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D Chevrier

Publications and source records attributed to D Chevrier.

At least 37 records · Page 2Linked to original sources
PubMed1993

PCR product quantification by non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells.

Oligonucleotides derived from IS6110, an insertion sequence from Mycobacterium tuberculosis, have been covalently immobilized on polystyrene Covalink NH microwells to develop a sandwich and a competitive non-radioactive hybridization assay for the quantitative determination of the DNA fragments obtained by polymerase chain reaction (PCR). Using the appropriate standard DNA, the method can be employed for the quantitative analysis of PCR fragments. The sandwich assay can detect as little as 3 fmol of target DNA per well and the standard curve may be used with quantities ranging from 3 to 300 fmol per well. The competitive hybridization assay is less sensitive since it is quantitative between 100 and 8000 fmol per well. We show here that both kinds of assays can be used to identify M. tuberculosis strains isolated from clinical samples. The non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells involve few and simple operations, and are thus suitable for routine diagnosis. Moreover, when stored at 5 degrees C, precoated strips can still be used for hybridization up to at least 10 months.

Base Sequence↗
PubMed1993

Use of nonradioactive DNA probes to identify a Campylobacter jejuni strain causing abortion.

A case of human abortion due to a Campylobacter infection is reported. Cultures revealed two morphologically different isolates with large and small colonies respectively. Using conventional methods of identification, the large colonies were identified as Campylobacter jejuni and the small colonies as Campylobacter coli. Dot blot hybridization and determination of rDNA restriction fragment patterns revealed that both colony types were the same strain of Campylobacter jejuni. This observation illustrates the need to use methods other than phenotypic methods when identifying strains of Campylobacter.

Abortion, Spontaneous↗
PubMed1993

Pro- and anti-inflammatory properties of human recombinant IL-1 beta during experimental arthritis in rats: 2. Period-dependent effect.

The systemic effects of human recombinant Interleukin-1 beta (HrIL-1 beta) on hindpaw edema were determined in arthritis induced by human native type II collagen (CII) with muramyl dipeptide (MDP) both injected on day 0. Daily treatment with HrIL-1 beta (0.2 microgram sc) pretreatment, from D-1 (the day before MDP and CII were injected) to D3 significantly delayed the secondary inflammation in the uninjected left hindpaw, whereas the same treatment from D6 to D10 at the end of the "primary" inflammation, enhanced the volume of the left hindpaw. Treatment from D13 to D17 did not affect the "secondary" edema in the left hindpaw. Thus, HrIL 1 beta administration produces pro- or anti-inflammatory effects on a developing polyarthritis depending on when treatment is started and is most effective as an anti-inflammatory molecule when started at the peak of the the inflammatory reaction, as previously described. In view of these early findings, we have compared the effect of adding HrIL-1 beta along with MDP in the sensitization procedure on the time-course of CII-induced arthritis. No adjuvant effect of HrIL-1 beta was observed. On the contrary, HrIL-1 beta significantly decreased the signs of inflammation in the injected hindpaw during the secondary inflammation. In addition, the immune response to type II collagen was less in the group receiving HrIL-1 beta, maybe because of nonspecific increase of antigen clearance. On the other hand, the MDP sensitization procedure enhanced the incidence of CII arthritis and significantly worsened the clinical parameters in both primary and secondary inflammations.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1993

Targeting of pro-opiomelanocortin to the regulated secretory pathway may involve cooperation between different protein domains.

The structure of pro-opiomelanocortin (POMC) can be divided into three main domains: an NH2-terminal domain formed by the NH2-terminal glycopeptide and the joining peptide, a central domain corresponding to the adrenocorticotropin sequences and a COOH-terminal domain containing the beta-lipotropin sequences. Expression of POMC in neuroendocrine cell lines such as the mouse neuroblastoma Neuro2A cells results in its targeting to the regulated secretory pathway of these cells. Intracellular targeting of proteins along non default pathways are widely believed to involve the recognition of specific structural features by a sorting machinery. To understand the nature of the signal involved in targeting prohormone to the regulated secretory pathway, we have constructed mutants of POMC in which sequences from the NH2-terminal, the central and the COOH-terminal domains were deleted and examined the sorting of these mutant POMC molecules in Neuro2A cells by immunofluorescence and immunoelectron microscopy. Our results indicate that POMC NH2-terminal glycopeptide or beta-LPH domain do not contain sufficient information for targeting to the regulated pathway since these peptides are not sorted to secretory vesicles when expressed in Neuro2A cells: Similarly, the ACTH domain does not contain essential targeting information since POMC mutants lacking these sequences were sorted to secretory vesicles. Mutant POMCs containing the sequences of more than one of the main protein domains were, however, correctly targeted to the regulated secretory pathway. Our results indicate that POMC is not targeted to the regulated secretory pathway through recognition of a unique continuous 'molecular address'.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1993

