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Biomedical subjects

J C Nicolas

Publications and source records attributed to J C Nicolas.

At least 55 records · Page 3Linked to original sources
PubMed1997

[Epstein-Barr virus and cellular immortalization].

Epstein-Barr virus has been associated to several forms of neoplasia including Burkitt's lymphoma, B lymphomas in immuno-compromised patients and undifferentiated nasopharyngeal carcinoma. Immunodepression, genetic and/or environmental factors and the expression of several viral genes (latent genes mainly) may contribute to these pathologies. In vitro, several latent proteins (EBNA 1, 2, 3A, 3C, 5 et LMP-1) directly or indirectly contribute to the initiation and maintenance of the transformation process. The role of these proteins is discussed in the present article.

Animals↗
PubMed1996

In vitro and in vivo interactions between nuclear receptors at estrogen response elements.

To study mechanisms involved in the antiestrogenic effect of retinoic acid (RA), previously described in mammalian cells, we used in vitro and in vivo approaches. One hypothesis was direct competition between nuclear receptors (ER, RAR and RXR) at the DNA level. We first showed in vitro that the RAR/RXR heterodimer could weakly bind an ERE and that retinoid receptors reduced binding of ER to an ERE. We next checked whether, in yeast, direct competition between receptors that recognize the same responsive element could be monitored in a reconstituted heterologous estrogen-responsive system, by determining the expression of a reporter gene. We then co-transformed RAR and RXR in an estrogenic responsive strain. This model demonstrated that, even though RAR/RXR was able to bind an ERE, the addition of retinoic acid had no inhibitory effect on estrogen-induced responses in this yeast system, unlike in mammalian cells. Interference between these receptors should require other factors than interactions at the ERE level. This model could be used to identify mammalian factors interacting with estrogen and retinoic acid receptors which could play a role in crosstalk between these receptors.

Animals↗
PubMed1996

Results of culture form colonoscopically obtained specimens for bacteria and fungi in HIV-infected patients with diarrhea.

BACKGROUND: The aim of our study was to determine the diagnostic yield of culture for bacteria and fungi from colonic biopsy specimens in 290 consecutive HIV-infected patients with diarrhea. METHODS: During each colonoscopy, three biopsy specimens were homogenized and cultured on media for Salmonella and Shigella and for Campylobacter and Yersinia, on Loewenstein medium and on Sabouraud medium. RESULTS: Cultures were found positive for one (n = 32) or two (n = 5) infectious agents in 37 cases, i.e., in 12.8% of the patients. Bacteria were isolated in 24 cases, and identified as Campylobacter jejunl-coli (n = 14), Salmonella (n = 2), Shigella (n = 1), or Pseudomonas aeruginosa (n = 7). Among the 14 patients with C. jejuni-coli intestinal infection, 11 had normal-appearing mucosa at colonoscopy, and 3 had a concomitant stool culture negative for Campylobacter. Mycobacterial cultures were positive for Mycobacterium avium intracellulare in 6 patients, who were already known as having a disseminated M. avium intracellulare infection from positive blood cultures. Fungal cultures were positive for Candida in 10 cases, without clear clinical significance. CONCLUSIONS: The overall yield of culture for bacterial pathogens from colonic tissue in HIV-infected patients with diarrhea is low, but some individual cases of C. jejuni-coli infections may be detected from colonic tissue culture and not diagnosed by concomitant stool culture.

AIDS-Related Opportunistic Infections↗
PubMed1996

Glucocorticoids induced down-regulation of glucocorticoid receptor mRNA expression in asthma.

Although their precise mechanism of action remains to be elucidated, glucocorticoids represent the most effective therapy in the treatment of asthma. Interactions between the glucocorticoid receptor and the AP-1 complex have been shown to regulate the transcription of some genes, including glucocorticoid receptor itself. The aim of the present study was to compare the expression of mRNA for glucocorticoid receptor in human blood monocytes obtained from seven unstable untreated asthmatic patients who were subsequently treated with high doses of parenteral corticosteroid (methyl prednisolone 120 mg/day) for 10 days. mRNA expression was identified after RNA extraction using RNAzol and analysed after reverse transcriptase, by polymerase chain reaction using a semiquantitative competitive hybridization assay. All asthmatic patients showed an improvement in their FEV1 values after corticosteroid treatment (per cent of predicted value 68.28 +/- 4.93 versus 95.57 +/- 6.41, P < 0.02), and a significant decrease for glucocorticoid receptor mRNA expression (P < 0.02) was observed in their monocytes. This is the first report of an ex vivo down-regulation for the glucocorticoid receptor mRNA expression, following corticosteroid treatment.

