Biomedical subjects

C Aubert

Publications and source records attributed to C Aubert.

At least 37 records · Page 2Linked to original sources

PubMed2000

Perfluoroalkyl ketones: novel derivatization products for the sensitive determination of fatty acids by gas chromatography/mass spectrometry in electron impact and negative chemical ionization modes.

Analytically useful pentafluoro ketone derivatives of fatty acids are described. The gas chromatographic/mass spectrometric characteristics of these new derivatives are compared with those of methyl, trimethylsilyl and pentafluorobenzyl esters. Pentafluoro ketones exhibit excellent chromatographic properties and significantly shorter chromatographic retention times than these other esters. The electron impact mass spectra of these new compounds show informative acylium ions, whose intensity decreases with the degree of unsaturation of the parent fatty acid. The formation of strong and informative fragment ions in negative chemical ionization (CH(4)) mass spectra of pentafluoro ketone derivatives allows the detection and the characterization (length of the chain and number of double bonds) of fatty acids at trace levels (femtomole), even in the case of polyunsaturated compounds. The scope and limitations of this new derivatization technique are also discussed.

Fatty Acids↗

PubMed2000

The effect of growth temperature on the long-chain alkenes composition in the marine coccolithophorid Emiliania huxleyi.

The hydrocarbon fraction of a pure culture of Emiliania huxleyi, composed of a mixture of C31, C33, C37 and C38 polyunsaturated n-alkenes, appeared strongly dependent on the growth temperature of the alga between 8 degrees C and 25 degrees C. The total hydrocarbon content increased linearly with decreasing temperatures. C37 and C38 alkenes (which accounted for more than 90% of the total hydrocarbons) showed distinct changes in distribution compared to C31 and C33 alkenes, suggesting different biological syntheses and/or functions for these two groups of compounds. C37 and C38 alkenes and C37 methyl ketones (alkenones) all showed a trend to lower proportions of the two diunsaturated isomers and to higher proportions of the corresponding trienes with decreasing temperature. Unlike the alkenone unsaturation ratio (U37k'), ratios based on the C37 and C38 alkadi- and trienes could be linearly related to the growth temperature of E. huxleyi only between 15 degrees C and 25 degrees C. The modifications in the distribution of alkenes induced by varying temperature appeared, however, to be twice as fast as the modifications undergone by the alkenones. Although structurally and biochemically related, the distinct evolutions of alkenes and alkenones in response to changes in growth temperature might indicate that these two classes of compounds play two distinct physiological functions. The non-systematic linearity of relationships to temperature of parameters based on alkenes distribution suggested that these compounds are of limited use as paleotemperature indicator in the marine environment in contrast with the alkenones.

Alkenes↗

PubMed2000

Enhanced antitumor activity of 5-fluorouracil in combination with 2'-deoxyinosine in human colorectal cell lines and human colon tumor xenografts.

We investigated the effects of 2'-deoxyinosine (d-Ino), a modulator yielding thymidine phosphorylase activity, on cellular pharmacology of 5-fluorouracil (FUra) in various human colorectal cell lines and its antitumoral activity when combined with FUra in human xenografts. Associating d-Ino with FUra increased by 38 up to 700 times the sensitivity of HT29 and FUra-resistant SW620 lines, respectively, but not of CaCO2 cells, although high levels of intracellular FdUMP and subsequent higher thymidylate synthase inhibition were observed. Cell death studies confirmed the ability of d-Ino to enhance both early and late apoptosis induced by FUra in HT29 and SW620 but not in CaCo2. Similarly, we showed that associating d-Ino increased by 68 up to 101% the Fas overexpression induced by FUra in HT29 and SW620 but not in CaCo2 cells. Anti-Fas and anti-FasL antibodies both partly reversed this increase of cell sensitivity, thus confirming the role Fas plays in the modulation of FUra toxicity by d-Ino. This Fas component could explain the discrepancy between the lines because CaCO2 has been described as insensitive to Fas-mediated apoptosis. Antitumor activity of the combination was next investigated in nude mice transplanted with SW620. Results showed that although FUra alone has little effect on SW620 xenografts (P > 0.05), associating d-Ino significantly reduced the tumor growth by 57% (P < 0.05). This study suggests that it is possible to reduce both in vitro and in vivo resistance to FUra by modulating the way the drug is converted after cellular uptake.

