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Biomedical subjects

J C Nicolas

Publications and source records attributed to J C Nicolas.

At least 73 records · Page 4Linked to original sources
PubMed1992

A multiple luminescent procedure for the detection of different papillomaviruses on dot blots.

We developed a method based on the use of various luminescent systems for identification of several nucleic acid sequences on the same dot blots. In a simultaneous assay, the target DNA molecules were hybridized with different DNA probes. These probes were labelled with biotin or digoxigenin or directly coupled to enzymes such as glucose-6-phosphate dehydrogenase, peroxidase or alkaline phosphatase. After hybridization, these labels were detected by luminescent reactions using an amplified camera. Rapid detection and specific identification of pathogenic agents such as human papillomaviruses (HPV) could be performed in a single step by this procedure. Polymerase chain reaction was carried out using general primers and virus types were identified on dot blots using short HPV 11, 16 and 18 specific oligonucleotides.

Base Sequence↗
PubMed1992

Quantitative and qualitative analysis of amplified DNA sequences by a competitive hybridization assay.

The presence of allelic sequence variations in DNA fragments can be easily detected by measuring the extent of DNA strand exchange between test double-stranded PCR products (target) and labeled standard double-stranded PCR products (probe). Under selected hybridization conditions, sequences identical to the probe decreased the formation of double-labeled hybrid, whereas differing sequences were not efficient enough to compete with the regeneration of the probe. A single base substitution in the target DNA increased the percentage of remaining double-labeled probe. A general procedure involving denaturation and hybridization in solution under different temperature conditions or using different probes enabled sequence identification. The degree of regeneration of double-labeled probe was determined using a bioluminescent assay. We evaluated the specificity of this method with two probes (108 and 131 bp) and several targets with different base substitutions.

Base Sequence↗
PubMed1992

Generic HLA-DRB1 gene oligotyping by a nonradioactive reverse dot-blot methodology.

HLA-DRB1 allelic specificities can be determined using SSOs annealing to their complementary PCR-amplified target DNA. To perform HLA-DR oligotyping routinely for donors and recipients of bone marrow transplantation, a "reverse" dot-blot technique has been developed that consists in the hybridization of labeled PCR-amplified target DNA to SSOs that have been first attached to nitrocellulose membranes. The 15 oligonucleotides chosen enabled the following HLA-DRB1 "generic" specificities to be defined: DR1, BON, 2, 3, 4, 11, 11 JVM, 12, 13, 13 HAG, 14, 7, 8, 9, 10. The genomic DNA was amplified by asymetric PCR with incorporation of biotinylated deoxynucleotides predominantly to generate labeled single-stranded DNA. Hybridization between specific immobilized oligoprobes and target DNA was nonradioactively detected by a colorimetric reaction using alkaline phosphatase. The reverse dot-blot methodology was successfully tested, first, for the determination of HLA-DR4 subspecificities, and then the procedure was routinely applied to the generic HLA-DR oligotyping of bone-marrow donors and recipients.

Base Sequence↗
PubMed1992

[Bioluminescence and creation of chimeric cellular models].

The response of a chimeric cellular model to a hormone has to be more specific, more sensitive, more easily measurable and quantifiable than the natural response. Hence, it can be used either in fundamental and applied studies, for example to analyse hormonal interactions, or to screen new hormone molecules. In the field of steroid hormones, we took benefit from the advantages of the firefly luciferase as receptor gene (simplicity and rapidity of the measurement, high sensitivity, handiness ...) to construct such chimaeric models. Such cell lines were obtained by developing another original use of this reporter gene based on an easy detection of the luciferase activity in intact living cells and using a photon counting camera. This was successfully adapted to obtain and to detect stable transfected cells.

Cell Line↗
PubMed1992

[Detection of nucleic acid sequences by bioluminescence. Importance of an internal reference system for qualitative and quantitative analysis].

Various luminescent detection systems were used to detect and quantify simultaneously several probes. For quantitative analysis, one probe was used as an internal standard to evaluate the amplification yield and the others to quantify the amplified target. After using hybridization in solution, accurate results were obtained. For a qualitative analysis, amplified sequences were hybridized to specific oligonucleotides which were labelled or immobilized. To compare amplified fragments to products of known sequences, we developed a more efficient technique based on DNA strand exchange occurring during hybridization in solution. This method allowed to detect a single base substitution in DNA fragments of 280 base pairs.

Base Sequence↗
PubMed1991

Detection of anti-Epstein-Barr-virus transactivator (ZEBRA) antibodies in sera from patients with nasopharyngeal carcinoma.

The Epstein-Barr virus (EBV) is a ubiquitous Herpes virus which causes infectious mononucleosis and is associated with such different neoplasms as Burkitt's lymphoma and nasopharyngeal carcinoma. EBV latently infects its target cells; nevertheless, evidence of viral replication in NPC tumours has been uncovered. Among the EBV transactivators, the ZEBRA protein plays a crucial role in switching the virus from a latent to a productive mode. ZEBRA protein was produced using a eukaryotic expression vector: the open reading frame containing the BZFLI cDNA has previously been inserted down-stream from the adenovirus major late promoter leading to expression of a 38-kDa nuclear protein. We performed serological studies by employing ZEBRA protein expressed in human cells for immunofluorescence and Western-blot assays. We were able to detect IgG anti-ZEBRA antibodies (IgG/ZEBRA) in 87% of NPC patients. These antibodies were absent in control sera; IgG/ZEBRA antibodies can be proposed as a useful marker for diagnosis of NPC tumors.

