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Biomedical subjects

A Nicolas

Publications and source records attributed to A Nicolas.

At least 73 records · Page 4Linked to original sources
PubMed1996

Advances of human core temperature minimum and maximal paradoxical sleep propensity by ambient thermal transients.

By using slow thermal transients of reduced amplitude (+/- 3 degrees C (thermoneutrality in humans sleeping nude) during only 1 night (experimental, EX), we have advanced the minimum of rectal temperature (Tre) and the peak of their paradoxical sleep propensity (PPSP) of sleeping subjects. During this EX night Tre minimum was significantly (P = 0.0001) advanced by 143 min versus that observed during baseline night spent at thermoneutrality. The advance of PPSP was objectivated by the more rapid cumulation of paradoxical sleep (P = 0.02) during the second half of EX night, i.e. strictly after the occurrence (around 0330 h) of the new Tre minimum, and by the earlier occurrence of its barycentric point (P = 0.04) between 0330 and 0700 h. The involvement of the central thermoregulatory system on phase-shifting mechanisms is discussed.

Adult↗
PubMed1996

Endo.SK1: an inducible site-specific endonuclease from yeast mitochondria.

Site-specific endonucleases have been found in various eukaryotic organelles such as mitochondria, chloroplasts and nuclei. These endonucleases initiate site-specific or homologous gene conversion in mitochondrial and nuclear DNA. Here, we report a new site-specific endonuclease activity, Endo.SK1, identified in mitochondria of strain SK1, a homothallic diploid strain of Saccharomyces cerevisiae. Nucleotide sequences around the Endo.SK1-cleavage sites are different from those of known yeast site-specific endonucleases. The Endo.SK1 activity is, at least partly, specified by a gene in the SK1-derived mitochondria. A novel feature of the Endo.SK1 activity is its inducibility: the endonuclease activity was induced by ca. 40-fold by transfer of cells from a glucose medium into an acetate medium, and was then repressed. This transient induction was independent of the ploidy level of the cells, and coincided with induction of fumarase, a mitochondrial enzyme involved in the TCA cycle. Co-induction and co-repression of the mitochondrial site-specific endonuclease activity and a respiration-related enzyme indicate that the endonuclease activity in regulated in response to physiological conditions, and suggest a possible role for the endonuclease in mitochondrial DNA metabolism.

Bacillus Phages↗
PubMed1996

Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state.

Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.

Amino Acid Sequence↗
PubMed1996

Dynamics of Fusarium solani cutinase investigated through structural comparison among different crystal forms of its variants.

In characterizing mutants and covalently inhibited complexes of Fusarium solani cutinase, which is a 197-residue lipolytic enzyme, 34 variant structures, crystallizing in 8 different crystal forms, have been determined, mostly at high resolution. Taking advantage of this considerable body of information, a structural comparative analysis was carried out to investigate the dynamics of cutinase. Surface loops were identified as the major flexible protein regions, particularly those forming the active-site groove, whereas the elements constituting the protein scaffold were found to retain the same conformation in all the cutinase variants studied. Flexibility turned out to be correlated with thermal motion. With a given crystal packing environment, a high flexibility turned out to be correlated with a low involvement in crystal packing contacts. The high degree of crystal polymorphism, which allowed different conformations with similar energy to be detected, made it possible to identify motions which would have remained unidentified if only a single crystal form had been available. Fairly good agreement was found to exist between the data obtained from the structural comparison and those from a molecular dynamics (MD) simulation carried out on the native enzyme. The crystallographic approach used in this study turned out to be a suitable tool for investigating cutinase dynamics. Because of the availability of a set of closely related proteins in different crystal environments, the intrinsic drawback of a crystallographic approach was bypassed. By combining several static pictures, the dynamics of the protein could be monitored much more realistically than what can be achieved on the basis of static pictures alone.

Carboxylic Ester Hydrolases↗
PubMed1996

Acyl glycerol hydrolases: inhibitors, interface and catalysis.

