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Biomedical subjects

A Nicolas

Publications and source records attributed to A Nicolas.

At least 55 records · Page 3Linked to original sources
PubMed1999

[Mechanisms and control of meiotic recombination in the yeast Saccharomyces cerevisiae].

Recent studies in Saccharomyces cerevisiae have provided new insights in our understanding of the molecular mechanisms of meiotic recombination. Meiosis-specific DNA double-strand breaks have been detected and have been shown to be the lesions that initiate recombination events. These are located mostly in promoter regions where the chromatin is in an open configuration, and cluster in domains along the chromosome. They are likely to be made by a topoisomerase II-like protein encoded by the SPO11 gene. Several DNA intermediates in the meiotic double strand-break repair pathway have been characterised and several multi-protein complexes have been identified and shown to be involved at different steps in the repair pathway. The conservation of these protein complexes in higher eukaryotes suggests that the meiotic recombination mechanism could be conserved. With the application of the well characterised genetical, molecular, cytological and biochemical techniques and the recently developed technology for genomic studies (biochips), we can expect a rapid increase in our comprehension of the meiotic recombination process.

Chromosomes, Fungal↗
PubMed1998

Determination of cellular thiols and glutathione-related enzyme activities: versatility of high-performance liquid chromatography-spectrofluorimetric detection.

A high-performance liquid chromatography (HPLC) method to determine the most important cellular thiols [reduced glutathione (GSH), cysteine, gamma-glutamylcysteine and cysteinylglycine] is described. Separation relies upon isocratic ion-pairing reversed-phase chromatography and detection is operated by spectrofluorimetry coupled with post-column derivatization reactions using either N-(1-pyrenyl)maleimide (NPM) or ortho-phthalaldehyde (OPA). When OPA is used without co-reagent, only GSH and gamma-glutamylcysteine are detected (heterobifunctional reaction). However, either the OPA reaction in the presence of glycine in the mobile phase (thiol-selective reaction) or NPM allows the detection of all the cited thiols. The HPLC system has been validated as concerning linearity, accuracy and precision. The low detection limits reached (in the pmol range for each thiol injected) allow the screening and the quantification of thiols (as NPM derivatives) in V79cl and V79HGGT cells as well as the measurement of two cytosolic enzymes related to the glutathione synthesis, using the heterobifunctional OPA reaction.

Animals↗
PubMed1998

Mutations in the MRE11, RAD50, XRS2, and MRE2 genes alter chromatin configuration at meiotic DNA double-stranded break sites in premeiotic and meiotic cells.

In the yeast Saccharomyces cerevisiae, meiotic recombination is initiated by DNA double-stranded breaks (DSBs) occurring in micrococcal nuclease (MNase)-hypersensitive regions of the chromatin. MNase-sensitive sites also undergo meiosis-specific alterations in chromatin structure prior to the appearance of DSBs. DSB formation requires the products of numerous genes. Herein we have examined the effects of mutations in four such genes, MRE11, RAD50, XRS2, and MRE2, on MNase sensitivity at DSB sites in premeiotic and meiotic cells. Disruption mutations in each of four genes confer greater than wild-type levels of MNase sensitivity in premeiotic cells. In meiotic prophase, all of these mutations affect MNase sensitivity at DSB sites and fall into two distinct phenotypic classes. The type 1 mutations (mre2 and mre11) confer a reduction in MNase sensitivity relative to the wild-type level. The type 2 mutations (rad50 and xrs2) permit a meiotic increase in the MNase sensitivity to reach a final level higher than that observed in wild-type cells. An mre11 disruption mutation (type 1) is epistatic to a rad50 null mutation (type 2) with respect to its meiotic effects on MNase sensitivity, suggesting that the events observed in the type 2 mutants during meiosis are dependent upon type 1 functions. One interpretation of these results is that Mre11, Rad50, Xrs2, and possibly Mer2 (whose splicing is Mre2-dependent) form a complex at recombination hot spots and establish a chromatin/DNA configuration favorable for the induction of DSBs.

