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Biomedical subjects

M Gervais

Publications and source records attributed to M Gervais.

At least 37 records · Page 2Linked to original sources
PubMed1993

Evidence for an interaction between the CYP1(HAP1) activator and a cellular factor during heme-dependent transcriptional regulation in the yeast Saccharomyces cerevisiae.

Previously, it was shown that the CYP1(HAP1) gene product mediates the transcription of several oxygen-regulated genes through a metabolic co-effector, heme, in the yeast Saccharomyces cerevisiae. This study investigates the overproduction of the CYP1 protein when the CYP1(HAP1) gene is placed under the control of the GAL10-CYC1 hybrid promoter (either at the locus of the CYP1(HAP1) gene or cloned in a high-copy-number plasmid). In these conditions, the CYP1 protein is detected by Western blot analysis and has a molecular mass in agreement with the open reading frame sequence. Band-shift experiments show that the CYP1(HAP1) protein is able to interact specifically with its target sequences in vitro without addition of hemin, and forms a large complex with one or several unidentified factors denoted as X. Addition of hemin allows the formation of a new complex which has a lower molecular mass. The internal deletion of the seven repeated amino acid sequences containing the KCPVDH motif in the CYP1(HAP1) protein modifies the heme responsiveness phenomenon observed in vitro in the band-shift experiments and in vivo in the transcription of the CYB2, CYC1, CYP3(CYC7) and ERG11 genes. On the basis of these data, we propose a new model for heme-induced activation of the CYP1 protein.

Amino Acid Sequence↗
PubMed1992

Effects of Pasteurella haemolytica leukotoxic culture supernatant on bovine neutrophil aggregation.

Pasteurella haemolytica A1 leukotoxic culture supernatant was evaluated for its ability to cause aggregation of bovine peripheral neutrophils. Neutrophils were isolated by a hypotonic lysis method and incubated with zymosan-activated plasma (ZAP), leukotoxic culture supernatant, antileukotoxin serum, calcium and magnesium-free media, p-bromophenacyl bromide and protein kinase C inhibitors. Aggregation was evaluated by changes in infrared light transmittance. Leukotoxic culture supernatant caused neutrophils to aggregate, and this effect was significantly removed by preincubation with antileukotoxin serum. Aggregation to ZAP and leukotoxin was dependent on the presence of extra-cellular calcium. Activation of protein kinase C by phorbol myristate acetate induced aggregation which was reduced by staurosporine; however, aggregation to leukotoxin did not involve protein kinase C activation. Phospholipase A2 inhibition did not alter the aggregation response to ZAP or to leukotoxin. The in vitro measurement of neutrophil aggregation induced by the leukotoxin of P. haemolytica reflects cytoskeletal and other activation events that may contribute to the intense inflammatory process which this organism induces in the lungs of cattle.

Acetophenones↗
PubMed1990

Cloning of the genome of a densovirus and rescue of infectious virions from recombinant plasmid in the insect host Spodoptera littoralis.

We have cloned an infectious genome of the Junonia coenia densonucleosis virus (JcDNV) into the bacterial plasmid pBR322. The viral genome could be rescued from the recombinant plasmid pBRJ by transfection of pBRJ DNA to sensitive Spodoptera littoralis larvae. pBRJ DNA produced a typical viral infection and a comparable percentage of larvae became infected following inoculation of equivalent amounts of purified virion DNA or cloned viral DNA. Virions extracted from transfected larvae were indistinguishable from wild-type (wt) virions with regard to their biophysical and biological properties. In particular, rescued virions were as infectious as wt virions and showed identical restriction profiles of their genome. In contrast, subcloning of JcDNV DNA deleted at both extremities of a sequence of ca 250 or ca 100 bp resulted in the inability of the recombinant plasmids to initiate a viral infection. These data suggest that, as for vertebrate parvoviruses, the inverted terminal repeats display essential functions in the rescue process and replicative cycle of densoviruses. This is the first report of the molecular cloning of the infectious genome from an insect parvovirus, and more generally from an invertebrate virus. pBRJ should provide an efficient tool to further define the organization of the JcDNV genome and compare it to other parvoviruses.