The last five years have witnessed the solution of a large number of lipase structures, which has led, among other insights, to the structural interpretation of the interfacial activation phenomenon in terms of 'lid' opening. This interpretation has been extended this year to include phospholipase A2. Recent structural studies on lipases have provided data on the detailed mechanisms underlying the behaviour of lipases: how they bind to inhibitors or substrates, and what interactions occur between their hydrophobic face and hydrophobic molecules, for example. In addition, studies on cutinase point mutants have shed some light on the role of the oxyanion hole in lipolytic catalysis.

Animals↗
PubMed1996

Sensing of DNA non-homology lowers the initiation of meiotic recombination in yeast.

BACKGROUND: Meiotic recombination between homologous chromosomes in the yeast Saccharomyces cerevisiae is initiated by the formation of DNA double-strand breaks (DSBs). The mechanism of DSB formation and the factors that determine their frequency and location have yet to be elucidated. Current studies of meiotic recombination are also concerned with the question of the functional relationship between DSB formation and the other meiotic processes of homology searching, pairing and synapsis of homologues. RESULTS: To test if DNA identity is required for high levels of DSBs and recombination, we have asked whether small DNA heterologies (140-547 bp) located within the well characterized ARG4 initiator of meiotic recombination, can affect DSB formation and gene conversion events in the ARG4 locus. The present physical and genetic analyses show that some heterologies reduced recombination frequencies without altering DSB formation, whereas others reduced both DSB and gene conversion frequencies. CONCLUSIONS: These results suggest that DNA heterologies overlapping a recombination initiator impair meiotic gene conversion at two levels. First, some heterologies affect the level of DSB formation, revealing the existence of an anti-initiation process sensing the presence of sequence non-homology between the homologous chromosomes. Second, heterologies can impair the successful processing of the recombination intermediates once DSBs are made. We present a model for interhomologue cross-talks involving chromosomal and DNA/DNA interactions.

Argininosuccinate Lyase↗
PubMed1996

[Method selected for the determination of creatinine in plasma or serum. Choice of optimal conditions of measurement].

The method selected by the SFBC (Société française de biologie clinique) is derived from the colorimetric reaction of creatinine with alkaline picrate, measured kinetically, without any pretreatment step. The key parameters of the reaction determining the quality of the results are studied, with special regard to samples including known interferents. The aims of the study were to gain an optimal analytical sensitivity and to reduce main interferences (acetoacetate, bilirubine, glucose, protein) which plague the Jaffé reaction, through a comprehensive study of the reagents, of their concentrations and of the analytical procedures. The selected concentrations (in the test) are: 150 mmol/L sodium hydroxide, 10 mmol/L picric acid and 2 g/L sodium dodecyl sulfate. Ten millilitres of a BRIJ solution (30% volvol) are added to the reagent. The operating procedures are as follow: sample ratio 0.07 to 0.08; wavelength 505 to 510 nm; temperature 37 degrees C; incubation of the specimen with the alkaline reagent 5 mn (at least), before starting the reaction with picric acid. A seric calibrator is recommended. The first measurement is taken 20 to 40 s after starting the reaction. Total measurement time is 120 to 150 seconds.

Blood Chemical Analysis↗
PubMed1996

[Validation and study of the transferability of the selected technique by SFBC for the determination of creatinine in plasma].

A selected method for the determination of creatinine in plasma, using the reaction with alkaline picrate without prior pretreatment has been proposed by the Commission 'Validation de techniques' in the SFBC (Société Française de biologie clinique). The transferability step was conducted in seven laboratories, equipped with different automatic analyzers, using analytical procedures derived from the recommended method. Its goal was to test whether the original analytical performances could be maintained and consistent results obtained. The validation step was designed to evaluate the linearity limits of the analytical range, the detection limit, to assess accuracy as compared to a high performance liquid chromatography and to investigate the effect of the main interferents. Linearity limits are 15 and 2000 mumol/L. The detection limit is 3 to 8 mumol/L according to the analytical systems. The selected method can fulfil the set imprecision goals: intralaboratory CV minus than 2% (within-run), minus than 4% (run-to-run), interlaboratory CV minus than 5% (for 100 mumol/L creatinine). Inaccuracy evaluated for the chosen control sera is 1 to 15% as compared to the chromatographic method, according to the sera and to the analytical systems. The results obtained with the selected method are more consistent with the HPLC than are those obtained with an alkaline picrate method without SDS or with an enzymatic method. No interference could be demonstrated for acetoacetate (up to 8 mmol/L), hemoglobin (up to 210 mumol/L), unconjugated bilirubin (up to 250 mumol/L), glucose (up to 30 mmol/L), IgG (up to 45 g/L), albumin (up to 60 g/L). The effect of cephalosporins depends on the molecule. The reagents are stable for at least 6 months when stored in closed vials at +20 degrees C. The alkaline reagent is stable 30 days at +4 degrees C. Reference limits (0.025 and 0.975 fractiles) have been established for healthy adults. They are respectively 73 to 126 mumol/L for men and 59 to 100 mumol/L for females.