Analysis of Variance↗

PubMed1999

Intraprotein electron transfer between tyrosine and tryptophan in DNA photolyase from Anacystis nidulans.

Light-induced electron transfer reactions leading to the fully reduced, catalytically competent state of the flavin adenine dinucleotide (FAD) cofactor have been studied by flash absorption spectroscopy in DNA photolyase from Anacystis nidulans. The protein, overproduced in Escherichia coli, was devoid of the antenna cofactor, and the FAD chromophore was present in the semireduced form, FADH., which is inactive for DNA repair. We show that after selective excitation of FADH. by a 7-ns laser flash, fully reduced FAD (FADH-) is formed in less than 500 ns by electron abstraction from a tryptophan residue. Subsequently, a tyrosine residue is oxidized by the tryptophanyl radical with t(1)/(2) = 50 microseconds. The amino acid radicals were identified by their characteristic absorption spectra, with maxima at 520 nm for Trp. and 410 nm for TyrO. The newly discovered electron transfer between tyrosine and tryptophan occurred for approximately 40% of the tryptophanyl radicals, whereas 60% decayed by charge recombination with FADH- (t(1)/(2) = 1 ms). The tyrosyl radical can also recombine with FADH- but at a much slower rate (t(1)/(2) = 76 ms) than Trp. In the presence of an external electron donor, however, TyrO. is rereduced efficiently in a bimolecular reaction that leaves FAD in the fully reduced state FADH-. These results show that electron transfer from tyrosine to Trp. is an essential step in the process leading to the active form of photolyase. They provide direct evidence that electron transfer between tyrosine and tryptophan occurs in a native biological reaction.

Cyanobacteria↗

PubMed1999

Interspecies variability and drug interactions of clozapine metabolism by microsomes.

Cytochrome P450 expression in liver is influenced by several factors, including species, sex and strain. We compared metabolism formation of clozapine in different species (rat, mouse, guinea-pig, dog, monkey and man) so as to choose between species to further validate interaction studies. Liver microsomes of male and female Sprague-Dawley rats, hairless rats, OF1 mice, Balb C mice and Dunkin-Hartley albino guinea-pigs, male beagle dogs, male cynomolgus monkeys and man were used to investigate in vitro metabolism of clozapine. This process was dependent on the presence of NADPH and on the presence of microsome protein. In addition, we observed the formation of desmethyl- and N-oxide metabolites, with the rate of formation of each of these compounds varying with species, sex and strain of microsomes incubated. The desmethyl- and N-oxide metabolites formed were statistically greater in male than in female rats, mice in the two strains studied, as well as for the guinea-pigs. Levels of desmethyl clozapine formed were high for the rats and no significant difference in clozapine biotransformation was observed between Sprague-Dawley and hairless rats. For man, the formation of metabolites of clozapine was comparable with guinea-pig, dog and monkey. In addition, we screened the effect of 52 molecules, representative of 11 different therapeutic classes, on the metabolism of clozapine by rat liver microsomes. We found that most of the calcium channel blockers (diltiazem, felodipine, isradipine, lacidipine, nicardipine and nitrendipine), antifungals (ketoconazole, miconazole) and two anticancer drugs (paclitaxel, teniposide) caused more than 50% inhibition of clozapine metabolism in vitro. The extent of inhibition was increased in a concentration-dependant manner. Complementary clinical and pharmacokinetic studies should be performed to confirm these results.

Animals↗

PubMed1998

Structural and kinetic studies of the Y73E mutant of octaheme cytochrome c3 (Mr = 26 000) from Desulfovibrio desulfuricans Norway.