Animals↗
PubMed1990

Actual usage of medical facilities by asthmatics in two French rural settings: a preliminary study.

Until recently the medical management of asthmatic patients with respect to their socio-cultural environment had only been superficially studied. Four hundred and fifty eight adult asthmatics were identified through a two-stage questionnaire in two rural districts of south west France. The study has mainly demonstrated: 1) the large number of subjects experiencing more than one attack per week (33% in Gironde, 40% in Lot et Garonne); 2) the large number of subjects feeling disabled in their occupational and day-to-day life (52% in Gironde, 54% in Lot et Garonne); 3) the absence of a relationship between the severity of the disease and the use of medical facilities in the community. Despite the frequency of the attacks and the perceived disability, 27% of the patients felt that their asthma did not require any treatment and up to 60% did not seek medical help even during an attack; 4) the role of population density, type of dwelling and social status in the management of asthma. We conclude that under-treatment is widespread in these two rural settings and is closely related with the association of at least two of these three characteristics: 1) living in a low population density area; 2) living in an isolated dwelling; and 3) being a farm owner.

Adult↗
PubMed1989

Flavin and heme structures in lactate:cytochrome c oxidoreductase: a resonance Raman study.

Resonance Raman spectra of Hansenula anomala L-lactate:cytochrome c oxidoreductase (or flavocytochrome b2), of its cytochrome b2 core, and of a bis(imidazole) iron-protoporphyrin complex were obtained at the Soret preresonance from the oxidized and reduced forms. Raman contributions from both the isoalloxazine ring of flavin mononucleotide (FMN) and the heme b2 were observed in the spectra of oxidized flavocytochrome b2. Raman diagrams showing frequency differences of selected FMN modes between aqueous and proteic environments were drawn for various flavoproteins. These diagrams were closely similar for flavocytochrome b2 and for flavodoxins. This showed that the FMN structure must be very similar in both types of proteins, despite their very different proteic pockets. However, the electron density at this macrocycle was found to be higher in flavocytochrome b2 than in these electron transferases. No significant difference was observed between the heme structures in flavocytochrome b2 and in cytochrome b2 core. The porphyrin center-N(pyrrole) distances in the oxidized and reduced heme b2 were estimated to be 1.990 and 2.022 A from frequencies of porphyrin skeletal modes, respectively. The frequency of the vinyl stretching mode of protoporphyrin was found to be very affected in resonance Raman spectra of flavocytochrome b2 and of cytochrome b2 core (1634-1636 cm-1) relative to those observed in the spectra of iron-protoporphyrin [bis(imidazole)] complexes (1620 cm-1). These specificities were interpreted as reflecting a near coplanarity of the vinyl groups of heme b2 with the pyrrole rings to which they are attached. The low-frequency regions of resonance Raman indicated that the iron atoms of the four hemes b2 are in the porphyrin plane whatever their oxidation state. The histidine-Fe-histidine symmetric stretching mode was located at 205 cm-1 in the spectra of flavocytochrome b2 and of cytochrome b2 core. It was insensitive to the iron oxidation state and indicated strong Fe-His bonds in both states.

Flavin Mononucleotide↗
PubMed1989

Nitrite reductase from Pseudomonas aeruginosa: sequence of the gene and the protein.

The gene coding for nitrite reductase of Pseudomonas aeruginosa has been cloned and its sequence determined. The coding region is 1707 bp long and contains information for a polypeptide chain of 568 amino acids. The sequence of the mature protein has been confirmed independently by extensive amino acid sequencing. The amino-terminus of the mature protein is located at Lys-26; the preceding 25 residue long extension shows the features typical of signal peptides. Therefore the enzyme is probably secreted into the periplasmic space. The mature protein is made of 543 amino acid residues and has a molecular mass of 60,204 Da. The c-heme-binding domain, which contains the only two Cys of the molecule, is located at the amino-terminal region. Analysis of the protein sequence in terms of hydrophobicity profile gives results consistent with the fact that the enzyme is fully water soluble and not membrane bound; the most hydrophilic region appears to correspond to the c-heme domain. Secondary structure predictions are in general agreement with previous analysis of circular dichroic data.