Characterization of Xanthomonas campestris Pathovars by rRNA Gene Restriction Patterns.

Genomic DNA of 191 strains of the family Pseudomonadaceae, including 187 strains of the genus Xanthomonas, was cleaved by EcoRI endonuclease. After hybridization of Southern transfer blots with 2-acetylamino-fluorene-labelled Escherichia coli 16+23S rRNA probe, 27 different patterns were obtained. The strains are clearly distinguishable at the genus, species, and pathovar levels. The variability of the rRNA gene restriction patterns was determined for four pathovars of Xanthomonas campestris species. The 16 strains of X. campestris pv. begoniae analyzed gave only one pattern. The variability of rRNA gene restriction patterns of X. campestris pv. manihotis strains could be related to ecotypes. In contrast, the variability of patterns observed for X. campestris pv. malvacearum was not correlated with pathogenicity or with the geographical origins of the strains. The highest degree of variability of DNA fingerprints was observed within X. campestris pv. dieffenbachiae, which is pathogenic to several hosts of the Araceae family. In this case, variability was related to both host plant and pathogenicity.

Journal Article↗
PubMed1992

Pro- and anti-inflammatory properties of human recombinant IL-1 beta during experimental arthritis in rats: 1. Dependence on dose and severity threshold.

The effects of human recombinant interleukin-1 beta (HrIL-1 beta) were investigated in arthritic rats sensitized with type II collagen (CII) and muramyl dipeptide (MDP). When administered subcutaneously (sc) daily during established arthritis, low (0.02 micrograms) and medium (0.2 micrograms) HrIL-1 beta doses exerted paradoxical beneficial properties on paws with moderate and severe inflammation, respectively. In contrast, the highest dose (2 micrograms) had a pejorative effect on developing arthritis. In addition, HrIL-1 beta attenuated paw volume and deterioration of the joints as assessed radiologically. Hence, paw inflammation response to IL-1 exposure depended on the dosage and the severity of previous arthritis prior to the IL-1 challenge. Some of these paradoxical activities may be due to the capacity of IL-1 to induce its own inhibitors or feedback loops thus counterbalancing its phlogistic properties.

Animals↗
PubMed1992

Enhancement of HIV-specific cytotoxic T lymphocyte responses by zidovudine (AZT) treatment.

Zidovudine or 3'-azido-2'-3'-dideoxy-thymidine (AZT) is an antiviral drug widely used to treat HIV-infected patients. Because cytotoxic T lymphocytes (CTL) are thought to contribute actively to resistance against HIV-induced disease, we studied sequentially 10 HIV-infected individuals under zidovudine treatment for a period of 6-12 months. For a given patient all lymphocyte suspensions corresponding to the complete zidovudine therapy period were tested on the same day and on the same target cells. Patients were selected for expression of HLA-A2 and/or HLA-A3 class I transplantation antigen. HLA-restricted cytotoxicity specific for env, gag and nef HIV proteins was quantified for each patient at 6 week intervals. The data clearly indicated that zidovudine has a beneficial effect on the CTL response during the first 6-12 weeks of treatment, inducing cytotoxicity levels up to 100-fold stronger than base line. This effect was usually short lived. However, patients who maintained strong levels of cytotoxicity had better clinical and survival outlook than patients who had lost all detectable cytotoxic lymphocytes. It is proposed that AZT, among other effects, delays the onset of disease in HIV-infected patients by contributing to the stimulation of the HIV-specific CTL response.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Lack of adjuvanticity of human recombinant interleukin-1 beta in collagen induced arthritis in rats.