Adult↗
PubMed1995

The nucleotide mapping of DNA double-strand breaks at the CYS3 initiation site of meiotic recombination in Saccharomyces cerevisiae.

Initiation of meiotic recombination in the yeast Saccharomyces cerevisiae occurs by localized DNA double-strand breaks (DSBs) at several locations in the genome, corresponding to hot spots for meiotic gene conversion and crossing over. The meiotic DSBs occur in regions of chromatin that are hypersensitive to nucleases. To gain insight into the molecular mechanism involved in the formation of these DSBs, we have determined their positions at the nucleotide level at the CYS3 hot spot of gene conversion on chromosome I. We found four major new features of these DSBs: (i) sites of DSBs are multiple with varying intensities and spacing within the promoter region of the CYS3 gene; (ii) no consensus sequence can be found at these sites, indicating that the activity involved in DSB formation has little or no sequence specificity; (iii) the breaks are generated by blunt cleavages; and (iv) the 5' ends are modified in rad50S mutant strains, where the processing of these ends is known to be prevented. We present a model for the initiation of meiotic recombination taking into account the implications of these results.

Base Sequence↗
PubMed1995

Influence of sample preparation on cellular glutathione recovery from adherent cells in culture.

During the last decade, the unbound glutathione content of cultured adherent cells has become a very important biological marker for many pharmacological and toxicological in vitro studies with regard to the protective role of the tripeptide in its reduced form (GSH). However, the literature does not provide extensive information on the influence of sample preparation on cellular GSH and thiol analyses. Using the fibroblast-like V79 cell line as model, we undertook a comparative study of the efficiency of different procedures reported in the literature with respect to GSH recovery. Depending on the preanalytical step, up to 10-fold discrepancies could be observed in the recovery of intracellular GSH. Different parameters that must be controlled in order to maximize GSH recovery are discussed. The optimal strategy consisted in rapid perchloric acid deproteinization performed directly in the dish, which was extremely valuable for preparing GSH samples from adherent cells, and especially from cells expressing elevated gamma-glutamyl transferase activity.

Animals↗
PubMed1995

Intracellular concentrations of fura-2 and fura-2/am in vascular smooth muscle cells following perfusion loading of fura-2/am in arterial segments.

A new method for the determination of tissue concentrations of Fura-2 and Fura-2/AM was developed based upon acetonitrile extraction followed by RP-HPLC separation (using tetrahexylammonium as counter-ion), post-column alkaline hydrolysis of Fura-2/AM, and fluorimetric detection. The detection limit was 1.2 nM and 1 nM for Fura-2 and Fura-2/AM, respectively. When this technique was applied to perfusion-loaded segments of the rat tail artery, intracellular concentrations of Fura-2 determined by tissue disruption were 10 times those obtained by comparing the increase in fluorescence at the isoemissive point (following loading), with a calibration curve for Fura-2. Loading conditions of 90 min at [Fura-2/AM]e = 5 microM were optimal in terms of [Fura-2]i which attained a concentration not significantly different from [Fura-2/AM]e. Under such conditions, however, Fura-2/AM also accumulated in the arterial wall. Although incompletely de-esterified, Fura-2/AM metabolites produced by in vitro incubation of Fura-2/AM with pig liver esterases could be easily detected, fluorescent forms of Fura-2 with a different sensitivity for calcium were not detected in arterial extracts.

Animals↗
PubMed1995

Ion-exchange liquid chromatography method with indirect UV detection for the assay of choline in pharmaceutical preparations.