Adult↗
PubMed1996

Interstrain variations in the cytomegalovirus (CMV) glycoprotein B gene sequence among CMV-infected children attending six day care centers.

Using the shell vial assay and sequence analysis of a variable region of the glycoprotein B (gB) gene, cytomegalovirus (CMV) excretion rates in urine and virus transmission were studied among 93 children from six day care centers (DCCs). During a 6-month period, excretion rates differed significantly between DCCs (P < .001). The 6 gB gene sequences, obtained from 24 CMV-infected children, were classified in four previously defined groups. In five DCCs, 2 or 3 strains cocirculated, and none was dominant. Infection could have been acquired outside the DCC for 2 children and inside it for 9. Two children from the same DCC had mixed infections. No differences in hygiene, child care practices, or experience and level of qualification of the staff could explain this wide variety of excretion rates between DCCs. The distribution of gB gene patterns observed does not suggest that 1 type was dominant or more efficiently transmitted.

Base Sequence↗
PubMed1996

Study of mother-to-child Epstein-Barr virus transmission by means of nested PCRs.

The aim of this study was to investigate the possibility of vertical Epstein-Barr virus (EBV) transmission. We developed two nested-PCR methods for amplifying distinct regions of EBV DNA (BNRF1 and BamHI W) in circulating lymphocytes. Nested PCR was applied to samples obtained from 67 mother-infant pairs within 1 week of birth. We also tested samples from 16 neonates born to human immunodeficiency virus (HIV)-infected mothers to determine whether HIV increases the potential risk of vertical EBV transmission. About half of the 67 women in the first population were positive by nested PCR. Two neonates born to EBV PCR-positive women were also PCR positive. One of the 16 neonates born to HIV-infected women was PCR positive for EBV. These results strongly support the possibility of EBV transmission in utero or during delivery but do not suggest that HIV infection increases this risk. Further studies are required to confirm these findings, to identify the precise mode of vertical EBV transmission, and to determine the outcome for infants who are positive at birth for EBV DNA by nested PCR.

Female↗
PubMed1996

Mutation of isoleucine 747 by a threonine alters the ligand responsiveness of the human glucocorticoid receptor.

Mutation of isoleucine 747 to threonine in the C-terminal part of the ligand-binding domain (LBD) of the human glucocorticoid receptor (GR) alters the ligand specificity for transactivation. Natural glucocorticoids such as cortisol or corticosterone were completely inactive with the mutant 1747T, whereas synthetic steroids like dexamethasone efficiently stimulated GR 1747T-mediated transactivation. However, the corresponding ligand dose-response curve for dexamethasone-induced transactivation was shifted to higher concentrations when compared with that obtained with the wild type GR. Neither this shift nor the inability of cortisol to activate the 1747T mutant was due to an altered in vitro ligand-binding affinity. In the canonical three-dimensional structure of nuclear receptor LBDs, isoleucine 747 is in the direct vicinity of residues that contribute to the ligand-binding pocket. Moreover, it is located in the C-terminal LBD region, which harbors the conserved core of the activation function AF-2 and undergoes a ligand-induce transconformation, required to generate the surface interacting with putative transcriptional intermediary factors/coactivators of AF-2. The phenotype of 1747T mutant is discussed in view of the possible consequences of the mutation on the various events which, according to the model, lead to a transcriptionally competent AF-2.

Amino Acid Sequence↗
PubMed1995

[Preimplantation diagnosis of delta F508 mutation of mucoviscidosis in transgenic mice].

