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P Roux

Publications and source records attributed to P Roux.

At least 55 records · Page 3Linked to original sources
PubMed1998

Update on Pneumocystis carinii f. sp. hominis typing based on nucleotide sequence variations in internal transcribed spacer regions of rRNA genes.

Pneumocystis carinii f. sp. hominis isolates from 207 clinical specimens from nine countries were typed based on nucleotide sequence variations in the internal transcribed spacer regions I and II (ITS1 and ITS2, respectively) of rRNA genes. The number of ITS1 nucleotides has been revised from the previously reported 157 bp to 161 bp. Likewise, the number of ITS2 nucleotides has been changed from 177 to 192 bp. The number of ITS1 sequence types has increased from 2 to 15, and that of ITS2 has increased from 3 to 14. The 15 ITS1 sequence types are designated types A through O, and the 14 ITS2 types are named types a through n. A total of 59 types of P. carinii f. sp. hominis were found in this study.

Base Sequence↗
PubMed1998

Identification of Pneumocystis carinii f. sp. hominis gene sequences in filtered air in hospital environments.

To evaluate the risk of a nosocomial spread of Pneumocystis carinii f. sp. hominis (P. carinii hominis), air filter samples from rooms of P. carinii pneumonia (PCP) patients, adjacent corridors, and other hospital environments have been investigated for the presence of P. carinii hominis. Amplified DNA from air filters and sputum or bronchoalveolar lavage samples from the PCP patients have been genotyped with the P. carinii hominis genes of the mitochondrial large-subunit (mtLSU) rRNA and the internal transcribed spacers (ITS1 and ITS2) of the rRNA. Genotypes of the two loci were identified by direct sequencing, and for site 85 of the mtLSU locus, three allele-specific PCR assays were used. P. carinii hominis DNA was identified in the air of five of seven PCP patient rooms and in the air of two of four air filtrations from the ward corridors. The P. carinii hominis genotypes were the same in four of the five room air samples as those in the corresponding patients, suggesting a risk of person-to-person transmission of P. carinii hominis from PCP patients. Three of 16 air samples collected in infectious disease wards without the presence of PCP patients and one sample from a cardiology unit in a separate hospital building were also positive, which further strengthens the possibility of acquisition of P. carinii hominis from the environment.

Air Microbiology↗
PubMed1998

[Pulmonary crytosporidium in a patient with AIDS].

Compared to digestive disorders, respiratory disease linked to cryptosporidium has not been very well documented in AIDS. We report a case of a patient suffering from AIDS who presented with chronic bronchial symptoms which became more and more incapacitating. Different respiratory specimens (broncho-alveolar lavage, expectoration and bronchial biopsy) revealed the presence of cryptosporidium species. The patient died in a few weeks from respiratory insufficiency without the parasite being eradicated from the bronchi. Although the autopsy confirmed the presence of cryptosporidium species, it was difficult to confirm its responsibility due to the fact that other pathogens were present in the bronchial tree.

AIDS-Related Opportunistic Infections↗
PubMed1997

Kinetics of secondary structure recovery during the refolding of reduced hen egg white lysozyme.

We have shown previously that, in less than 4 ms, the unfolded/oxidized hen lysozyme recovered its native secondary structure, while the reduced protein remained fully unfolded. To investigate the role played by disulfide bridges in the acquisition of the secondary structure at later stages of the renaturation/oxidation, the complete refolding of reduced lysozyme was studied. This was done in a renaturation buffer containing 0.5 M guanidinium chloride, 60 microM oxidized glutathione, and 20 microM reduced dithiothreitol, in which the aggregation of lysozyme was minimized and where a renaturation yield of 80% was obtained. The refolded protein could not be distinguished from the native lysozyme by activity, compactness, stability, and several spectroscopic measurements. The kinetics of renaturation were then studied by following the reactivation and the changes in fluorescence and circular dichroism signals. When bi- or triphasic sequential models were fitted to the experimental data, the first two phases had the same calculated rate constants for all the signals showing that, within the time resolution of these experiments, the folding/oxidation of hen lysozyme is highly cooperative, with the secondary structure, the tertiary structure, and the integrity of the active site appearing simultaneously.

Animals↗
PubMed1997

The small GTPases Cdc42Hs, Rac1 and RhoG delineate Raf-independent pathways that cooperate to transform NIH3T3 cells.

