Biomedical subjects

C Aubert

Publications and source records attributed to C Aubert.

At least 19 recordsLinked to original sources

PubMed2005

Characterization of isomeric allylic diols resulting from chlorophyll phytyl side-chain photo- and autoxidation by electron ionization gas chromatography/mass spectrometry.

The electron ionization (EI) mass spectral fragmentation of the trimethylsilyl derivatives of 3-methylidene-7,11,15-trimethylhexadecane-1,2-diol, Z- and E-3,7,11,15-tetramethylhexadec-3-ene-1,2-diols and Z- and E-3,7,11,15-tetramethylhexadec-2-ene-1,4-diols resulting from chlorophyll phytyl side-chain photo- and autoxidation was investigated. Different pathways (substantiated by deuterium labelling) were proposed in order to explain the main fragmentation observed. Then, some sufficiently specific fragment ions were selected and used to characterize these compounds in natural environmental samples.

Chlorophyll↗

PubMed2005

Electron ionization mass spectral fragmentation of cholestane-3beta,4alpha,5alpha-triol and cholestane-3beta,5alpha,6alpha/beta-triol bis- and tris-trimethylsilyl derivatives.

The electron ionization (EI) mass spectral fragmentation of the bis- and tris-trimethylsilyl derivatives of cholestane-3beta,4alpha,5alpha-triol, cholestane-3beta,5alpha,6beta-triol and cholestane-3beta,5alpha,6alpha-triol was investigated. The EI mass spectrum of the 3beta,4alpha-bis-trimethylsilyl derivative of cholestane-3beta,4alpha,5alpha-triol exhibits interesting fragment ions at m/z 142 and 332 resulting from the initial loss of TMSOH between the carbons 2 and 3 and subsequent retro-Diels-Alder (RDA) cleavage of the ring A. Trimethylsilyl transfer between the 4alpha- and the 5alpha-hydroxy groups acts significantly before RDA cleavage affording an ion at m/z 404. Complete silylation of cholestane-3beta,4alpha,5alpha-triol strongly stabilizes the molecule, affording an abundant molecular ion at m/z 636 and decreasing the abundance of the RDA cleavage. Loss of water (from the non-silylated 5alpha-hydroxy group) plays a very important role during the decomposition of the molecular ion of 3beta,6alpha/beta-bis-trimethylsilyl derivatives of cholestane-3beta,5alpha,6alpha/beta-triols. These derivatives appear to be very useful in assigning the configuration of the carbon 6. This assignment is based on the abundance of the fragment ions at m/z 321, 367 and 403, which are more prominent in the EI mass spectrum of the beta-isomer. In contrast, EI mass spectra of the tris-trimethylsilyl derivatives of cholestane-3beta,5alpha,6beta-triol and cholestane-3beta,5alpha,6alpha-triol differ only slightly and appear to be poorly informative.

Cholestanols↗

PubMed2005

Hydrogen metabolism in the hyperthermophilic bacterium Aquifex aeolicus.

Aquifex aeolicus is a microaerophilic, hydrogen-oxidizing, hyperthermophilic bacterium containing three [NiFe] hydrogenases. Two of these three enzymes (one membrane-bound and one soluble) have been purified and characterized. The Aquifex hydrogenases are thermostable and tolerant to oxygen. A cellular function for the three hydrogenases has been proposed. The two membrane-bound periplasmic hydrogenases may function in energy conservation, whereas the soluble cytoplasmic hydrogenase is probably involved in the CO(2) fixation pathway.

Bacteria↗

PubMed2004

Electron ionization mass spectral fragmentation of derivatized 4,5- and 5,6-epoxysterols.

The electron ionization (EI) mass spectral fragmentation of derivatized 4,5- and 5,6-epoxysterols was investigated. Interesting fragmentation processes involving a transannular cleavage of the epoxide ring after transfer of the trimethylsilyl group are significant in the case of 4,5-epoxysterol trimethylsilyl ethers (affording abundant fragment ions at m/z 403 and 404). Different pathways, which have been substantiated by deuterium labelling, are proposed in order to explain the formation of these ions. In contrast, this transfer is not significant in the case of 5,6-epoxysterol trimethylsilyl ethers. The EI mass spectra of these latter compounds appear to be very complex and to differ slightly according to the stereochemistry of the epoxy group. Acetate and trifluoroacetate derivatives of 4,5-epoxysterols display interesting EI mass spectra dominated by a fragment ion at m/z 332 resulting from cleavage of the steroid ring A.