A combination of structural, kinetic, and interaction experiments has been used to study the role of a highly conserved aromatic residue, Tyr73, parallel to the sixth heme axial ligand of heme 4 in multiheme cytochrome c3 (Mr = 26 000), also called cytochrome cc3 or octaheme cytochrome, from Desulfovibrio desulfuricans Norway. This residue is expected to be involved in intermolecular electron transfer and protein-protein interaction, since heme 4 is described to be the interaction site between physiological partners. The kinetic experiments show that the Y73E replacement provokes no significant change in the electron-transfer reaction with the physiological partner, the [NiFeSe] hydrogenase, but that the protein-protein interaction between cytochrome c3 (Mr = 26 000) and hydrogenase is strongly affected by the mutation. The aromatic residue does not play a role in maintaining the axial heme ligand in a particular orientation, since the mutation did not affect the orientation of histidine 77, the sixth axial ligand of heme 4. The structural analysis by X-ray crystallography clearly shows that a rearrangement of the charged residues in the vicinity of the mutation site is responsible for the change in protein-protein interaction, which is of an electrostatic nature. Lys22 and Arg66, residues which are located at the interacting surface, are twisted toward the mutated position Glu73 in order to compensate for the negative charge and therefore are no longer accessible for the docking with a physiological partner. Tyr73 has instead a structural function and probably a role in maintaining the hydrophobic environment of the heme 4 cavity rather than a function in the intermolecular electron transfer with the physiological partners.

Binding Sites↗

PubMed1998

Characterization of the cytochromes C from Desulfovibrio desulfuricans G201.

A monoheme cytochrome c553 and a hexadecaheme high molecular weight cytochrome (Hmc) have been isolated and characterized from the sulfate-reducing bacteria Desulfovibrio desulfuricans G201, in addition to the tetraheme cytochrome c3 (Mr 13000) that has been previously described. Both cytochromes are homologous with respect to several biochemical properties to the corresponding cytochromes found in other Desulfovibrio species. However, they are acidic proteins while the corresponding molecules, isolated from other Desulfovibrio species, are relatively more basic. The D. desulfuricans cytochrome content appears identical to that of D. vulgaris Hildenborough. Isolation of these cytochromes from a Desulfovibrio desulfuricans strain is of great interest in order to get more insight on the physiological function of these molecules.

Amino Acid Sequence↗

PubMed1998

The Desulfuromonas acetoxidans triheme cytochrome c7 produced in Desulfovibrio desulfuricans retains its metal reductase activity.

Multiheme cytochrome c proteins that belong to class III have been recently shown to exhibit a metal reductase activity, which could be of great environmental interest, especially in metal bioremediation. To get a better understanding of these activities, the gene encoding cytochrome c7 from the sulfur-reducing bacterium Desulfuromonas acetoxidans was cloned from genomic DNA by PCR and expressed in Desulfovibrio desulfuricans G201. The expression system was based on the cyc transcription unit from Desulfovibrio vulgaris Hildenborough and led to the synthesis of holocytochrome c7 when transferred by electrotransformation into the sulfate reducer Desulfovibrio desulfuricans G201. The produced cytochrome was indistinguishable from the protein purified from Desulfuromonas acetoxidans cells with respect to several biochemical and biophysical criteria and exhibited the same metal reductase activities as determined from electrochemical experiments. This suggests that the molecule was correctly folded in the host organism. Desulfovibrio desulfuricans produces functional multiheme c-type cytochromes from bacteria belonging to a different genus and may be considered a suitable host for the heterologous biogenesis of multiheme c-type cytochromes for either structural or engineering studies. This report, which presents the first example of the transformation of a Desulfovibrio desulfuricans strain by electrotransformation, describes work that is the first necessary step of a protein engineering program that aims to specify the structural features that are responsible for the metal reductase activities of multiheme cytochrome c7.

Amino Acid Sequence↗

PubMed1997

A single mutation in the heme 4 environment of Desulfovibrio desulfuricans Norway cytochrome c3 (Mr 26,000) greatly affects the molecule reactivity.