BACKGROUND: Ras-mediated transformation of mammalian cells has been shown to activate multiple signalling pathways, including those involving mitogen-activated protein kinases and the small GTPase Rho. Members of the Rho family affect cell morphology by controlling the formation of actin-dependent structures: specifically, filopodia are induced by Cdc42Hs, lamellipodia and ruffles by Rac, and stress fibers by RhoA. In addition, Rho GTPases are involved in progression through the G1 phase of the cell cycle, and Rac1 and RhoA have recently been directly implicated in the morphogenic and mitogenic responses to transformation by oncogenic Ras. In order to examine the cross-talk between Ras and Rho proteins, we investigated the effects on focus-forming activity and cell growth of the Rho-family members Cdc42Hs, Rac1 and RhoG by expressing constitutively active or dominant-negative forms in NIH3T3 cells. RESULTS: Expression of Rac1 or RhoG modulated the saturation density to which the cells grew, probably by affecting the level of contact inhibition. Although all three GTPases were required for cell transformation mediated by Ras but not by constitutively active Raf, the selective activation of each GTPase was not sufficient to induce the formation of foci. The coordinated activation of Cdc42Hs, RhoG and Rac1, however, elicited a high focus-forming activity, independent of the mitogen-activated ERK and JNK protein kinase pathways. CONCLUSIONS: Ras-mediated transformation induces extensive changes in cell morphology which require the activity of members of the Rho family of GTPases. Our data show that the pattern of coordinated Rho family activation that elicits a focus-forming activity in NIH3T3 cells is distinct from the regulatory cascade that has been proposed for the control of actin-dependent structures in Swiss 3T3 cells.

3T3 Cells↗
PubMed1997

A simple luciferase assay for signal transduction activity detection of epidermal growth factor displayed on phage.

Studies on receptor-ligand interactions are important for the design of agonists or antagonists of natural ligands. We developed a luciferase reporter assay to screen epidermal growth factor receptor (EGFR) binding molecules rapidly for their ability to stimulate or inhibit signal transduction. Human EGF displayed on fd filamentous phage presented an activity similar to soluble EGF when tested for binding to the EGFR, for induction of cell cycle progression or in the luciferase assay. Two libraries of human EGF variants displayed on phage were constructed in which the aspartic acid residue at position 46 or the arginine residue at position 41 were randomised. EGF mutants displayed on phage were screened in parallel for binding to the EGFR using an ELISA assay and for transducing activity using the luciferase assay. Regarding the 46 position, most of the mutants retained the ability to bind the EGFR and their transducing activity corresponded perfectly with their binding. For the more crucial 41 position, only the wild-type EGF was able to bind the EGFR. Our approach allowed a simple determination of crucial positions and paved the way for identification of agonists with altered transduction activity.

3T3 Cells↗
PubMed1997

DNA curvature controls termination of plus strand DNA synthesis at the centre of HIV-1 genome.

In vivo and in vitro, reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) terminates plus strand synthesis at the centre of the viral genome. The central termination sequence (CTS) contains curved DNA fragments located upstream of each terminator site. Two different models, relying either on the A-tract or general sequence roll assumptions, were used to predict the extent and the direction of this curvature as well as to design mutants, which abolished it. Straightening of each curved element abolished termination at the site located immediately downstream from the curvature. When synthesis was performed on the other strand and in the opposite direction, the two curved elements C1 and C2 associated with the two termination sites Ter1 and Ter2, led again to termination of DNA synthesis. Therefore, termination occurred as a nascent bent duplex was synthesized within the template primer binding cleft of RT, even when putative strand-specific motifs have been removed by the inversion. Computation of DNA paths upstream of other known arrest sites suggested that this feature was of general relevance for termination. At the CTS, termination occurred more precisely at the 3' end of an AnTm motif (n + m = 7). The possible structures, adopted by this motif, are discussed and confronted with the present crystallographic and biochemical data obtained on HIV-1 RT-DNA interactions and on HIV-1 RT processivity.

Base Sequence↗
PubMed1997

Biodiversity of Pneumocystis carinii hominis: typing with different DNA regions.

The purpose of this study was to identify the most useful gene for the detection of biodiversity of Pneumocystis carinii hominis isolates and to compare samples from French and Italian subjects. We studied 20 bronchoalveolar lavage fluid specimens from 20 human immunodeficiency virus-infected patients (10 French and 10 Italian patients) with Pneumocystis carinii pneumonia by DNA sequencing of the thymidylate synthase (TS), 5S rRNA, large-subunit mitochondrial rRNA (mt LSU rRNA), and internal transcribed spacer (ITS1 and ITS2) genes. Thirteen of the 20 sequenced samples had the prototype TS gene sequence. Fourteen of the 20 samples showed the prototype sequence of the 5S rRNA gene, and 6 had variant sequences of the 5S rRNA gene. The mt LSU rRNA gene was sequenced for 18 of the 20 samples; all sequences were different from the prototype sequence and were classified into four groups. Thirteen of the 20 ITS1 and ITS2 sequences were analyzed, and all the sequences were found to be different from the prototype sequence and were classified into 10 groups. The internal transcribed spacer regions thus appear to be the most discriminatory region of DNA for analysis of the biodiversity of P. carinii hominis isolates.

