Biomedical subjects

A Nicolas

Publications and source records attributed to A Nicolas.

At least 91 records · Page 5Linked to original sources

PubMed1994

Initiation of recombination in Saccharomyces cerevisiae haploid meiosis.

In most eukaryotes during prophase I of meiosis, homologous chromosomes pair and recombine by coordinated molecular and cellular processes. To directly test whether or not the early steps of the initiation of recombination depend on the presence of a homologous chromosome, we have examined the formation and processing of DNA double-strand breaks (DSBs, the earliest physical landmark of recombination initiation) in various haploid Saccharomyces cerevisiae strains capable of entering meiosis. We find that DSBs occur in haploid meiosis, showing that the presence of a homolog is not required for DSB formation. DSBs occur at the same positions in haploid and diploid meioses. However, these two types of meiosis exhibit subtle differences with respect to the timing of formation and levels of DSBs. In haploid meiosis, a slower rate of DSB formation and a reduction in the frequency of DSB (at one of the three sites analyzed) were observed. These results might indicate that interactions between homologs play a role in the formation of meiotic DSBs. Furthermore, haploid strains exhibit a pronounced delay in the disappearance of meiotic DSBs compared to diploid strains, which suggests that sister chromatid interactions for DSB repair are inhibited in haploid meiosis.

Crosses, Genetic↗

PubMed1994

Changes in chromatin structure at recombination initiation sites during yeast meiosis.

Transient double-strand breaks (DSBs) occur during Saccharomyces cerevisiae meiosis at recombination hot spots and are thought to initiate most, if not all, homologous recombination between chromosomes. To uncover the regulatory mechanisms active in DSB formation, we have monitored the change in local chromatin structure at the ARG4 and CYS3 recombination hot spots over the course of meiosis. Micrococcal nuclease (MNase) digestion of isolated meiotic chromatin followed by indirect end-labeling revealed that the DSB sites in both loci are hypersensitive to MNase and that their sensitivity increases 2- to 4-fold prior to the appearance of meiotic DSBs and recombination products. Other sensitive sites are not significantly altered. The study of hyper- and hypo-recombinogenic constructs at the ARG4 locus, also revealed that the MNase sensitivity at the DSB site correlates with both the extent of DSBs and the rate of gene conversion. These results suggest that the local chromatin structure and its modification in early meiosis play an important role in the positioning and frequency of meiotic DSBs, leading to meiotic recombination.

Argininosuccinate Lyase↗

PubMed1994

Human core temperature minimum can be modified by ambient thermal transients.

By using slow linear thermal transients (+/- 0.025 degree C/min) of reduced amplitude (+/- 3 degrees C around thermoneutrality), we were able to advance the minimum of human internal temperature (Ti) during nocturnal sleep. During experimental night the minimum of esophageal (Tes) and rectal (Tre) temperature were respectively advanced by 1.6 h (P < 0.01) and 2.6 h (P < 0.001) in comparison to reference night spent at thermoneutrality. It must be emphasized that the provoked advance of nocturnal Ti minimum was not accompanied by any change in sleep latency, efficiency, SWS and REM sleep percentages. The result shows that appropriate ambient temperature transient changes could be used to modify the course of human Ti one of the major biological rhythms usually considered as resistant to sleep-wake rhythm manipulation.

Adult↗

PubMed1994

High-performance liquid chromatographic enantioselective assay for the measurement of ketoprofen glucuronidation by liver microsomes.