OBJECTIVE: The aim of our study was to master preimplantation diagnosis in transgenic mice. METHODS: Stage 4 or 8 cells preembryos were collected from C57BL6xCBAF1 mice, three days after mating with transgenic C57BL6xSJLF1 mice homozygous for the human gene delta F508 mutation of cystic fibrosis. A single blastomere was sampled by microbiopsy and the preembryos transferred in Swiss mice foster mothers. Molecular biology for delta F508 mutation on a single blastomere was performed with a double PCR technique on a Perkin Elmer Cetus 9600. RESULTS: The success rate of biopsy on the 122 preembryos with 4 cells was 90.2%, and 93.8% on the 128 prembryos with 8 cells. The rate of in vitro hatching was 84.6% after biopsy on 52 preembryos with 4 cells (92.2% for controls without biopsy, p > 0.05), and 90.0% for 50 preembryos with 8 cells (94.4% for controls without biopsy, p > 0.05). The rate of birth after biopsy and transfer of 42 preembryos with 4 cells was 66.7% (72.5% for controls transferred without biopsy, p > 0.05) and 70.3% for 37 preembryos with 8 cells (71.9% for controls, p > 0.05). No difference was evidenced for births weight or organ weight at 3 weeks between mice born from biopsied embryos and controls. Thirty-two double PCR were performed for the diagnosis of the cystic fibrosis delta F508 mutation, 20 on a single blastomere obtained by microbiopsy and 12 for various negative controls, with 100% specificity and 100% sensitivity. CONCLUSIONS: Microbiopsy of preembryos with 4 cells or 8 cells does not alter their viability or further development. The double PCR technique on a single blastomere for the cystic fibrosis delta F508 mutation is reliable with our current methods of investigation.

Animals↗
PubMed1995

Nonradioactive quantification of glucocorticoid receptor expression during differentiation of human monocytic cells (U937).

We describe a method for relative quantification of specific mRNA using a nonradioactive assay based on DNA strand competition between identical sequences of biotin- and fluorescein-labeled amplicon (probe) and unlabeled amplicon (target) during hybridization. As the target quantity increased, that of the double-labeled probe decreased in accordance with the mass action law. This technique was successfully applied to evaluate differences in glucocorticoid receptor expression in U937 cells before and after the addition of potent differentiation inducers: 12-O-tetradecanoylphorbol 13-acetate (TPA) and a combination of all-trans retinoic acid (RA) and 1,25-dihydroxyvitamin D2 (VD). We observed that TPA treatment was associated with an increase in specific binding of [3H]dexamethasone and up-regulation of GR mRNA while no enhanced GR expression was perceived with RA/VD treatment.

Base Sequence↗
PubMed1995

[Applications of luminescence reactions in life science].

Bioluminescence and chemiluminescence have emerged in the last decade as a major tool for biochemical and biological studies. Several very sensitive assays have been developed in our laboratory such as enzymatic assays, immunoassays and detection of nucleic acids. The use of a new instrumentation allowing to dimensional photon counting has permit the emergence of new investigations at the cellular level. In addition to the advantages of sensitivity and the real-time non-invasive nature of this detection system, the imaging potential of using low-light and photon counting video cameras has been particularly influential in establishing its ascendence over more traditional systems. This review provides a reflection in this field through several applications in life sciences.

Adenosine Triphosphate↗
PubMed1995

[Modulation of cellular response expression during prolonged treatment with antiestrogens].

The effects of a prolonged antiestrogen treatment on two estrogen-dependent responses and an AP-1 response were studied on two cell lines derived from MCF-7 cells. 1) Hydroxytamoxifen specifically provoked an irreversible inactivation of a chimeric estrogen-dependent gene expression in less than 30 days. This process was estrogen receptor mediated and led to a cellular heterogeneity that was induced by the treatment and was not due to a cell selection process. A similar heterogeneity was also observed for the progesterone receptor expression but after a longer treatment time. The mechanism underlying this phenomenon is currently investigated. 2) After a four day treatment of cells with an antiestrogen, the phorbol ester inducible expression of a chimeric AP-1 response was stimulated by a factor 3-4. This stimulation was antiestrogen dose-dependent and suppressed by the presence of estradiol, which strongly suggested that estrogen receptor was involved. This was confirmed by the fact that the phenomenon was not observed in a cell line devoid of estrogen receptor. This result suggests a yet unknown mechanism by which an antiestrogen could have an agonistic property allowing hormone independence to appear. Both these phenomena show that new activities of antiestrogens may be evidenced after prolonged treatments with unexpected consequences on endocrine therapy.