We investigated the influence of human recombinant interleukin-1 beta (hrIL-1 beta) on the time-course of collagen induced arthritis (CIA) when injected concomitantly with the arthritogenic emulsion. Three sensitizing procedures were compared. The control group received type II collagen only. The other groups differed by the adjunction of demonstrated (MDP) or potential (IL-1 beta) adjuvant. No adjuvant effect of IL-1 was observed as judged on clinical or radiological scores. On the contrary, MDP significantly worsened the lesions of the injected right hindpaw, and increased the incidence of CIA. Surprisingly, humoral response to type II collagen was decreased in the group receiving IL-1 beta. This might be explained by a non specific increase of antigen clearance.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1991

Assay of circulating hyaluronic acid in the rat: study of diurnal variation and effect of anesthesia.

Serum hyaluronic acid (HA) may provide a good marker for the severity of joint disease in the rat since a positive correlation was observed in experimental models of arthritis. However, little is known about its physiological variation in rats. In the present work, we do not find any circadian rhythm of HA in healthy Sprague-Dawley rats in contrast to that observed in humans, whose serum levels vary during daytime. Furthermore, the influence of blood sampling conditions on HA concentrations was evaluated in conscious animals and by using different anesthetics. The greater reproducibility for the assay of HA is observed with the intracardiac puncture under ether inhalation. Blood sample collection in the absence of anesthesia leads to a significant increase in serum levels of HA, which could be attributed partly to enhanced joint movements generated by psychological stress.

Analysis of Variance↗
PubMed1991

Investigation of a possible role of the amino-terminal pro-region of proopiomelanocortin in its processing and targeting to secretory granules.

Proopiomelanocortin (POMC) is a polyprotein which is targeted to the regulated secretory pathway of neuroendocrine cells where it undergoes tissue-specific proteolysis to yield peptides such as adrenocorticotropic hormone, beta-lipotropin and beta-endorphin. The pro-region of POMC is 49 amino acid long with two disulfide bonds between cysteine residues 2 and 24 and 8 and 20. These cysteine residues are conserved across the species. The pro-region contains no known hormonal sequence. Sorting to the regulated secretory pathway is thought to involve targeting signals encoded in the structure of secretory proteins. In the present study, we have examined the possibility that the disulfide bridges located in the NH2-terminal portion of the pro-region of POMC are essential for maintaining a determinant involved in the sorting of POMC to the regulated secretory pathway. Using site-directed and deletion mutagenesis of the porcine POMC cDNA, we created mutants in which one or both disulfide bridges were disrupted or in which the first 26 amino acid residues of the pro-region were deleted. Recombinant retroviruses carrying the mutated POMC cDNAs were used to infect Neuro2A cells. Immunofluorescence and immunoelectron microscopy studies performed on infected cells revealed that the unmutated and mutated POMC-immunoreactive peptides were localized in dense-core vesicles at the tips of cellular extensions. Analysis of the POMC-immunoreactive peptides extracted from the infected Neuro2A cells indicated that the mutated precursors in which one disulfide bridge was disrupted (POMC-S2 or POMC-S8) were stored and processed as efficiently as the unmutated POMC. By contrast, the mutated precursor in which both disulfide bridges were disrupted (POMC-S2,8) did not accumulate in intracellular compartments to the same extent as unmutated POMC. Moreover, this mutant was very inefficiently processed and no release could be observed upon stimulation of the cells with K+/Ca2+. These results suggest that POMC-S2,8 entered the regulated secretory pathway less efficiently than the unmutated precursor. However, when both disulfide bridges were removed from the precursor from the precursor by deletion of the first 26 amino acid residues of POMC, the truncated precursor (POMC delta 1-26) behaved as the unmutated POMC. Taken together our results indicate that the NH2-terminal portion of the pro-region including both disulfide bridges can be deleted without affecting the targeting of the molecule to secretory granules. However, when the entire POMC sequence is expressed in Neuro2A cells, the proper folding of the NH2-terminal region might be important for efficient processing and targeting.

Amino Acid Sequence↗
PubMed1991

Expression of porcine pro-opiomelanocortin in mouse neuroblastoma (Neuro2A) cells: targeting of the foreign neuropeptide to dense-core vesicles.