Cholestanol↗

PubMed2004

Trimethylsilyl transfer during electron ionization mass spectral fragmentation of some omega-hydroxycarboxylic and omega-dicarboxylic acid trimethylsilyl derivatives and the effect of chain length.

The electron ionization (EI) mass spectral fragmentation of omega-hydroxycarboxylic and omega-dicarboxylic acid trimethylsilyl derivatives was investigated. The mass spectra of these compounds exhibited fragment ions resulting from classical fragmentation of the trimethylsilyl ether and ester groups, and others resulting from the interactions between the two functionalities (m/z 147, 204, 217, [M-31](+) and [M-105](+) in the case of omega-hydroxycarboxylic acid derivatives and m/z 147, 204, 217 and [M-131](+) in the case of omega-dicarboxylic acid derivatives). Several fragmentation pathways were proposed to explain the formation of these different fragment ions. It is proposed that the ions at m/z 204 and 217 are formed via an initial trimethylsilyl transfer between the ether and the ester group or between the two ester groups. This transfer appeared to be more favoured in the case of omega-dicarboxylic acid derivatives and to be dependent on the chain length. A more efficient transfer was in fact observed for compounds with a relatively long alkyl chain. In the case of shorter omega-hydroxycarboxylic and omega-dicarboxylic acid trimethylsilyl derivatives the formation of the ions at m/z 204 and 217 suffers strongly from competition from production of the ion at m/z 147.

Carboxylic Acids↗

PubMed2003

Electron ionization mass spectral fragmentation of C19 isoprenoid aldehydes and carboxylic acid methyl and trimethylsilyl esters.

The electron ionization (EI) mass spectra of saturated and alpha,beta-unsaturated C(19) isoprenoid aldehydes and carboxylic acid methyl and trimethylsilyl esters are reported. Different pathways are proposed in order to explain the main fragmentations observed. The conjugated double bond migrates more or less readily before gamma-hydrogen rearrangement according to the structure of the considered compound. Configurations of the double bond of alpha,beta-unsaturated C(19) isoprenoid aldehydes and fatty acid methyl and trimethylsilyl esters can be easily determined thanks to the peaks at m/z 97, 127 and 185, respectively, which are much more abundant in the mass spectra of the Z isomers owing to the formation of a cyclic ion. In the case of trimethylsilyl esters, subsequent fragmentation of the cyclic ion at m/z 185 affords two other diagnostic ions at m/z 95 and 169.

Journal Article↗

PubMed2003

Fas/FasL expression in tumor biopsies: a prognostic response factor to fluoropyrimidines?

Various studies suggested that cytotoxicity induced by 5-fluorouracil (5-FU) is an apoptotic mechanism possibly mediated by the Fas/FasL system. In this preliminary work, we studied retrospectively the role the Fas/FasL expression as a predictive response factor with fluoropyrimidine-based chemotherapies. We developed a real-time PCR method for measuring Fas and FasL expression in various biopsies from patients treated with a FUFOL-like protocol. No correlation was found between Fas or FasL expression and overall survival or partial response. However, the PCR assay was simple and convenient to use for quantitation of Fas/FasL in tumor biopsies.

Adult↗

PubMed2003

Drug interactions of paclitaxel metabolism in human liver microsomes.

The human liver metabolism of paclitaxel (Taxol), an anticancer drug, leads to three metabolites: 6alpha-hydroxypaclitaxel, 3'-p-hydroxypaclitaxel and 6alpha,3'-p-dihydroxypaclitaxel. The inter-individual variability of paclitaxel metabolism was investigated first in vitro using 22 human liver microsomes. Three metabolites have been detected by HPLC. This preliminary work revealed marked inter-individual differences in paclitaxel metabolism. The amount of major metabolite 6alpha-hydroxypaclitaxel formed varied 16-fold (0.7 to 11.5 nmol/mg/h). We next studied the effect of 29 compounds (antineoplastics, antiemetics, histamine-2 receptor antagonist, antalgics, antifungals, antivirals, psychotropics, antibiotic, corticoid, antiarrhythmic, calcium channel blocker) on paclitaxel metabolism in human liver microsomes. Among the compounds studied, quercetin, antifungal drugs such as ketoconazole and miconazole, and the antineoplastic drug doxorubicin inhibited formation of 6alpha-hydroxypaclitaxel. Dixon plots indicated that quercetin and doxorubicin inhibited 6alpha-hydroxypaclitaxel formation through a competitive mechanism with a Ki of 10.1 microM and 64.8 microM, respectively. The inhibition of this metabolite by ketoconazole was through a noncompetitive mechanism with a Ki of 11.8 microM. Our data thus suggest that special attention should be paid when these drugs are combined in clinical practice.