A high-performance liquid chromatography (HPLC) method coupled with indirect UV spectrophotometric detection was developed for the routine control of pharmaceutical preparations containing choline. Optimization of the HPLC system using silica-based cation exchange columns was focused on the combination in the mobile phase between a UV transparent co-ion and a UV active cationic probe. The selected mobile phase consisted of a mixture of ethanol-ammonium acetate buffer (100 mM, pH 5.0) (20:80, v/v) containing 0.3 mM of 3-hydroxytyramine as chromophoric probe; UV detection was operated at 280 nm. The HPLC system provided full selectivity with regard to the multi-ingredient pharmaceutical matrices tested. Quantitative analysis was validated by testing linearity of the method between 50 and 150% of the theoretical content (coefficient of correlation greater than 0.99) and repeatability (relative standard deviation less than 2.5%; n = 5).

Choline↗
PubMed1995

Electrochemical oxidation of CBS-113 A, a new anti-inflammatory drug: applicability to liquid chromatography-electrochemical detection.

The electrochemical properties of a new non-steroidal anti-inflammatory drug (2-hydroxy-4-methylphenyl-2-aminothiazole hydrochloride; CBS-113 A) have been studied using voltammetry in direct current and cyclic modes at glassy carbon disk electrodes. The results show an oxidative process with a diffusion-controlled and a reversible mechanism; these data agree with those obtained in a reversed-phase high-performance liquid chromatography (HPLC) system coupled either with amperometric (single glassy carbon electrode) or coulometric detection (two porous graphite electrodes in series) set at potentials of +0.65 V (vs. Ag/AgCl) and +0.4 V (vs. Pd/H2), respectively. Similar electrochemical properties were found for 2-hydroxyphenyl-2-amino-5'-methylthiazole hydrochloride (RD-1546) which is a potential internal standard. An HPLC system coupled with a UV detector (lambda = 272 nm) and an amperometric detector (+0.65 V) showed a gain in sensitivity of about 10 using electrochemical detection (ED) for the determination of CBS-113 A in human plasma. Linearity range, precision and accuracy were calculated and showed the potential application of HPLC-ED to pharmacokinetic studies of CBS-113 A in plasma.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1995

Implementation of napping in industry and the problem of sleep inertia.

It seems reasonable to believe that in specific situations napping at the work place would be possible and used if authorized and encouraged. Very short naps could have very positive long-term effects on biological functions. Training someone to sleep for short periods appears feasible if there is a high motivation to do so. Sleep inertia can be considered as one of the main limiting factors in napping strategy. Sleep inertia depends on different factors such as sleep stage preceding the awakening, temporal placement of the nap, duration of nap and wakefulness preceding it, etc. The effects of sleep inertia might be different depending on the type of task, and a reactivation technique applied immediately after awakening may remove it. Despite the fact that its implementation in industry raises some practical issues, napping can be considered as a possible strategy to increase the vigilance level of night workers.

Journal Article↗
PubMed1995

Blood and plasma glutathione measured in healthy subjects by HPLC: relation to sex, aging, biological variables, and life habits.

We report an HPLC method for measuring the concentrations of reduced (GSH) and total (GSHt) free glutathione in human plasma and whole blood. The chromatographic step was coupled with a postcolumn derivatization reaction and fluorometric detection. The linear range was 0.81-13.02 mumol/L, and the detection limit was 0.13 mumol/L. In healthy adults (ages 18-73 years), mean concentrations were 941 +/- 155 mumol/L for GSHt and 849 +/- 63 mumol/L for GSH in blood (107 men, 94 women), and 3.39 +/- 1.04 mumol/L for GSH in plasma (66 men, 58 women). Blood GSHt but not GSH was significantly lower in children (32 boys, 32 girls: 872 +/- 157 mumol/L) than in adults. Blood GSHt and GSH appeared to be correlated positively with the number of cigarettes smoked per day and the regular practice of physical exercise, and negatively with alcohol abstinence. We observed positive correlations between blood GSHt and cholesterol and calcium concentrations, and between blood GSH and cholesterol concentration.

Adolescent↗