Pro-opiomelanocortin (POMC) is the precursor to several pituitary hormones and neuropeptides including adrenocorticotropic hormone (ACTH) and beta-endorphin (beta-END). In neuroendocrine cells, peptide hormones and neuropeptides are targeted to the dense-core vesicles of the regulated secretory pathway. These vesicles are transported to the ends of cellular extensions where they are stored until they release their content upon external stimulation of the cell. In order to study the cellular mechanisms involved in targeting of neuropeptides, we have expressed POMC in Neuro2A cells, a cell line of neural origin. Using immunofluorescence labeling and immunoelectron microscopy we show that in Neuro2A cells POMC is packaged in dense-core vesicles which accumulate at the tips of cellular processes. Intracellular accumulation of POMC was not observed in NIH 3T3 fibroblasts. When a soluble form of an integral membrane protein, neutral endopeptidase (E.C. 3.4.24.11) (secNEP), was expressed in Neuro2A cells, the protein was found to be constitutively secreted without prior accumulation in dense-core vesicles. Our results suggest that in Neuro2A cells, targeting to the regulated secretory pathway is restricted to peptide hormones and neuropeptides and establish this cell line as a valid model for studying the molecular events involved in neuropeptide sorting into the regulated secretory pathway.

Animals↗
PubMed1991

Production of monoclonal antibodies against the N-terminal glycopeptide of porcine pro-opiomelanocortin: their use for solid-phase radioimmunoassay, western blotting, and immunogold cytochemistry.

We have prepared two monoclonal antibodies for the N-terminal glycopeptide of pro-opiomelanocortin 1-77 (N-POMC(1-77)) purified from porcine pituitaries. Antibody 1-244 recognizes an epitope located within the gamma 3-melanotropin (gamma 3-MSH or POMC(51-77)) sequence, whereas antibody 2-197 binds specifically to a determinant in the 1-49 region of N-POMC. These monoclonal antibodies were used to construct a two-site solid-phase radioimmunoassay that can detect as little as 50 pg of N-POMC(1-77). The assay is linear between 0.5 and 5 ng of porcine peptide and recognizes equally well the homologous peptides purified from human and bovine pituitaries. The assay has been used to analyze reversed-phase high pressure liquid chromatography fractions of crude bovine pituitary extracts and detected a peptide with chromatographic properties identical to those of N-POMC(1-77). When used to stain immunoblots of bovine intermediate pituitary extracts, both the 2-197 and 1-244 antibodies could recognize a major peptide comigrating with purified N-POMC(1-77). In addition, antibody 2-197 also detected a peptide with a mobility similar to that of standard N-POMC(1-49). When used in conjunction with a second anti-mouse antibody coupled to colloidal gold particles, antibody 2-197 stained N-POMC immunoreactive material located in granules in thin sections of pituitary.

Animals↗
PubMed1991

[Nonradioactive molecular hybridization].

Nucleic acid hybridization techniques have been used for several decades in basic research to isolate genes, to determine their structure or analyse their mechanisms. The new technology of non-radioactive probes (cold probes) allows the routine use of this method outside of the specialized molecular biology laboratory. Hybridization makes use of a nucleic acid probe to detect a complementary nucleic acid target present in biological fluid or in a biopsy tissue. Hybridization leads to the formation of a double-stranded molecule called hybrid or duplex, which can be detected with a great sensitivity by using high-energy radioisotope. However, the use of radioisotope labeled probes is limited by the short half-life of the isotope, radiolysis of the probe and the radioactive hazards. In order to overcome autoradiography delay and the drawbacks of techniques employing radioisotopes, various non-radioactive labels have been proposed. Labeling and detection systems are currently designed in two ways: The label molecule can be attached directly to the DNA probe (direct labelling) or it can be attached to a molecule which binds either to a modified probe or specifically to the duplex (indirect labeling). Various substances has been used to label directly a nucleic acid probe. The assay detection limit depends largely on the detection limit of the label, therefore, probe assay based on label which provides signal amplification (e.g., enzymes) is likely to be more sensitive than an assay using a label which provides only a single signal per molecule (e.g., fluorochromes). In the indirect labeling, the probe cannot be detected alone; rather, it requires the addition of a detection system.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗
PubMed1990

Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood.