Adult↗

PubMed2002

Declared hepatitis C screening strategies in blood recipients in French hospitals.

AIMS OF THE STUDY: In spite of official recommendations and measures in France, screening strategies of hepatitis C performed in the field of transfusion are not clearly known. The aim of this study is to describe the screening strategies before and after the current year of the transfusion in blood recipients in several French medical departments and hospitals. MATERIALS AND METHODS: A qualitative study using the key informant technique was carried out. A sample of 179 departments and 64 hospitals in charge of patients transfused with low or high-volumes of homologous blood products was constituted. The key informants were asked about the number of homologous blood products, the number of recipients transfused in the hospital, the volume of transfusion performed, the existence of a single defined screening strategy, the time of prescription of the biological tests (before or after transfusion), the tests performed on cryopreserved blood samples, and the indications of the transfusion. RESULTS: The main screening strategy was HCV serology (second or third generation of enzyme immunoassays) with transaminase assessments before and after transfusion in 14% of the declared screening strategies. Screening tests were more frequently prescribed after transfusion, in at least 64% of the declared screening strategies according to the volume of transfusion. HCV serology was the common test prescribed in 61 and 50% of the screening strategies for low and high-volume transfusion respectively. The screening strategies showed a large heterogeneity combining HCV serology, transaminase assessment, before or after transfusion. CONCLUSION: A great heterogeneity of screening strategies was found. The most frequent was HCV serology with transaminase assessment before and after transfusion. Recommendations on screening strategies are needed in order to limit practice heterogeneity. This study will help building a cost-efficacy model in order to guide public health decision making.

Alanine Transaminase↗

PubMed2001

Cytochromes c555 from the hyperthermophilic bacterium Aquifex aeolicus (VF5). 1. Characterization of two highly homologous, soluble and membranous, cytochromes c555.

Two distinct class I (monoheme) c-type cytochromes from the hyperthermophilic bacterium Aquifex aeolicus were studied by biochemical and biophysical methods (i.e., optical and EPR spectroscopy, electrochemistry). The sequences of these two heme proteins (encoded by the cycB1 and cycB2 genes) are close to identical (85% identity in the common part of the protein) apart from the presence of an N-terminal stretch of 62 amino acid residues present only in the cycB1 gene. A soluble cytochrome was purified and identified by N-terminal sequencing as the cycB2 gene product. It showed an alpha-peak at 555 nm, an E(m) value of +220 mV, and electron paramagnetic resonance parameters of gz = 2.89, gy = 2.287, and gx = 1.52. A firmly membrane-bound cytochrome characterized by nearly identical properties was detected and attributed to the cycB1 gene product. The very high degree of homology of its N-terminal part to cytochrome c553 from Heliobacterium gestii strongly suggests it to be anchored to the membrane via N-terminally attached lipid molecules. The two heme proteins were named cytochrome c555s (soluble) and cytochrome c555m (membranous). Electron paramagnetic resonance on partially ordered membrane multilayers suggests that the solvent-exposed heme domain of cytochrome c555m is flexible with respect to the membrane plane. Possible functional roles for both cytochromes are discussed.

Adaptation, Biological↗

PubMed2001

Cytochromes c555 from the hyperthermophilic bacterium Aquifex aeolicus. 2. Heterologous production of soluble cytochrome c555s and investigation of the role of methionine residues.

The cycB2 gene encoding the soluble cytochrome c555s from Aquifex aeolicus, an hyperthermophilic organism, has been cloned and expressed using Escherichia coli as the host organism. The cytochrome was successfully produced in the periplasm of an E. coli strain coexpressing the ccmABCDEFGH genes involved in the cytochrome c maturation process. Comparison of native and recombinant cytochrome c555s shows that both proteins are indistinguishable in terms of spectroscopic and physicochemical properties. Since two different methionine residues are present in the sequence stretch usually providing the sixth ligand to the heme iron, site-directed mutagenesis has been performed in order to identify the methionine serving as the axial ligand. Two single mutations were introduced, leading to the replacement of each methionine by a histidine residue. Characterization of both mutants, M78H and M84H cytochromes c555s, using biochemical and biophysical techniques has been carried out. The M84H mutant exhibits spectral features identical to those of native cytochrome. Its redox midpoint potential is decreased by 40 mV. By contrast, substitution of methionine 78 by a histidine residue strongly alters the structural and physicochemical properties of the molecule which exhibits characteristics of His/His iron coordination type rather than His/Met. These results allow us to identify methionine 78 as the sixth ligand of cytochrome c555s heme iron. Preliminary results on the thermostability of the native and mutant cytochromes c555 are also reported.