Estrogen Antagonists↗
PubMed1994

Hydroxytamoxifen induces a rapid and irreversible inactivation of an estrogenic response in an MCF-7-derived cell line.

The MVLN cell line was established in our laboratory from MCF-7 cells by stable transfection with the luciferase gene under the control of an estrogen-responsive element from the Xenopus vitellogenin A2 gene. This cell line allowed us to visualize the induction by hydroxytamoxifen of a heterogeneity in the cell population with regard to the expression of the luciferase gene. Treated cells lost their estradiol-inducible luciferase activity, progressively and irreversibly; the luciferase expression of 80% of the cells was irreversibly inactivated by a 12-day hydroxytamoxifen treatment. We showed that this inactivation process was specific for an estrogenic response and was mediated by the estrogen receptor. Tamoxifen itself gave rise to such an inactivation, whereas other compounds belonging to the triphenylethylenic family but differently substituted on the ethylenic carbon and the ICI 164,384 compound were not as efficient. This irreversible inactivation was accompanied by a sharp decrease in the luciferase mRNA level; however, the estrogen receptor function and the cellular transcriptional machinery were not affected by the treatment. Although this antiestrogen treatment neither affected the estrogen-dependent cell growth nor irreversibly inhibited the expression of the natural pS2 gene, these results highly suggest that long-term antiestrogen therapy may lead to some heterogeneity in tumor cells throughout the course of patient treatment.

Base Sequence↗
PubMed1994

Antiestrogenic effects of all-trans-retinoic acid and 1,25-dihydroxyvitamin D3 in breast cancer cells occur at the estrogen response element level but through different molecular mechanisms.

Most breast tumors show estrogen-dependent growth and are thus susceptible to antiestrogenic therapy. MCF-7 cells, obtained from a human estrogen-dependent breast carcinoma, are widely used for studying the modulation of estrogenic responses by different effectors. All-trans-retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (Vit D3) inhibited estrogen-induced growth of MCF-7 cells and their effect was potentiated by the classical antiestrogen, hydroxytamoxifen. In MCF-7 cells, we found that RA and Vit D3 also inhibited estrogen-induced transcription; this was shown both for an endogenous gene (pS2) and for various exogenous transfected genes. Their inhibitory effect could not be reversed by increasing estradiol concentrations, showing that contrary to classical antiestrogens, they did not compete with estradiol to bind the estrogen receptor (ER). Analysis of the inhibitory mechanisms indicates that RA and Vit D3 receptors can directly or indirectly impair the binding of ER to the estrogen responsive element. The antagonist effect of RA would be found especially at DNA level since it seems to essentially involve an estrogen responsive element. The antagonist effect of Vit D3 would be found especially at the ER level since it seems to concern estrogen binding and dimerization domains of ER. We conclude that the antiestrogenic effects of RA and Vit D3 are similar since they can, via their receptors, interfere with estrogenic action at the estrogen responsive element level but that they are not identical since different molecular mechanisms are involved.

Breast Neoplasms↗
PubMed1994

The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes.

Throughout the purification of the mdm-2 or mdm-2-p53 protein complexes, a protein with a molecular weight of 34,000 was observed to copurify with these proteins. Several monoclonal antibodies directed against distinct epitopes in the mdm-2 or p53 protein coimmunoprecipitated this 34,000-molecular-weight protein, which did not react to p53 or mdm-2 polyclonal antisera in a Western immunoblot. The N-terminal amino acid sequence of this 34,000-molecular-weight protein demonstrated that the first 40 amino acids were identical to the ribosomal L5 protein, found in the large rRNA subunit and bound to 5S RNA. Partial peptide maps of the authentic L5 protein and the 34,000-molecular-weight protein were identical. mdm-2-L5 and mdm-2-L5-p53 complexes were shown to bind 5S RNA specifically, presumably through the known specificity of L5 protein for 5S RNA. In 5S RNA-L5-mdm-2-p53 ribonucleoprotein complexes, it was also possible to detect the 5.8S RNA which has been suggested to be covalently linked to a percentage of the p53 protein in a cell. These experiments have identified a unique ribonucleoprotein complex composed of 5S RNA, L5 protein, mdm-2 proteins, p53 protein, and possibly the 5.8S RNA. While the function of such a ribonucleoprotein complex is not yet clear, the identity of its component parts suggests a role for these proteins and RNA species in ribosomal biogenesis, ribosomal transport from the nucleus to the cytoplasm, or translational regulation in the cell.