Amino Acid Sequence↗
PubMed1989

Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis by an H. anomala proteinase.

The protomeric chain of Hansenula anomala flavocytochrome b2 was previously shown to be built as the covalent association of two functional domains: an L-lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone. This paper concerns the specific cleavage of this latter zone with a H. anomala proteinase(s) preparation and the purification of the resulting L-lactate dehydrogenase moiety of the molecule with at least 25% recovery, (i.e. one order of magnitude more than for the previously published method). A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer (Mr = 4 x 39000) containing FMN as expected and not heme. It has high L-lactate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b2) and the same Km for L(+)-lactate as flavocytochrome b2, but it has no L-lactate:cytochrome c oxidoreductase activity. Its flavin semiquinone is stabilized in the presence of pyruvate as in flavocytochrome b2. The subcellular origin of the H. anomala proteinase in the preparation has not yet been elucidated.

Chromatography, DEAE-Cellulose↗
PubMed1987

Fusion of negatively charged liposomes with clathrin-uncoated vesicles.

The interaction of lipid vesicles with uncoated vesicles from bovine brain has been studied by fluorescence energy transfer between fluorescent lipid analogs (NBD-PE, Rh-DOPE), by loss of fluorescence self-quenching (NBD-PE, carboxyfluorescein) and by freeze-fracture electron microscopy. The fluorescence techniques monitor the mixing of membranous lipids and the induced release of encapsulated material. The results demonstrate a mixing of the negatively charged lipid (PA, PS) vesicles with the uncoated vesicles. In parallel with the lipid mixing a release of intravesicularly encapsulated material takes place. Lipid vesicles composed of zwitterionic lipids (PC, DOPC, PC:PE) do not specifically interact with uncoated vesicles. The electron micrographs reveal single fusion events. Studies on the kinetics are consistent with a fusional mechanism of the negatively charged lipid vesicles with uncoated vesicles.

Animals↗
PubMed1986

Kinetics of electron transfer between two Hansenula anomala flavocytochrome b2 derivatives and two simple copper proteins (azurin and stellacyanin).

Two derivatives of Hansenula anomala flavocytochrome b2 have been prepared, one deprived of the flavin prosthetic group (deflavocytochrome b2), and the other consisting of the heme-b-carrying globule (b2 core). The redox potential of the heme in the two derivatives is -5 (+/- 5) mV and -10 (+/- 5) mV respectively, fairly similar to the value of -20 (+/- 5) mV reported for the holoenzyme, indicating a minor effect of the flavin and of the flavodehydrogenase domain on heme potential. The kinetics of azurin and stellacyanin reduction by both derivatives have been investigated. At pH 7.0, I = 0.2 M and 20 degrees C the second-order rate constants are: k = 8 X 10(5) M-1 S-1 for azurin reduction by deflavocytochrome b2; k = 1.6 X 10(6) M-1 S-1 for azurin reduction by b2 core; k = 1 X 10(7) M-1 S-1 for stellacyanin reduction by deflavocytochrome b2; k = 3 X 10(7) M-1 S-1 for stellacyanin reduction by b2 core. The change in pH markedly affects the kinetics in the case of azurin, but has no effect on stellacyanin reduction. The change in ionic strength has a significant effect when deflavocytochrome b2 is the reductant, indicating that the flavodehydrogenase domain plays a role in the stabilization of the transient kinetic complex by means of electrostatic interactions. The kinetic results are discussed in the framework of the Marcus theory.

Azurin↗
PubMed1984

Liposomes injected intravenously into mice associate with liver mitochondria.