Polymerase chain reaction (PCR) was developed for the in vitro amplification of dengue virus RNA via cDNA. A fraction of the N-terminus gene of the envelope protein in the four dengue serotypes was amplified using synthetic oligonucleotide primer pairs. Amplified products were cloned and used as dengue type-specific probes in gel electrophoresis and dot-blot hybridization. We detected and characterized dengue virus serotypes in blood samples by the three-step procedure DNA-PAH consisting in cDNA priming (P), DNA amplification (A) and hybridization (H) using specific non-radiolabelled probes. Our findings showed that DNA-PAH was more rapid and sensitive in the identification of the infecting serotype than the mosquito cell cultures. Moreover, the failure of cultures to detect virus particles in sera containing few copies of viral genome or anti-dengue antibodies justified the approach of DNA-PAH to the dengue identification in clinical specimens.

Base Sequence↗
PubMed1990

An automatic modified polymerase chain reaction procedure for hepatitis B virus DNA detection.

In order to perform an efficient and reproducible diagnostic test for hepatitis B virus (HBV) infection using the polymerase chain reaction (PCR), sixteen primer couples specific for the HBV genome were selected. Primers 15-31 nucleotides in length containing between 31-73% GC permitted amplification of fragments corresponding to the whole HBV genome. The specificity and efficiency of PCR amplification were studied in detail using DNA extracted from either a viral particle preparation or from the liver of a patient with chronic active hepatitis. Three primer couples in the X, C and PreS regions, i.e. MD24/MD26, MD27/MD31 and MD19/MD18, respectively, gave satisfactory results and performed efficiently under highly stringent hybridization conditions. A modified PCR procedure was then developed using only two thermal steps with a temperature shift of 16 degrees C. This simple method was as efficient as conventional PCR and permitted detection of a single HBV DNA molecule with the X region specific primer couple. The automatization of this PCR-based procedure permitted 40 amplification cycles in 105 min.

Autoanalysis↗
PubMed1989

Monoclonal anti-nucleoside antibodies. Characterization and application in an enzyme immunoassay of single-stranded DNA.

Two mouse monoclonal antibodies were raised against adenosine and guanosine coupled to bovine serum albumin (BSA) by periodate oxidation. They were named A-16 and G-K21 respectively and selected for their ability to recognize single-stranded DNA. Their epitope specificities were assessed and their dissociation constants determined by an indirect ELISA method. The KD values for adenosine and guanosine coupled to BSA were 9.9 X 10(-7) M and 1.1 X 10(-10) M for G-K21 respectively, and 2.5 X 10(-8) M and 1.0 X 10(-6) M for A-16. These monoclonal anti-nucleoside antibodies were used to develop a sandwich enzyme immunoassay for single-stranded DNA. The purified IgG antibodies were coupled to beta-galactosidase and alkaline phosphatase by the one-step glutaraldehyde method and used in a test optimized for pH, saturating proteins, coating antibody, nature of the conjugate and protein concentrations. Less than 100 pg/well of single-stranded DNA could be detected, and the detection was linear over a DNA concentration ranging from 0.34 to 34 ng/ml. The assay could quantitate single-stranded DNAs of differing origin, but not RNAs. The test was compared to another titration method, and used to calibrate target DNA amounts in non-radioactive hybridization experiments.

Animals↗
PubMed1989

A non-radioactive diagnostic test for the detection of HBV DNA sequences in serum at the single molecule level.

A non-radioactive diagnostic test, using an acetylaminofluorene-labelled DNA probe, was developed to detect HBV DNA sequences in serum. In vitro enzymatic amplification was employed to increase the amount of HBV DNA sequences, and an amplification rate up to 1.5 x 10(7) was observed. When a dot-blot was performed after amplification with the Klenow fragment for 32 cycles, the detection limit was 3-30 particles. Sera from 20 blood donors and 10 HBs-Ag carriers were screened simultaneously, with the non-radioactive test performed after 28 amplification cycles, and with the classical radioactive test without amplification. An acceptable correlation was obtained between these two techniques. In Southern blot analysis of samples amplified with the thermoresistant DNA polymerase (Taq polymerase) for 40 cycles, a single DNA molecule was detected. Thermal treatment at 115 degrees C efficiently disrupted purified viral particles and allowed the detection of a single viral particle. Applied to crude serum, a kinetic study showed that this treatment was optimal after an incubation time of up to 10 min. Under these conditions, the detection limit was approximately 2 x 10(5) viral particles, after 40 amplification cycles performed with the Taq polymerase.

Base Sequence↗