Chromatin↗
PubMed1998

Immobilization tests and periodic leg movements in sleep for the diagnosis of restless leg syndrome.

Patients with restless leg syndrome (RLS) complain of motor restlessness, usually occurring while they rest in the evening. Two immobilization tests have been described to assess leg restlessness in these patients. In the first test, the patient sits in bed with his or her legs outstretched while electromyograms are recorded from right and left anterior tibialis muscles for an hour (Suggested Immobilization Test [SIT]); in the second test, the legs are immobilized in a stretcher (Forced Immobilization Test [FIT]). In the current study, the SIT and the FIT were compared in patients with RLS and normal control subjects matched for age and sex. More leg movements were seen in patients than in controls during immobilization tests, especially the SIT. These movements were periodic, occurring at a frequency of approximately one every 12 seconds. The SIT (index > 40) was found to discriminate between RLS and control subjects better than the FIT (index > 25). Patients were also recorded during two consecutive nights to measure periodic leg movements in sleep (PLMS). A SIT index greater than 40 and a PLMS index greater than 11 (highest PLMS index of 2 consecutive nights) were found to discriminate patients with RLS from control subjects with similar power. With each of these two measures, the clinical diagnosis was correctly predicted in 81% of patients and 81% of the control subjects. The SIT has several advantages over the measure of the PLMS index; it does not require an all-night polygraphic recording and can be administered several times a day to measure circadian fluctuation of motor restlessness.

Adult↗
PubMed1998

Mitotic recombination and localized DNA double-strand breaks are induced after 8-methoxypsoralen and UVA irradiation in Saccharomyces cerevisiae.

Mitotic recombination within the ARG4 gene of Saccharomyces cerevisiae was analysed after treatment of cells with the recombinogenic agent 8-methoxypsoralen (8-MOP) plus UVA. The appearance of DNA double-strand breaks (DSBs) in the ARG4 region during post-treatment incubation was also tested. The results obtained after 8-MOP plus UVA treatment indicate that in mitotic cells: (1) recombination at the ARG4 locus is increased 30 - 500 fold per survivor depending on the strains and the doses employed, (2) the increase of recombination results essentially from gene conversion events which involve the RV site located in the 5' region of the ARG4 gene twice as often as the Bgl site at the 3' end, (3) depending on 8-MOP/UVA dose, ectopic gene conversion is associated with reciprocal translocation, (4) DSBs occur preferentially in the ARG 5' region during post-treatment incubation, as well as in other intergenic regions containing both promoters or/and terminators of transcription, and (5) changes in sequence content in the 5' region of ARG4, which influences positions and frequencies of DSBs formed during repair, are correlated with a modification of the local chromatin structure.

Argininosuccinate Lyase↗
PubMed1998

Recombination at work for meiosis.

In sexually reproducing organisms, homologous recombination increases genetic diversity in gametes and ensures proper chromosome segregation. Recent publications have provided details of the molecular intermediates and proteins involved, the control of the distribution of recombination events at the chromosomal level, and the surveillance mechanisms that coordinate recombination with the meiotic cell cycle.

Aneuploidy↗
PubMed1998

Is excessive daytime sleepiness with periodic leg movements during sleep a specific diagnostic category?