A stereoselective high-performance liquid chromatographic (HPLC) method was developed to study the in vitro glucuronidation of ketoprofen enantiomers by liver microsomes. The HPLC system consisted of a Superspher 100 RP 18 end-capped column eluted with a mixture of acetonitrile and 10 mM tetrabutylammonium bromide in 1 mM potassium phosphate adjusted to pH 4.3 (30:70, v/v). Ultraviolet detection was performed at a wavelength of 254 nm. The capacity factors of S-ketoprofen glucuronide, R-ketoprofen glucuronide and R,S-ketoprofen were 12.8, 14.5 and 18.1, respectively. Sample pretreatment consisted of protein precipitation in microsomal incubation suspensions and further purification on a Sep Pak C18 cartridge before injection onto the HPLC system. Quantitation was performed with standard glucuronides biosynthetized with immobilized microsomes and purified by semi-preparative HPLC. The linearity of the method between 1.25 and 25.0 micrograms ml-1 (coefficient of correlation greater than 0.999), the repeatability (coefficient of variation = 1.2%; n = 5), and recovery (within 85%) were tested. The limit of detection was 10 ng for each glucuronide injected. The in vitro glucuronidation of R- and S-ketoprofen was measured in liver microsomes from man and from various animal species (dog, rat, rabbit). For both enantiomers, dog presented the highest specific activity. In contrast, the lowest activity was found in rabbit. On the other hand, the formation ratio of the S- and R-glucuronides of ketoprofen was close to 1 in man, rat and rabbit, but was 4.5 in dog, thus indicating that the reaction was stereoselective in this species.

Animals↗

PubMed1994

Polarity of meiotic gene conversion in fungi: contrasting views.

The frequency of meiotic gene conversion often varies linearly from one end of the gene to the other. This phenomenon has been called 'polarity'. In this review, we will primarily studies of polarity that have been done in the yeast Saccharomyces cerevisiae (ARG4 and HIS4 loci) and in Ascobolus (b2 locus) with an emphasis on possible mechanisms. The genetic and physical data obtained at these 'hotspots' of recombination strongly suggests that the formation of a polarity gradient reflects both the frequency of heteroduplex formation and the processing of this recombination intermediate by mismatch-repair-dependent processes.

Ascomycota↗

PubMed1994

Cutinase, a lipolytic enzyme with a preformed oxyanion hole.

Cutinases, a group of cutin degrading enzymes with molecular masses of around 22-25 kDa (Kolattukudy, 1984), are also able to efficiently hydrolyse triglycerides (De Geus et al., 1989; Lauwereys et al., 1991), but without exhibiting the interfacial activation phenomenom (Sarda et al., 1958). They belong to a class of proteins with a common structural framework, called the alpha/beta hydrolase fold (Martinez et al., 1992; Ollis et al., 1992). We describe herein the structure of cutinase covalently inhibited by diethyl-p-nitrophenyl phosphate (E600) and refined at 1.9-A resolution. Contrary to what has previously been reported with lipases (Brzozowski et al., 1991; Derewenda et al., 1992; Van Tilbeurgh et al., 1993), no significant structural rearrangement was observed here in cutinase upon the inhibitor binding. Moreover, the structure of the active site machinery, consisting of a catalytic triad (S120, H188, D175) and an oxyanion hole (Q121 and S42), was found to be identical to that of the native enzyme, whereas the oxyanion hole of Rhizomucor lipase (Brzozowski et al., 1991; Derewenda et al., 1992), like that of pancreatic lipase (van Tilbeurgh et al., 1993), is formed only upon enzyme-ligand complex formation. The fact that cutinase does not display interfacial activation cannot therefore only be due to the absence of a lid but might also be attributable to the presence of a preformed oxyanion hole.

Amino Acid Sequence↗

PubMed1994

Local sweating responses during recovery sleep after sleep deprivation in humans.

Changes in the central control of sweating were investigated in five sleep-deprived subjects (kept awake for 40 h) during their recovery sleep under warm ambient conditions [operative temperature (T(o)) was either 35 or 38 degrees C]. Oesophageal (T(oes)) and mean skin (Tsk) temperatures, chest sweat rate (msw,ch), and concomitant electro-encephalographic data were recorded. Throughout the night at 35 or 38 degrees C T(o), msw,ch changes were measured at a constant local chest skin temperature (Tch) of 35.5 degrees C. The results showed that body temperatures (T(oes) and Tsk) of sleep-deprived subjects were influenced by thermal and hypnogogic conditions. The msw,ch levels correlated positively with T(oes) in the subjects studied during sleep stage 1-2 (light sleep: LS), sleep stage 3-4 (slow wave sleep: SWS) and rapid eye movement (REM) sleep. Contrary to what has been reported in normal sleep, firstly, the T(oes) threshold for sweating onset differed between REM sleep and both LS and SWS, and, secondly, the slopes of the msw,ch versus T(oes) relationships were unchanged between REM and non-REM (i.e. LS or SWS) sleep. The changes observed after sleep deprivation were hypothesized to be due to alterations in the functioning of the central nervous system controller.