AIDS-Related Opportunistic Infections↗
PubMed1997

Study of internal transcribed spacer and mitochondrial large-subunit genes of Pneumocystis carinii hominis isolated by repeated bronchoalveolar lavage from human immunodeficiency virus-infected patients during one or several episodes of pneumonia.

The objective of this study was to type, analyze, and compare Pneumocystis carinii hominis strains obtained from different samples during a given or recurrent episodes of P. carinii pneumonia (PCP) for epidemiologic purposes. We studied 36 bronchoalveolar lavage (BAL) or induced sputum (IS) samples from 16 human immunodeficiency virus-infected patients with one or several episodes of PCP. PCR amplification and direct sequencing were performed on the two internal transcribed spacers (ITS1 and ITS2) of P. carinii hominis rRNA genes by using DNA extracted from BAL or IS samples, and the sequences were compared to the mitochondrial large-subunit (mt LSU) gene sequence determined in a previous study in our laboratory. The studies of the mt LSU and ITS sequences showed that some patients (n = 10) were infected with the same strains of P. carinii hominis during a given episode of PCP. In one patient infected with strains with identical sequences in several episodes, the recurrence could have been due to reactivation of organisms not eliminated by treatment during the first episode or to de novo infection by an identical strain. In five patients infected with strains with different sequences in each episode, recurrence was due to de novo infection. Sequence analysis of these two P. carinii hominis gene regions showed that de novo infection can occur in AIDS patients with recurrent PCP.

AIDS-Related Opportunistic Infections↗
PubMed1997

[Pulmonary mucormycosis in a leukemia patient. Diagnostic and therapeutic difficulties].

The observation of pulmonary mucormycosis occurring in a patient presenting with aplasia induced therapeutically during treatment for acute myeloblastic leukaemia, has led to a review of the characteristics of this rare opportunistic fungal infection: it occurs in a particular condition; the clinical manifestations are characterised by the thrombotic character and the rapidly necrosing nature of the histological lesions; the diagnosis is usually very difficult to make and is linked to the rarity of the pathology and the frequently negative mycological specimens apart from tissue biopsies; the value of a medicosurgical therapeutic strategy on which the prognosis of the infection depends.

Amphotericin B↗
PubMed1996

Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence. 1. Tryptophanyl residues of the carrier can be mutated without impairing protein activity.

During the transport process the mitochondrial adenine nucleotide carrier (Ancp) undergoes conformational changes which result in modifications of the intrinsic fluorescence of the carrier. To further study these changes by a fluorometric approach, the three tryptophanyl residues (Trp87, Trp126, and Trp235) of the Saccharomyces cerevisiae Anc2p were individually mutated to their tyrosine counterparts. The resulting mutated genes (two-Trp, one-Trp or Trp-less variants) were integrated at the ANC2 locus. A prerequisite for such studies is that all the engineered carrier molecules are still able to catalyze ADP/ATP exchange. The cellular characteristics of the strains expressing the mutated Anc2p and the biochemical properties of the variant Anc2p in mitochondria were examined. Although Trp87 is absolutely conserved in all 30 available Ancp sequences, none of the tryptophanyl residues is essential to the carrier protein folding and the transport activity. The mutated and wild-type Anc2p were expressed to the same level, as evidenced by both ligand binding and immunochemical analyses. When isolated in the presence of detergent, all the variant Anc2p preparations contained ergosterol in similar amounts (9 mol/mol of 35 kDa Anc2p) but no specific interaction was revealed. Our results show that the tryptophanmutated Anc2p are suitable for fluorescence studies, which are reported in the accompanying paper by Roux et al. [(1996) Biochemistry 35, 16125-16131].

Atractyloside↗
PubMed1996

Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence. 2. Assignment of tryptophanyl residues of the carrier to the responses to specific ligands.