Thirty-four patients who presented with excessive daytime sleepiness (EDS) and who showed an elevated number of periodic leg movements during sleep (PLMS) were studied. None of these patients reported other symptoms or presented sleep laboratory manifestations of narcolepsy or of breathing disorders during sleep. A diagnosis of restless leg syndrome, head trauma or a past history of psychopathology or infectious diseases known to cause EDS were also ruled out. In addition, none of the patients reported a history of drug or alcohol abuse, chronic sleep deprivation or irregular sleep-wake schedule and none were taking medications known to influence sleep at the time of the study. Results of the present study showed no correlation between PLMS index and poor sleep efficiency or daytime sleepiness as measured by the multiple sleep latency test (MSLT). However, a significant negative correlation was found between sleep efficiency at night and the mean sleep latency on the MSLT. These results suggest not only that PLMS and nocturnal sleep disruption are not the primary cause of EDS, but that these sleepy patients have a high propensity to sleep both at night and during the daytime. Therefore, the presence of PLMS during nocturnal sleep recording should not preclude the diagnosis of idiopathic hypersomnia.

Adult↗
PubMed1998

Increased expression of prolactin receptor gene assessed by quantitative polymerase chain reaction in human breast tumors versus normal breast tissues.

The role of PRL in human breast tumorigenesis is not well understood. One of the limitations is the difficulty of accurately measuring PRL receptors (PRLR) in human tissues. We established a quantitative PCR method (Q-PCR) in T-47D human breast cancer cells and applied it to 29 patients, 25 of whom presented with either cancer or fibroadenoma. Four patients underwent a mammoplasty, and normal epithelial cells were cultured before Q-PCR. In T-47D cells, 31 x 10(6) messenger RNA molecules were detected per microgram of total RNA. In all patients, expression of the PRLR gene was detected, varying from 1500 to 1 x 10(6) molecules/microgram of RNA in normal tissues and from 4500 to 34.7 x 10(6) molecules/microgram of RNA in tumors. PRLR expression was always greater in tumor than in normal contiguous tissue and similar in cultured mammary epithelial cells and normal breast tissues. Estradiol and progesterone receptor-negative tumors expressed low levels of PRLR transcripts, similar to normal breast tissue from menopausal women. Immunocytochemical analysis of PRLR confirmed stronger staining in almost all tumor samples compared with normal tissues. A messenger RNA encoding locally produced human PRL was also identified by RT-PCR in every sample tested. Our results confirm PRLR gene expression in all tissues studied, and moreover, indicate that this expression is increased in human breast tumors vs. normal contiguous tissues.

Adult↗
PubMed1997

Primary sequence of the human, lysine-rich, ribosomal protein RPL38 and detection of an unusual RPL38 processed pseudogene in the promoter region of the type-1 angiotensin II receptor gene.

We have isolated a cDNA clone encoding the human ribosomal protein L38 (HSRPL38). The longest ORF of the cDNA predicts a lysine-rich small polypeptide identical to the rat RPL38 protein (100% identity), and sharing a 84% of identity to the tomato RPL38 protein sequence. Northern blot analysis of a number of epithelial cell lines showed that the HSRPL38 is encoded by a mRNA ubiquitously expressed. Southern blot analysis of human genomic DNA suggested that the RPL38 does not constitute a multigene family but it is encoded by a reduced set of active genes, among which we have also found a RPL38 processed pseudogene located in the promoter region of the human type-1 angiotensin II receptor gene. This RPL38 pseudogene is very unusual among processed pseudogenes in that the poly A tail and the entire 5'-UTR of the original RPL38 mRNA were deleted during the retrotransposition process.

Amino Acid Sequence↗
PubMed1997

Atomic resolution (1.0 A) crystal structure of Fusarium solani cutinase: stereochemical analysis.

X-ray data have been recorded to 1.0 A resolution from a crystal of Fusarium solani cutinase using synchrotron radiation and an imaging-plate scanner. The anisotropic treatment of thermal motion led to a fivefold increase in accuracy and to a considerable quality improvement in the electron density maps with respect to an intermediate isotropic model. The final model has an R-factor of 9.4%, with a mean coordinate error of 0.021 A, as estimated from inversion of the least-squares matrix. The availability of an accurate structure at atomic resolution and of meaningful estimates of the errors in its atomic parameters, allowed an extensive analysis of several stereochemical parameters, such as peptide planarity, main-chain and some side-chain bond distances. The hydrogen atoms could be clearly identified in the electron density, thus providing unambiguous evidence on the protonation state of the catalytic histidine residue. The atomic resolution revealed an appreciable extent of flexibility in the cutinase active site, which might be correlated with a possible adaptation to different substrates. The anisotropic treatment of thermal factors provided insights into the anisotropic nature of motions. The analysis of these motions in the two loops delimiting the catalytic crevice pointed out a "breath-like" movement in the substrate binding region of cutinase.