Amino Acid Sequence↗
PubMed1993

Detection of single base substitutions in polynucleotides by capture with immobilized oligonucleotides.

We have improved a sandwich hybridization assay to detect single base substitutions in polymerase chain reaction (PCR) amplified DNA sequences. The target DNA was captured by an immobilized oligonucleotide and revealed using a second oligonucleotide coupled to an enzyme. Short oligonucleotides (13, 15 bases) were used to obtain specific hybridization at 37 degrees C. We developed two different assay formats for rapid identification of PCR products: a microtitration plate format with oligonucleotides bound to polystyrene and a channelling assay using oligonucleotides immobilized on Sepharose, which did not require any separation step. The specificity and advantages of both methods are described.

Alkaline Phosphatase↗
PubMed1993

Virus isolation, polymerase chain reaction and in vitro antibody production for the diagnosis of pediatric human immunodeficiency virus infection.

Viral culture (VC), polymerase chain reaction (PCR) and in vitro antibody production (IVAP) by peripheral blood mononuclear cells were compared for the early diagnosis of HIV-1 infection in 46 infants born to HIV-1 seropositive mothers. The ten children considered infected on the basis of clinical signs and persistence of anti-HIV-1 antibodies had at least one positive viral culture and seven were always positive in both PCR and IVAP tests. PCR and IVAP tests were occasionally negative in three infected children. Among 30 healthy children who became seronegative and were always negative for viral culture, 22 (73.3%) were also repeatedly negative in PCR and IVAP. We report 6 cases of children classified as P2A at the term of this study but who had lost anti-HIV-1 antibodies. They presented at least one positive viral culture and occasional positive PCR and/or IVAP results. The results indicate that the combination of viral culture, PCR and IVAP tests improves the early diagnosis of pediatric HIV infection.

Antibody Formation↗
PubMed1993

The DNA-binding domain of two bZIP transcription factors, the Epstein-Barr virus switch gene product EB1 and Jun, is a bipartite nuclear targeting sequence.

The Epstein-Barr virus BZLF1 gene product EB1 (also called ZEBRA and Zta), is a transcription factor belonging to the bZIP (basic domain leucine zipper) family of nuclear proteins. Translocation to the nucleus of EB1 (J. Becker, U. Leser, M. Marschall, A. Langford, W. Jilg, H. Gelderblom, P. Reichart, and H. Wolf, Proc. Natl. Acad. Sci. USA 88:8332-8336, 1991) and of two other bZIP proteins, c-Jun and c-Fos (P. Roux, J.-M. Blanchard, A. Fernandez, N. Lamb, P. Jeanteur, and M. Piechaczyk, Cell 63:341-351, 1990), has been shown to be subject to regulation. We show here that for both EB1 and Jun the nuclear targeting signals (NTS) in the proteins' primary sequences are two clusters of positively charged amino acids. These clusters, called BRA and BRB, are necessary and sufficient to direct beta-galactosidase to the nuclear compartment and act as a bipartite NTS. They are conserved among all the bZIP proteins, and although they are not identical, they probably share the same function. Site-directed mutagenesis studies made on these basic clusters suggest that they also act as a bipartite NTS in the EB1 protein. Our results also demonstrate that in EB1 and Jun, these bipartite NTS are superimposed with bipartite DNA-binding domains, since BRA and BRB are required in vitro for direct and specific contact between these proteins and their DNA-binding sites.

Amino Acid Sequence↗