The gene encoding Desulfovibrio desulfuricans Norway cytochrome c3 (Mr 26,000), a dimeric octaheme cytochrome belonging to the polyheme cytochrome c3 superfamily, has been cloned and successfully expressed in another sulfate reducing bacteria, D. desulfuricans G201. The gene, named cycD, is monocistronic and encodes a cytochrome precursor of 135 amino acids with an extension at the NH2 terminus of 24 amino acids. This extension acts as a signal sequence which allows export across the cytoplasmic membrane into the periplasmic space. Tyrosine 73, which is in a close contact with the histidine sixth axial ligand to the heme 4 iron atom, has been replaced by a glutamate residue using site-directed mutagenesis. The cytochrome mutant when expressed in D. desulfuricans G201, is correctly folded and matured. A global increase of the oxidoreduction potentials of about 50 mV is measured for the Y73E cytochrome. The mutation also has a strong influence on the interaction of the cytochrome with its redox partner, the hydrogenase. This suggests, like the tetraheme cytochrome c3 (Mr 13, 000), heme 4 is the interactive heme in the cytochrome-hydrogenase complex and that alteration of the heme 4 environment can greatly affect the electron transfer reaction with its redox partner.

Amino Acid Sequence↗

PubMed1996

An in vitro nucleoside analog screening method for cancer gene therapy.

Suicide genes that sensitize cells to drugs that are normally nontoxic at therapeutic levels represent an important approach in human gene therapy research. We have developed an in vitro screening assay to assess the modulation of nucleoside analogs after transfection of a vector expressing the herpes simplex virus thymidine kinase gene (HSV-TK). The thymidine kinase gene enhances nucleoside phosphorylation to nucleotides that kill cells by blocking DNA elongation. Cells lines used are 3T3-NIH fibroblasts (parental cells) and 3T3-TKc3 (HSV-TK gene-transfected 3T3-NIH). Two types of analysis are performed: a cytotoxicity assay, the neutral red uptake assay to assess the IC50 on the two cell lines, and an HPLC analysis coupled to a radiochemical flow detector to evaluate metabolic profiles after incubation of cells with tritiated analogs. Results show that cells expressing the HSV-TK gene are more sensitive than the parent cells to the effect of acyclovir or ganciclovir, the reference purine analog drugs, and also to the effect of pyrimidine analogs, bromodeoxyuridine, bromovinyldeoxyuridine, and ethyldeoxyuridine. Promising nucleoside analogs for gene therapy that can be achieved by HSV-TK could be evaluated using this model.

3T3 Cells↗

PubMed1995

Theiler's virus infection of 129Sv mice that lack the interferon alpha/beta or interferon gamma receptors.

The Daniels strain of Theiler's virus causes a persistent infection of the white matter of spinal cord of susceptible mice, with chronic inflammation and primary demyelination. Inbred 129Sv mice are resistant to this infection; they present with mild encephalomyelitis and clear the infection within a matter of days. A very different outcome was observed with inbred 129Sv mice whose receptors for interferon alpha/beta or interferon gamma had been inactivated by homologous recombination. The former presented severe encephalomyelitis with acute infection of neurons, particularly in brain and hippocampus, and extensive infection with necrosis of the choroid plexus. Most animals died of this acute disease. The latter, presented the same early encephalomyelitis as the control 129Sv mice. However, they remained persistently infected and developed a very severe late infection of the white matter with extensive primary demyelination. This late disease looked like an exacerbated form of the chronic demyelinating disease observed in susceptible inbred mice such as the SJL/J or FVB strains. Our results show that the two interferon systems play nonredundant roles in the resistance of the 129Sv mouse to the infection by Theiler's virus. They also lend support to the notion that the Ifg gene is involved in the resistance/susceptibility of inbred strains of mice to persistent infection by this picornavirus.

Animals↗

PubMed1995

Early infection of the central nervous system by the GDVII and DA strains of Theiler's virus.