Anisotropy↗
PubMed1997

Clustering of meiotic double-strand breaks on yeast chromosome III.

In the yeast Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-strand breaks (DSBs) that are repaired by interaction of the broken chromosome with its homologue. To identify a large number of DSB sites and gain insight into the control of DSB formation at both the local and the whole chromosomal levels, we have determined at high resolution the distribution of meiotic DSBs along the 340 kb of chromosome III. We have found 76 DSB regions, mostly located in intergenic promoter-containing intervals. The frequency of DSBs varies at least 50-fold from one region to another. The global distribution of DSB regions along chromosome III is nonrandom, defining large (39-105 kb) chromosomal domains, both hot and cold. The distribution of these localized DSBs indicates that they are likely to initiate most crossovers along chromosome III, but some discrepancies remain to be explained.

Chromosome Mapping↗
PubMed1997

An atypical topoisomerase II from Archaea with implications for meiotic recombination.

Type II topoisomerases help regulate DNA topology during transcription, replication and recombination by catalysing DNA strand transfer through transient double-stranded breaks. All type II topoisomerases described so far are members of a single protein family. We have cloned and sequenced the genes encoding the A and B subunits of topoisomerase II from the archaeon Sulfolobus shibatae. This enzyme is the first of a new family. It has no similarity with other type II topoisomerases, except for three motifs in the B subunit probably involved in ATP binding and hydrolysis. We also found these motifs in proteins of the Hsp90 and MutL families. The A subunit has similarities with four proteins of unknown function. One of them, the Saccharomyces cerevisiae Spo11 protein, is required for the initiation of meiotic recombination. Mutagenesis, performed on SPO11, of the single tyrosine conserved between the five homologues shows that this amino acid is essential for Spo11 activity. By analogy with the mechanism of action of known type II topoisomerases, we suggest that Spo11 catalyses the formation of double-strand breaks that initiate meiotic recombination in S. cerevisiae.

Amino Acid Sequence↗
PubMed1997

[Limitations of the protein profile for diagnostic orientation in initial internal medicine consultation. Prospective study on 76 patients].

Determination of the protein profile of orientation (PPO) is now considered by some authors as a means of improving the diagnosis in internal medicine. The feasibility of systematizing this practice was investigated in 76 outpatients (79 included, three excluded secondarily) seen for pathology of undetermined diagnosis. The 79 patients (mean age: 52 years) underwent the classical biological explorations plus PPO. The physicians were divided into two groups (seniors and assistants). Two complete clinical files were established for each patient, with one difference concerning inflammatory and immunologic data: one file included the minimum number of tests considered necessary by the physician and the other the complete PPO (nine proteins). Each file (with or without PPO) was randomly distributed to one of two physicians in the same group. Each physician filled in a diagnostic evaluation sheet indicating whether there was organic pathology or not, the main diagnosis (inflammatory, neoplastic, infectious or other), the secondary diagnosis and the hypothesis of probability. The relevance of the clinical opinion was analyzed by an internal medicine specialist from outside the department with 40 years of clinical experience. The duration of symptoms before the medical visit was from 3 weeks to 5 years (mean 6 months). A diagnosis of organic pathology was reached for three out of four patients. Sixty-seven patients were seen again after a minimum of 6 months, and nine were lost to follow-up. Diagnostic efficiency was no greater for cases with PPO, which appears to be a biological examination of second intention. We suggest that the term "protein profile of orientation" be replaced by "broad protein profile."