Adult↗

PubMed1994

Determination of josamycin residues in porcine tissues using high-performance liquid chromatography with pre-column derivatization and spectrofluorimetric detection.

A simple, selective and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the measurement of josamycin residues in four porcine tissues (i.e., muscle, liver, kidney and fat). The sample preparation consisted of a homogenization step in an acetonitrile-10 mmol l-1 phosphate buffer mixture, pH 6.0 (35 + 65), centrifugation and a liquid-liquid extractive clean-up of the resulting supernatant with isooctane. Pre-column derivatization of josamycin was performed using cyclohexa-1,3-dione in ammonium acetate buffer, pH 5.0 (90 degrees C for 2 h). The derivative was chromatographed in an isocratic reversed-phase HPLC system. A LiChrospher RP 18 end-capped (5 microns) column was eluted with an acetonitrile-methanol-10 mmol l-1 phosphate buffer mixture, pH 6.0 (45 + 5 + 50). The capacity factor of the josamycin derivative was 17.5. Detection was achieved using spectrofluorimetry (lambda ex = 375 nm; lambda em = 450 nm). The structure of the derivative was assessed by using mass spectrometry. Full selectivity was obtained in the HPLC system versus other macrolide antibiotics (tylosin, spiramycin and erythromycin), aldehydes (formaldehyde, acetaldehyde and benzaldehyde) and endogenous compounds. Linearity and repeatability were tested. Correlation coefficients, for calibration curves in the range of 0.1-3.2 micrograms g-1, were greater than 0.999 for all tissues and the relative standard deviation (S(r)) was 4.9% (1.6 micrograms g-1; n = 6); recovery was higher than 88%.

Adipose Tissue↗

PubMed1994

Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae.

Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining. To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters. We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation [100 ng or less], recombination between similar but not identical [homeologous] P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions (the rare transformants which are obtained contain plasmids resulting from deletion-forming intramolecular events involving little or no sequence homology); (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain. The recombinant junctions of the rare illegitimate events obtained with S. cerevisiae are very similar to those observed in the repair of DSB in mammalian cells. Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products. We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product.

Animals↗

PubMed1993

A structural analysis of the bent kinetoplast DNA from Crithidia fasciculata by high resolution chemical probing.

The chemical probes potassium permanganate (KMnO4) and diethylpyrocarbonate (DEPC) have been used to study the conformation of bent kinetoplast DNA from Crithidia fasciculata at different temperatures. Chemical reactivity data shows that the numerous short A-tracts of this bent DNA adopt a similar structure at 43 degrees C. This conformation appears to be very similar to the conformation of A-tracts in DNA exhibiting normal gel mobility. The A-tract structure detected by chemical probing is characterized by a high degree of base stacking on the thymine strand, and by an abrupt conformational change at the 3' end of the adenine strand. In general, no major alteration of this A-tract specific structure was detected between 4-53 degrees C. However, probing with KMnO4 revealed two unusual features of the C. fasciculata sequence that may contribute to the highly aberrant gel mobility of this DNA: 1) the B DNA/A-tract junction 5' dC/A3-6 3'. 5' dT3-6/G 3' is disproportionately represented and is conformationally distinct from other 5' end junctions, and 2) low temperature favors a novel strand-specific conformational distortion over a 20 base pair region of the bent kinetoplast DNA. Presence of the minor groove binding drug distamycin had little detectable effect on the A-tract conformation. However, distamycin did inhibit formation of the novel KMnO4 sensitive low temperature structure and partially eliminated the anomalous gel mobility of the kinetoplast DNA. Finally, we describe a simple and reproducible procedure for the production of an adenine-specific chemical DNA sequence ladder.