Amino Acid Substitution↗

PubMed2001

Cobalt(I)-mediated cycloisomerization of enynes: mechanistic insights.

[CpCo(CO)2] catalyzes the cycloisomerization of 1,n-enynes to afford selectively five- and six-membered ring systems in high yields. The factors governing the cyclization have been explored and we have discovered that the reaction associates two different, but complementary, reactivities of the cobalt(I) complexes. By a judicious choice of the substitution of the enyne, it was also possible to isolate a cyclobutene that arises from a cobaltcyclopentene

Journal Article↗

PubMed2001

Enzymatic reduction of chromate: comparative studies using sulfate-reducing bacteria. Key role of polyheme cytochromes c and hydrogenases.

Various sulfate-reducing bacteria of the genera Desulfovibrio and Desulfomicrobium were tested and compared for enzymatic reduction of chromate. Our study demonstrated that the ability to reduce chromate is widespread among sulfate-reducing bacteria. Among them, Desulfomicrobium norvegicum reduced Cr(VI) with the highest reaction rate. This strain grew in the presence of up to 500 microM chromate, but Cr(VI) reduction in the absence of sulfate was not associated with growth. The presence of chromate induced morphological changes and leakage of periplasmic proteins into the medium. The ability of isolated polyheme cytochromes c from sulfate- and sulfur-reducing bacteria to reduce chromate was also analyzed. Tetraheme cytochrome c3(Mr. 13,000) from Desulfomicrobium norvegicum showed twice as much activity as either tetraheme cytochrome c3 from Desulfovibrio vulgaris strain Hildenborough or triheme cytochrome c7 from Desulfuromonas acetoxidans. Results with cytochromes c3 and other c-type cytochromes altered by site-directed mutagenesis indicated that negative redox potential hemes are crucial for metal reductase activity. The present study also demonstrated that the (Fe) hydrogenase from sulfate-reducing bacteria could reduce chromate.

Chromates↗

PubMed2001

Combination of thymidine phosphorylase gene transfer and deoxyinosine treatment greatly enhances 5-fluorouracil antitumor activity in vitro and in vivo.

We reported previously that 5-fluorouracil (FUra) efficacy could be enhanced by increasing tumoral thymidine phosphorylase (TP) activity. Potentiated TP yield was achieved by either transfecting cells with human TP gene (A. Evrard et al., Br. J. Cancer, 80: 1726-1733, 1999) or associating FUra with 2'-deoxyinosine (d-Ino), a modulator providing the tumors with TP cofactor deoxyribose 1-phosphate (J. Ciccolini et al., Clin. Cancer Res., 6: 1529-1535, 2000). The purpose of the present work was to study the effects of a combined modulation (TP gene transfer + use of d-Ino) on the sensitivity to FUra of the LS174T human colorectal cell line. Results showed a near 4000 times increase of cell sensitivity in vitro after double (genetic + biochemical) modulation. This potentiation of tumor response was accompanied by a total change in the FUra anabolic pathway with a 5000% increase of cytosolic fluorodeoxyuridine monophosphate, a stronger and longer inhibition of thymidylate synthase, and 300% augmentation of DNA damage. Besides, whereas thymidine failed to inhibit FUra cytotoxicity in LS174T wild-type cells, the potentiation of the antitumor activity observed in the modulating regimen was partly reversed by thymidine, indicative of thymidylate synthase as the main drug target. The impact of this double modulation was next investigated in xenograft-bearing nude mice. Results showed that whereas FUra alone was completely ineffective on wild-type tumor growth, the size of TP-transfected tumors in animals treated with the FUra/d-Ino combination was reduced by 80% (P < 0.05). Our results suggest that FUra exhibits stronger antiproliferative activity when activated via TP through the DNA pathway and that high tumoral TP activity therefore leads to enhanced sensitivity to fluoropyrimidines.

