steps occur with an inactivator during the incubation of a ternary enzyme-substrate-inactivator mixture, the rate of the enzyme-catalysed reaction progressively decreases. Even under these conditions, the present computer simulations investigations show that apparently linear Lineweaver-Burk, Hanes, Eadie-Hofstee and Dixon graphs can be obtained when the amount of product formed is mistakenly assumed to represent the true initial rate. Moreover, the observed pattern can change with time, going for instance from non-competitive to competitive. "Ki's" measured under these conditions also vary with time and bear little relationship to the true constants involved in the interaction.

Anti-Bacterial Agents↗