Animals↗

PubMed1993

The control in cis of the position and the amount of the ARG4 meiotic double-strand break of Saccharomyces cerevisiae.

During meiosis, a transient DNA double-strand break (DSB) occurs in the promoter region (positions -200/-185) of the Saccharomyces cerevisiae ARG4 gene and is a likely intermediate in the initiation of meiotic gene conversion events in this region. We report here a functional analysis of the ARG4 DSB based on the study of various deletions in this chromosomal region. We have identified several cis-acting elements located within the -465/+3 region of the ARG4 promoter that control the formation of this DSB. The -465/-317 region includes a transcription terminator and is necessary for a normal amount of ARG4 DSB, but not for its positioning. The -316/-140 region can be replaced by an unrelated DNA sequence where a meiotic DSB then occurs, suggesting that the site of DSB is not sequence-specific, but is positioned at a fixed distance from the adjacent -139/+3 region. Also, in all strains constructed, the amount of meiotic DSB is correlated with the frequency of gene conversion in ARG4, which provides a strong argument for the initiation of gene conversion by a DSB in this region of the yeast genome.

DNA Mutational Analysis↗

PubMed1993

Identification of two divergently transcribed genes centromere-proximal to the ARG4 locus on chromosome VIII of Saccharomyces cerevisiae.

We have sequenced a 3296 bp segment of the chromosome VIII adjacent to the 3' end of the ARG4 gene. This segment contains two divergently oriented open reading frames (YSC83 and YSC84). Northern blot analysis showed the presence of transcripts corresponding to these two open reading frames in vegetative cells. Levels of these transcripts increase five to ten-fold during sporulation. These two genes are not essential for vegetative growth or sporulation. Analysis of the putative protein products on the SwissProt database revealed that the C-terminal region of the Ysc84 protein contains a putative SH3 domain.

Amino Acid Sequence↗

PubMed1993

Electroencephalogram and cardiovascular responses to noise during daytime sleep in shiftworkers.

Intermittent noise occurring during sleep has been found to induce heart rate, peripheral vasomotor and electroencephalogram (EEG) changes. This study analysed these responses during the daytime and night-time sleep of shiftworkers doing a three shift system, to determine the influence of the inversion of the sleep-wake cycle on the sensitivity to noise. A group of 14 shiftworkers [aged 37 (SD 5) years] underwent an habituation daytime sleep, two experimental daytime sleeps and a night-time sleep. Traffic noises were presented during sleep [truck, 71 dB(A); motorbike, 67 dB(A); and car, 64 dB(A)] at a rate of nine each hour. The EEG measurements of sleep, electrocardiogram and finger pulse amplitude were recorded continuously. The results were expressed by computing the percentage of observed cardiac response (%HRR) and vasoconstrictive response (%FPR), magnitude of heart rate variation (heart rate response; HRR), percentage of reduction of the digital blood flow (finger pulse response, FPR), cardiac cost (CC = % HRR x HRR) and vasomotor cost (VC = % FPR x FPR). The results showed that, compared to night-time sleep, there was change in the structure of daytime sleep, that is an increase in slow wave sleep (SWS), especially stage 4 sleep decrease of stage 2 and rapid eye movement (REM) sleep latencies, and an earlier SWS and REM sleep barycentric point. During daytime sleep the % FPR was significantly smaller in SWS than in stage 2 or REM sleep. Large differences were observed in % HRR, HRR and CC between daytime sleep stages (SWS less than stage 2 less than REM sleep).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1993

Interaction of the alerting effect of noise with partial sleep deprivation and circadian rhythmicity of vigilance.

Only a few studies have been devoted to the interaction of noise with sleep deprivation and time of day. In a previous study we demonstrated that noise had an alerting effect on cognitive performance during the early night but not during the late night. However, it was not clear whether these different effects of noise were related to prior sleep debt or to time of day as both factors varied simultaneously. In the present experiment, we further studied this issue to identify which of these two factors was responsible for the noise effects. Analysis showed that, when performance was tested at different times with an equivalent prior sleep debt, noise improved speed of response at 0500 but not at 0800 at which variability of response time increased. Noise had no effect on errors. It is suggested that the effect of noise depends on the underlying arousal and raises arousal from its low level due either to time of day or to partial sleep deprivation.