Tryptophanyl substitution of the Saccharomyces cerevisiae adenine nucleotide carrier (Anc2p isoform) was not deleterious for the transport activity or the folding of the carrier [preceding paper by Le Saux et al. (1996) Biochemistry 35, 16116-16124]. Conformational changes of the isolated wild-type and Trp-substituted Anc2p variants, induced upon binding of specific substrates [adenosine triphosphate (ATP) or diphosphate (ADP)] or inhibitors [carboxyatractyloside (CATR) or bongkrekic acid (BA)], were studied by measurement of intrinsic fluorescence. Titration of CATR and BA binding sites ended in the same number of sites, namely, 6-7 nmol/mg of wild-type and variant Anc2p. Isolated Anc2p in detergent presented similar emission spectra, suggesting that all tryptophanyl residues were in environments of similar hydrophobicity. Trp87 and Trp126 contributed largely and to a similar extent to the fluorescence enhancement observed in response to ATP binding, while Trp235 contributed negatively and to a small extent to the fluorescence change. Both Trp126 and Trp235, and to a lower extent Trp87, participate in the CATR-induced fluorescence decrease of Anc2p. Responses to BA binding were observed only in the presence of ATP; they consisted of a further fluorescence increase of the Anc2p.ATP complex, which was mainly due to Trp87 and Trp126, Trp235 being much less responsive. The different fluorescence responses of the three Trp residues of Anc2 variants to ATP, CATR, and BA are in agreement with distinct binding sites for these ligands and distinct conformations of the carrier protein recognizing specifically CATR or BA. A mechanistic model is proposed to interpret the transitions between the different conformational states of Anc2p.

Adenosine Diphosphate↗
PubMed1996

Fluorometric titration of the mitochondrial ADP/ATP carrier protein in muscle homogenate with atractyloside derivatives.

We describe here the chemical synthesis of the novel methylanthraniloyl (Mant-) derivative of atractyloside (ATR), which is a specific inhibitor of the mitochondrial ADP/ATP carrier. The spectral properties of Mant-ATR and naphthoyl-ATR (N-ATR) are analyzed. Both derivatives bind to the membrane-bound ADP/ATP carrier at the same sites as ATR and carboxyatractyloside (CATR). When Mant-ATR and N-ATR are displaced by CATR, their fluorescence emissions are decreased and increased, respectively. These fluorescence changes allow the titration of the CATR binding sites and therefore the quantitation of the amount of ADP/ATP carrier protein in a biological preparation. The validity of the fluorometric titration was tested with beef heart mitochondria and confirmed by binding assays using radioactive ATR. The fluorometric method was applied to rabbit skeletal muscle homogenate and the results of titration were confirmed by binding assays of radioactive ATR. The reliability of the fluorometric method was assessed by comparing the amounts of CATR binding sites and the content of heme aa3 in muscle homogenates and in isolated mitochondria from the same homogenates. Because of its high sensitivity, the fluorometric titration of the ADP/ATP carrier requires small amounts of tissue. Mant-ATR and N-ATR can therefore be considered as convenient, reliable, and sensitive probes to quantify the amount of ADP/ATP carrier and detect a putative carrier protein deficiency in biopsy samples from human patients suffering from myopathies with no clear identified etiology.

Animals↗
PubMed1996

Radioimagers as an alternative to film autoradiography for in situ quantitative analysis of 125I-ligand receptor binding and pharmacological studies.

Three radioimagers, the mu-imager, the beta-imager and the phosphorimager, were tested as alternatives to quantitative autoradiography on film, for receptor imaging and pharmacological in situ quantitative analysis. Two iodinated ligands 125I-interleukin-1 alpha and 125I-gonadotropin releasing hormone agonist were used for receptor characterization in mouse brain and pituitary sections. Due to the high number of the agonist receptors in rat pituitary gland, this tissue was used to compare measurements obtained from digital autoradiograms with classical gamma detector determination. This permits the evaluation of radioimager efficiency and absolute quantification. Radioimagers represent an improvement in terms of time of image acquisition. All the radioimagers are more sensitive than film for the detection of low levels of radioactivity. The spatial resolution provided by the mu-imager compares favourably with that obtained on film autoradiograms while digital autoradiograms from the phosphorimager and beta-imager did not show precise definition under our experimental conditions. Superimposition of histological structures from the stained sections with radiolabelled areas in the autoradiograms remains, at this time, the unique advantage of film. In conclusion, radioimagers represent an alternative to autoradiography on film or emulsion for in situ quantitative studies on tissue sections. They combine precise imaging for in situ binding studies with easy and direct access to counts in cpm. The improvement in radioimaging technology has, therefore, brought in situ analysis of iodinated ligand binding to the level of accuracy that is obtained with classical detectors of radioactivity.

Animals↗