Adult↗
PubMed1997

Hyphenated liquid chromatographic method for the determination of colistin residues in bovine tissues.

A selective and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the measurement of colistin residues in milk and in four bovine tissues (i.e., muscle, liver, kidney, and fat). The sample treatment consists of protein precipitation using 10% (w/v) trichloroacetic acid, solid-phase purification on C18 cartridges, and precolumn derivatization of colistin with ortho-phthalaldehyde and 2-mercaptoethanol in borate buffer (pH 10.5). This latter step is performed automatically, and the resulting reaction mixture is injected into a switching HPLC system including a precolumn and an analytical column packed with end-capped LiChrospher RP18 (5 microns). Washing the precolumn and final elution onto the analytical column are conducted using acetonitrile-0.01M phosphate buffer (pH 7.0) mixtures with respective proportions of 65:35 and 68:32 (v/v). Detection is carried out by spectrofluorometry (excitation wavelength, 340 nm; emission wavelength, 440 nm). The retention times of the derivatives corresponding to the two main components of colistin (i.e., polymyxins E2 and E1) are approximately 14 and 18 min, respectively. The structural study of the derivatives corresponding to polymyxins E1 and E2 is carried out by HPLC coupled with electrospray mass spectrometry; data obtained confirms that the derivatization process occurs with the five amino groups of the analytes. Selectivity is obtained in the HPLC system versus other coadministered anti-infective drugs (beta-lactams, aminoglycosides, tetracyclines, and sulphonamides) and endogenous compounds. Quantitation is performed using the sum of the peak areas of polymyxin E1 and polymyxin E2 derivatives. Testing linearity affords correlation coefficients greater than 0.990 for calibration curves in the range of 10-500 microL/L for milk, 50-1000 micrograms/kg for muscle and fat, and 100-1000 micrograms/kg for kidney and liver. Relative standard deviation values are less than 10% at a concentration of 25 micrograms/L in milk and 100 micrograms/kg in tissues (six replicates); recoveries are higher than 60%.

Adipose Tissue↗
PubMed1997

Topographical distribution of spindles and K-complexes in normal subjects.

To assess the topographical distribution of sleep spindles and K-complexes, four 15-minute samples of stage 2 sleep in a group of eight healthy young adults were analyzed. Results show that a majority of spindles generated are detected over central regions, and that K-complexes are markedly predominant over prefrontal and frontal regions. These findings are consistent with the single-spindle generator hypothesis and raise questions concerning the Rechtschaffen and Kales rules for scoring K-complexes.

Adolescent↗
PubMed1997

Changes in EEG power density during sleep laboratory adaptation.

First- and second-night effects on the electroencephalogram (EEG) were investigated by means of polygraphic sleep recordings and all-night spectral analysis. Eighteen normal subjects were studied for three consecutive nights in a hospital sleep laboratory. Visual sleep scoring showed that there was a first-night effect in normal subjects similar to that reported previously [increased wakefulness; decreased total sleep time, sleep efficiency, and rapid eye movement (REM) sleep]. Spectral analysis of the sleep EEG revealed important changes, most of which occurred in REM sleep. Increased delta, theta, and beta1 power densities accompanied by decreased mean frequency were seen in REM sleep in the second night. On the basis of REM sleep deprivation results previously published, our data suggest that the second night could be affected by partial REM sleep deprivation that occurred in the first night. Delta and theta power density values decreased in the first non-rapid eye movement episode of nights 1 and 2; this could result from increased REM sleep pressure. The overall consistency of spectral data in the first and second night with REM sleep findings derived from visual scoring in the first night lends further support to this hypothesis. The sleep disturbance experienced during the first night in a sleep laboratory may be a useful and valid model of transient insomnia. Therefore, we conclude that data from all nights recorded should be included in assessing a subject's sleep.

Adult↗