Liposomes encapsulating uranyl acetate or ferritin were injected intravenously into mice. At periods of 20 min, 1 h and 4 h post-injection, animals were killed, and livers were excised. Transmission electron micrographs of liver tissue showed association of oligolamellar liposomes with mitochondria for each time period. At 1 h post-injection, an average of one out of ten mitochondria was associated with liposomes. In most cases, the liposomes were clearly enclosed in a cytoplasmic vacuole. Phagocytosis by Kupffer cells as well as fusion with primary lysosomes and inclusion in secondary lysosomes was observed. No difference in intracellular fate was observed when lactosylceramide was incorporated in the liposome bilayers, suggesting that the differences observed in biochemical studies are at the level of liposome-plasma membrane interaction. When liposomes containing uranyl acetate were intravenously injected and hepatocytes were isolated by collagenase perfusion one hour later, transmission EM revealed the presence of liposomes in these cells, in cytoplasmic vacuoles in the cytoplasm and in association with mitochondria. A freeze-fracture-etching analysis of liver tissue excised 20 min after injection of liposomes encapsulating ferritin, further supported the observation that liposomes associate with mitochondria in the liver.

Animals↗
PubMed1983

Study of the Hansenula anomala yeast flavocytochrome-b2-cytochrome-c complex 2. Localization of the main association area.

The reversible association of the Zn2+-substituted Hansenula anomala cytochrome c dimer (Thomas et al., preceding paper in this issue) to flavocytochrome b2 in oxidized or lactate-reduced state has been investigated by fluorimetry. The same method has been used for the determination of Zn-cytochrome c complexing to defined proteolytic fragments of flavocytochrome b2, either heme-b2-containing monomers or a flavin-linked tetramer. All these fragments but the isolated cytochrome b2 core showed binding stoichiometries, Kd values and ionic strength dependences quite similar to those found for native flavocytochrome b2. These data allowed localization of the single high-affinity binding site of cytochrome c on a particular globule in the dehydrogenase domain of the flavocytochrome b2 protomers. Quenching of the Zn-porphyrin c fluorescence in the various complexes occurred with only minor changes of the fluorescence lifetime and did not show any direct relationship to the presence or the redox state of the heme b2 group.

Ascomycota↗
PubMed1983

Proteolytic cleavage of Hansenula anomala flavocytochrome b2 into its two functional domains. Isolation of a highly active flavodehydrogenase and a cytochrome b2 core.

In a previous work, we have described the tryptic cleavage of yeast flavocytochrome b2 into its two functional domains: a cytochrome b2 core and a flavodehydrogenase. The lactate dehydrogenase efficiency of the latter was, however, dramatically low, only about 1% that of intact flavocytochrome b2. Our present study concerns a new flavodehydrogenase derivative of Hansenula anomala flavocytochrome b2 which spontaneously dissociates from the cytochrome domain when the polypeptide bridge connecting them is cleaved by Staphylococcus aureus V8 protease I. This flavodehydrogenase was purified and some of its functional and structural properties were studied. It presents an exceptionally high lactate dehydrogenase activity, about 80% that of flavocytochrome b2. This result clearly demonstrates that the cytochrome domain is not necessary for the lactate dehydrogenase function and suggests an autonomous folding for both domains. Our results are discussed in terms of 'gene fusion'.

Ascomycota↗
PubMed1982

Glycan conformational change in mesomorphic structures of ternary systems: liposaccharide/phospholipid/water.

An amphipatic liposaccharide, beta 16, has been synthesized by condensation of the glycoamino acid beta of ovomucoid with the palmitic acid to serve as a model on which the properties of the saccharide chains can be studied. This paper reports the ternary system beta 16/dipalmitoylphosphatidylcholine (DPPC)/water. Using X-ray diffraction and freeze-fracture electron microscopy, it was shown that the ternary system exhibits mesomorphic structures in the temperature range over which the aliphatic chains of the DPPC are in a liquid-like conformation. A phase diagram of the system was drawn at 75 degrees C in terms of the water concentration and of the beta 16 content. As the molar fraction in beta 16 increases from about 0.08 to 1, the ternary system displays successively two lamellar structures analogous to that exhibited by the system DPPC/H2O, then a hexagonal structure similar to that exhibited by the system beta 16/H2O. The two types of lamellar structure were shown to differ by the T or Y conformation adopted by their saccharide chains.