The DA strain of Theiler's virus, a murine picornavirus, causes a persistent infection of glial cells of the white matter of the spinal cord, associated with chronic inflammation and primary demyelination. The GDVII strain causes an acute fatal grey matter encephalomyelitis. We characterized the target cells of GDVII and DA viruses 4 days following intracerebral inoculation, and we compared the levels of viral RNA within these cells. GDVII virus infected approximately 10 times more cells than DA virus. Whereas GDVII virus infected neurons exclusively, DA virus infected also astrocytes and possible macrophage-microglial cells. The levels of viral RNA in neurons infected with GDVII and DA viruses were of the same order. These results show that DA virus infects glial cells already at the beginning of the disease and that the more efficient spread of GDVII virus is probably not due to a higher level of RNA replication per cell.

Animals↗

PubMed1995

Metastatic variants of the B16 melanoma: metastasis is related to environmental conditions. Phenotypic changes in vitro and metastatic colonization potential in nude mice.

Variants of B16 melanoma exhibit strikingly different metastatic potential in AY/a (YB16 tumours) and a/a C57BL/6J (MB16 tumours) syngeneic mice. This study focused on relative pigmentation and metastatic potential in eight subline cultures initiated from B16 control and YB16 and MB16 tumours. During 6 months of in vitro growth in minimal essential medium, cells displayed a continuous decrease in their ability to form spontaneous lung colonies in 140 syngeneic mice with only persistence of enhanced metastasis-related characteristics depending on genetic change in yellow AY/a mice. Conversely, in a parallel experiment in 101 syngeneic mice in vitro, cells had a greater capacity to generate experimental metastases; this might be related to successive different environmental factors. In order to compare these prior results obtained in syngeneic mice, the above eight secondary cell lines were inoculated subcutaneously into Swiss nude mice. The primary tumours thus obtained were then serially transplanted monthly during 4 months. The new results obtained in a total of 277 mice showed that metastatic properties of cells were enhanced or restored in nude mice. Various tumour cell environments seem to be responsible for selective pressures that determine the melanoma metastatic potential. New, enhanced, heritable, metastasis-related characteristics can occur in melanoma cells as a result of genetic and metabolic changes and immunologic deficiency of the host. Apparent tumour-host relationship should not be neglected, since it has a clear influence on neoplastic diversity and malignant behaviour.

Animals↗

PubMed1994

Metastatic variants of the B16 melanoma: metastasis is related to environmental conditions. Genetic and metabolic effects.

Using variant B16 metastatic melanomas, attempts were made to explore the effects of both genetic and metabolic environmental factors in vivo as well as in vitro, on specific steps in the metastatic process. Metastases of the progenitor tumour in black C57BL/6J mice arose from a stable, heritable, non-random, pre-existing subpopulation of metastatic cells. In vitro, the environment influenced both pigmentary and metastatic phenotypes of variant B16 metastatic melanomas. The possibility that cells from different strains may express a phenotype following reinoculation into syngeneic mice was also explored. The experimental results suggested that new, enhanced, heritable, metastasis-related characteristics can develop as a result of genetic changes in yellow Ay/a and C57BL/6J mice that are accompanied by pigmentary changes. Such genetic changes can determine unstable non-random and random epigenetic metastatic effects with enhancement in vivo or disappearance in vitro of spontaneous metastatic ability and of the number of metastases, as monitored by serial transfers of tumour cells in vivo.

Animals↗

PubMed1994

Chimeric Theiler's virus with altered tropism for the central nervous system.