Animals↗

PubMed2000

Escherichia coli is able to produce heterologous tetraheme cytochrome c(3) when the ccm genes are co-expressed.

The production of Desulfovibrio vulgaris Hildenborough cytochrome c(3) (M(r) 13000), which is a tetraheme cytochrome, in Escherichia coli was examined. This cytochrome was successfully produced in an E. coli strain co-expressing the ccmABCDEFGH genes involved in the cytochrome c maturation process. The apocytochrome c(3) was matured in either anaerobic or aerobic conditions, but aerobic growth in the presence of delta-aminolevulinic acid was found to be best for cytochrome c(3) production. Site-directed mutagenesis was performed to investigate the effect of the presence of four amino acids in between the two cysteines of the heme binding sites 2 and 4 on the maturation of holocytochrome c(3) in E. coli.

Aminolevulinic Acid↗

PubMed2000

Comparison and improvement of the determinations of actinide low activities using several alpha liquid scintillation spectrometers

We applied three procedures using two a liquid scintillation spectrometers (PERALS and TRI-CARB) and two scintillation cocktails (Alphaex and Ultima Gold LLT) for the determination of alpha-emitter low activities. For each procedure, the limit of detection, the resolution, the separation factor, and the Fischer coefficient were determined in order to perform 232U-234U-238U isotopic measurements. The deconvolution usually performed is clean when the PERALS spectrometer is used. This is not possible for the TRI-CARB spectrometer using the Ultima Gold LLT scintillation cocktail. This problem was solved by combining the advantages of both techniques using the Alphaex scintillation cocktail in the TRI-CARB spectrometer. Under these conditions, the limit of detection was improved, the resolution decreased from 500-800 to 420-590 keV, and the separation factor increased from 0.9 to 1.1-1.2. This third procedure was applied with success for 232U-234U-238U isotopic experiments.

Journal Article↗

PubMed2000

Intraprotein radical transfer during photoactivation of DNA photolyase.

Amino-acid radicals play key roles in many enzymatic reactions. Catalysis often involves transfer of a radical character within the protein, as in class I ribonucleotide reductase where radical transfer occurs over 35 A, from a tyrosyl radical to a cysteine. It is currently debated whether this kind of long-range transfer occurs by electron transfer, followed by proton release to create a neutral radical, or by H-atom transfer, that is, simultaneous transfer of electrons and protons. The latter mechanism avoids the energetic cost of charge formation in the low dielectric protein, but it is less robust to structural changes than is electron transfer. Available experimental data do not clearly discriminate between these proposals. We have studied the mechanism of photoactivation (light-induced reduction of the flavin adenine dinucleotide cofactor) of Escherichia coli DNA photolyase using time-resolved absorption spectroscopy. Here we show that the excited flavin adenine dinucleotide radical abstracts an electron from a nearby tryptophan in 30 ps. After subsequent electron transfer along a chain of three tryptophans, the most remote tryptophan (as a cation radical) releases a proton to the solvent in about 300 ns, showing that electron transfer occurs before proton dissociation. A similar process may take place in photolyase-like blue-light receptors.

DNA Repair↗

PubMed2000

A sequential electron transfer from hydrogenases to cytochromes in sulfate-reducing bacteria.

A central step in the energy metabolism of sulfate-reducing bacteria is the oxidation of molecular hydrogen, catalyzed by a periplasmic hydrogenase. The resulting electrons are then transferred to various electron transport chains and used for cytoplasmic sulfate reduction. The complex formation between [NiFeSe] hydrogenase and the soluble periplasmic polyheme cytochromes from Desulfomicrobium norvegicum was characterized by cross-linking experiments, BIAcore and kinetics analysis. Analysis of electron transfer between [NiFeSe] hydrogenase and octaheme cytochrome c(3) (M(r) 26¿ omitted¿000) pointed out that this cytochrome is reduced faster in the presence of catalytic amounts of tetraheme cytochrome c(3) (M(r) 13¿ omitted¿000) isolated from the same organism. The activation of the hydrogenase-dependent reduction of polyheme cytochromes by cytochrome c(3) (M(r) 13¿ omitted¿000), which is now described in both Desulfovibrio and Desulfomicrobium, is proposed as a general mechanism. During this process, cytochrome c(3) (M(r) 13¿ omitted¿000) would act as an electron shuttle in between hydrogenase and the polyheme cytochromes and its conductivity appears to be an important factor.

Cytochrome c Group↗