Animals↗
PubMed1982

How the loss of several residues, at the level of one interglobule junction, modulates the lactate dehydrogenase activity of yeast flavocytochrome b2: a study of the nicked enzymes resulting from clostripain and trypsin action.

The native chain of flavocytochrome b2 is folded into three globules linked together by two protease-sensitive bridges "a" and "cd". We show in this paper that zone "a" of H-flavocytochrome b2 is the first to be cleaved under clostripain action. The alpha c and beta c fragments thus formed are homologous to alpha T and beta'T trypsic fragments. The remaining activities of the resulting (alpha c beta c) and alpha T beta'T) forms are only 25 per cent and 4 per cent of the native flavocytochrome b2 one. The study of the catalytic properties of (alpha c beta'T) and (alpha T beta c) species resulting from the crossed reassociation of the isolated fragments show that the beta type fragment plays a critical role in the catalytic process. A dramatic activity decrease may be correlated with the loss of 6 amino acid residues at the N-terminal of beta c. Our best hypothesis is that these amino acids are involved in the active site, which may be located in the contact zone between alpha and beta. These results are in agreement with previous results obtained in this laboratory which showed the necessity of both alpha T and beta'T fragments for the correct conformation of the flavin binding site.

Amino Acid Sequence↗
PubMed1982

Transcription of the hepatitis B surface antigen gene in mouse cells transformed with cloned viral DNA.

Mouse L cells transformed with recombinant plasmids carrying hepatitis B virus (HBV) DNA fragments were used to study the transcription of the viral surface antigen gene (gene S). An HBV-specific, polyadenylated, 2.3-kilobase RNA was mapped on the HBV genome. This RNA hybridized with approximately 75% of the genome and excluded the region of the HBV core antigen gene (gene C). The 2.3-kilobase RNA species was present only in cell lines that produced hepatitis B surface antigen. An HBV-specific 2.3-kilobase RNA was also detected in human hepatoma cell line PLC/PRF/5 which produced hepatitis B surface antigen. A study of gene S expression in the transformed mouse L cells allowed us to localize the regions of initiation and termination of gene S transcription. Our results strongly suggest that the 2.3-kilobase RNA molecule is the mRNA of the major polypeptide of the envelope, which carries the viral surface antigen determinants.

Animals↗
PubMed1981

Structure and expression of the hepatitis B virus genome.

By fusion of the hepatitis B virus (HBV) surface antigen (HBsAg) gene to that of the E. coli lac Z gene carried by a phage lambda derivative, expression of HBsAg antigenic determinants was obtained and carried by a 138,000 dalton fusion polypeptide. Such a protein could be ultimately useful for second generation vaccine production. HBsAg gene expression was studied in eukaryotic cells using the mouse L cell (tk-) system. Cotransformation using a plasmid carrying two copies of the HBV genome in a tandem, head-to-tail arrangement (pCP10) and the cloned HSV-1 tk gene resulted in the excretion of 22 nm HBsAg particles in the supernatant. No other HBV markers were detected. These particles possess the same characteristics as the human serum particles (morphology, diameter, density, antigenicity). The purified HBsAg particles from L cells were found to be highly immunogenic in mice. HBV mRNA transcripts from these cells were analysed by Northern blotting. A major species of 2,300 bases was detected. This was mapped on the genome by hybridization with subgenomic fragments and in the L cell system using a series of plasmid derivatives carrying insertions at specific sites in the HBV genome and assaying for HBsAg expression. Thus the HBsAg gene promotor was localized between positions 2,400-2,800. Indeed there is only one TATA like sequence in this region, starting at position 2,776.

Animals↗