Theiler's virus is a neurotropic murine picornavirus which, depending on the strain, causes either an acute encephalitis or a persistent demyelinating disease. Following intracranial inoculation, the demyelinating strains infect sequentially the grey matter of the brain, the grey matter of the spinal cord, and finally the white matter of the spinal cord, where they persist and cause chronic demyelination. The neurovirulent strains cause a generally fatal encephalitis with lytic infection of neurons. The study of chimeric Theiler's viruses, obtained by recombining the genomes of demyelinating and neurovirulent strains, has shown that the viral capsid contains determinants for persistence and demyelination. In this article we describe the recombinant virus R5, in which the capsid protein VP1 and a small portion of protein 2A come from the neurovirulent GDVII strain and the rest of the genome comes from the persistent DA strain. The capsid of virus R5 also contains one mutation at amino acid 34 of VP3 (Asn-->His). Virus R5 does not persist in the central nervous system (CNS) of immunocompetent SJL/J or BALB/c mice. However, it replicates efficiently and persists in the CNS of BALB/c nu/nu mice, showing that its growth in the CNS is not impaired. In BALB/c nu/nu mice, whereas virus DA causes mortality with large amounts of viral antigens in the white matter of the spinal cord, virus R5 does not kill the animals, persists in the neurons of the grey matter of the brain, and never reaches the white matter of the spinal cord. This phenotype is due to the chimerism of the capsid and/or to the mutation in VP3. These results indicate that the capsid plays an important role in the characteristic migration of Theiler's virus within the CNS.

Animals↗

PubMed1993

Establishment and characterization of human ocular melanoma cell lines.

Five continuous cell lines have been established from 29 ocular melanomas and maintained for periods ranging from 3 to 9 years in medium identical to that in which 3 concomitantly studied lines of cutaneous melanoma cells were cultured as controls. The long-term problems to be overcome in establishing uveal cell lines are related to cell-doubling times which ranged from 72 to 432 hr, and plating efficiency, which ranged from 0.5%-6.5%. Tumors and cell lines were found to contain melanosomes. The morphology of uveal cells during the early subcultures exhibited multiple changes. Two different established cell lines were obtained from one ciliary-body tumor. Biochemical studies revealed markers of melanogenesis and neuroendocrine compounds. Cytogenetic studies revealed chromosomal abnormalities that differed between uveal and conjunctival melanomas.

Adult↗

PubMed1993

Analysis of proteolipid protein and P0 transcripts in mice infected with Theiler's virus.

Inbred strains of mice vary in their susceptibility to the chronic inflammatory and demyelinating disease caused by Theiler's virus. The severity of this disease depends on the extent of demyelination but also on the occurrence of remyelination. We studied the extent of demyelination and remyelination in mice with different genetic backgrounds. To estimate the extent of demyelination, we analyzed the abundance of proteolipid protein (PLP) transcripts in the spinal cord 45 days after inoculation. RNA transcripts were quantitated at the single cell level using in situ hybridization. We observed a correlation between the reduction of PLP mRNA and the extent of inflammation in the SJL/J, C3H, F1 SJL x C3H and C3H.Q strains. Remyelination by Schwann cells was estimated by the detection of P0 mRNA and was observed in C3H mice only. It is likely that intense inflammation in highly susceptible strains prevents the migration of Schwann cells into the central nervous system (CNS).

Animals↗

PubMed1993

Theiler's virus infection of beta 2-microglobulin-deficient mice.

Theiler's virus, a murine picornavirus, persists in the central nervous systems of susceptible mice and induces a chronic demyelinating disease. Susceptibility or resistance to this disease is controlled in part by the H2-D locus of the major histocompatibility complex (MHC). For this reason, it has been proposed that CD8+ class I-restricted cytotoxic T cells play a main role in the pathogenesis of this viral infection. We recently reported the existence of anti-virus CD8+ cytotoxic T cells in the course of Theiler's virus infection. In the present study, we examined the role of these effector cells in mice in which the beta 2-microglobulin gene had been disrupted. These mice fail to express class I MHC molecules and therefore lack CD8+ T cells. The mice are derived from a C57BL/6 x 129/Ola cross and are H-2b, a haplotype associated with resistance to Theiler's virus infection. beta 2-Microglobulin-deficient mice (beta 2m-/-mice) failed to clear the virus, developed demyelination, and, interestingly, did not succumb to early infection. These results demonstrate that CD8+ T cells are required to clear Theiler's virus infection. In contrast with a current hypothesis, they also demonstrate that CD8+ T cells are not major mediators of the demyelinating disease.

Animals↗