Adult↗

PubMed1993

[T.O.M. ointment, breast feeding and serum estradiol in newborn infants. A double-blind, randomized controlled study].

OBJECTIVE: To study whether there is an iatrogenic transfer of oestradiol through the use of T.O.M. ointment administered to prevent or to cure trophic changes in the breast while breastfeeding and what the dangers of plasma levels of oestradiol in the newborn will be. TYPE OF STUDY: Controlled, randomised, double blind. SUBJECTS AND METHODS: 80 mother/infant couples were included in the study: 40 received the ointment containing oestradiol (the treated group), and 40 received an ointment without oestradiol (the non-treated group); the ointment was applied before each breastfeeding from the 2nd to the 6th or 7th day of life. Mothers' milk and blood from the newborn babies were taken for estimating the levels of oestradiol using a radio-immunological technique. The total quantity of ointment was measured for each subject. RESULTS: The mean level of oestradiol in the treated mothers' milk was significantly higher (p. 0.0014) than in the milk of the mothers who had not been treated. The concentration of oestradiol in the plasma of the newborn dropped from the first to the last day of the trial without any difference in the treated or non-treated groups no matter what the sex of the child was, nor the quantity of ointment used.

Breast Diseases↗

PubMed1992

The Saccharomyces cerevisiae ARG4 initiator of meiotic gene conversion and its associated double-strand DNA breaks can be inhibited by transcriptional interference.

In the yeast Saccharomyces cerevisiae, as in other eukaryotes, some regions of the genome have a much higher level of meiotic gene conversion than others. Previous deletion analysis indicated that the sequence necessary for the high level of gene conversion within the ARG4 region defined an initiation site located between positions -316 and -37 [relative to the first base pair (+1) of the ARG4 coding sequence] of the ARG4 promoter. To test whether this sequence is sufficient to promote gene conversion in a novel chromosomal context, we inverted on the chromosome various DNA fragments including the implicated region and the ARG4 coding sequence. Surprisingly, these inversions resulted in the loss of the normal recombination properties and double-strand-break formation associated with this process. By Northern analysis, we found that a transcript traverses the ARG4 initiation site in these inversion mutants but not in the wild type. When transcription through this region was prevented by a transcription terminator, the activity of the initiation site and the formation of double-strand breaks were restored. From these results and from complementary deletion analysis in the normal ARG4 orientation, we conclude that the activity of the ARG4 initiation site requires protection from transcriptional interference.

Chromosome Inversion↗

PubMed1992

A multisite integrative cassette for the yeast Saccharomyces cerevisiae.

We have developed a cassette for the integration of cloned DNA sequences at multiple sites in the Saccharomyces cerevisiae genome, taking advantage of the naturally repeated sigma sequences. This cassette contains one engineered sigma element which allows the targeting of an embedded gene at different genomic sigma elements by gene replacement. Two yeast genes, ARG4 and URA3, were thus integrated in the absence of any bacterial sequences, individually or sequentially on twelve chromosomes. Consequently, these studies led to the genetical tagging of individual members of the sigma family.

Base Sequence↗

PubMed1992

Recombination between similar but not identical DNA sequences during yeast transformation occurs within short stretches of identity.

Interactions between similar but not identical (homeologous) DNA sequences play an important biological role in the evolution of genes and genomes. To gain insight into the underlying molecular mechanism(s) of genetic recombination, we have studied inter- and intramolecular homeologous recombination in S. cerevisiae during transformation. We found that homeologous DNAs recombine efficiently. Hybrid sequences were obtained between two mammalian cytochrome P450 cDNAs, sharing 73% identity, and between the yeast ARG4 gene and its human homeologous cDNA, sharing 52% identity. Sequencing data showed that the preferred recombination events are those corresponding to the overall alignment of the DNA sequences and that the junctions are within stretches of identity of variable length (2-21 nt). We suggest that these events occur by a conventional homologous recombination mechanism.

Base Sequence↗