Antibodies, Viral↗
PubMed1991

Quantification of DNA sequences obtained by polymerase chain reaction using a bioluminescent adsorbent.

We studied various parameters affecting the sensitivity of assays that use nucleic acid hybridization in solution followed by capture of the hybrid on a solid phase. Sensitivity is limited not only by nonspecific binding of the detection components but also by reannealing of the target or probe to itself. To perform sensitive assays, the probe concentration must be low enough to reduce high nonspecific binding. Under these conditions, however, the strand displacement reaction or the reannealing of the target to itself drastically decreases the hybridization yield, particularly when the target and the probes are different sizes. To improve DNA detection, we propose a sandwich method based on hybridization of oligonucleotides with a single-strand DNA obtained by polymerase chain reaction under asymmetric conditions. The assay can be performed in one step using a bioluminescent detection procedure which does not require any separation step. The specificity of the method is sufficient to perform a rapid detection and quantification of papillomavirus in biological samples.

Adsorption↗
PubMed1991

Transcriptional interference between the EBV transcription factors EB1 and R: both DNA-binding and activation domains of EB1 are required.

The switch from latency to a productive infection in EBV-infected B cells is linked to the expression of two viral sequence-specific DNA-binding transcription factors called EB1 and R. EB1 shares sequence homologies with the bZIP family of proteins in the basic region required for specific DNA interaction. Here, we provide evidence that EB1 and R can synergistically activate specific transcription, and that overexpressed, unbound EB1, represses the R-induced transcription ('squelching'). In order to identify the EB1 domains involved in transcriptional activation, transcriptional synergy and transcriptional repression, we performed extensive mutagenesis of the EB1 protein. Results show that five segments (region 1 to region 5), localized at the N-terminus of EB1 exhibit characteristics of activating domains, since they are required for full transcriptional activity, without obvious role in DNA-binding, or the nuclear localization. Two domains rich in basic amino-acids are required for the nuclear localization of EB1. One domain is within the basic region B, also necessary for specific and stable interaction between EB1 and its cognate DNA sequences. It is also shown that the 'activation' domain, and more surprisingly the DNA-binding domain of EB1, may interact with a factor(s), essential for R-induced activation, and probably required for synergy between EB1 and R.

Amino Acid Sequence↗
PubMed1991

Detection of anti-Epstein-Barr virus trans-activator (ZEBRA) antibodies in sera from patients with human immunodeficiency virus.

Patients with AIDS and AIDS-related complex often show symptoms of Epstein-Barr virus (EBV) reactivation. Several EBV-encoded trans-acting factors activate the EBV lytic cycle, and one, ZEBRA (BamHI Z EBV replication activator; also called EB1), switches EBV from its latent to productive cycle. Indirect immunofluorescence studies were done using human cells transfected with a recombinant DNA-harboring cDNA sequence spanning BZLF1 (BamHI Z left frame 1) that was inserted downstream of the adenovirus major late promoter. IgG anti-ZEBRA antibodies were detected in a high proportion of asymptomatic HIV carriers and in AIDS patients but were absent in healthy control individuals. The presence of anti-ZEBRA antibodies in the sera of HIV-positive patients favors the hypothesis that EBV reactivates in such subjects. This finding may be of practical importance in the prognostication of AIDS development.

Adult↗
PubMed1991

Analysis of the spontaneous in vitro anti-HIV-1 antibody secretion by peripheral blood mononuclear cells in HIV-1 infection.

We studied the spontaneous in vitro secretion of anti-HIV-1 antibodies by peripheral blood mononuclear cells (PBMC) from HIV-1-infected patients. Specific antibody production was detected in supernatants of PBMC cultures using an ELISA; HIV-1 specificity was confirmed by antigen adsorption and Western blotting. This antibody secretion was found to be an active phenomenon and was not due to a release of plasma antibodies passively adsorbed onto the cell membranes. In all positive supernatants, anti-HIV-1-secreted antibodies were directed against env-encoded antigens and many supernatants also contained antibodies to pol- and gag-encoded antigens. PBMC from all HIV-1-infected patients tested (140 adults and 18 infants) secreted anti-HIV-1 antibodies. This production was found during all the clinical stages of HIV-1 infection. Our results suggest that this spontaneous HIV-1-specific antibody secretion represents a marker of HIV-1 infection. Detection of these antibodies could be a valuable tool for early confirmation of HIV-1 infection in neonates born to HIV-1-seropositive mothers.

Adolescent↗
PubMed1991

[Retinoic acid has an antiestrogenic effect on different regulated estrogen genes in different cellular types].

In this paper, we confirmed that retinoic acid is an antiestrogenic compound with respect to different chimaeric estrogenic responses and with respect to different cellular types. This was shown by transient transfection of MCF-7 cells with plasmids driving the chloramphenicol acetyl-transferase gene via different estrogenic regulatory part (pS2) and the first promotor of the progesterone receptor gene (PR1); an identical conclusion was obtained in HeLa cells by cotransfecting a plasmid expressing the estrogen receptor. In addition, the inhibitory effect of retinoic acid was not observed for genes regulated by the progesterone receptor and the glucocorticoid receptor. As the antiestrogenic effect of retinoic acid was increased by cotransfecting acid receptor(s) RAR alpha, beta, gamma, we concluded that RAR(s) is(are) involved in the antiestrogenic effect of retinoic acid.

Estrogen Antagonists↗
PubMed1990

Restriction fragment pattern analysis of genomes from French isolates of suis herpes virus 1 (Aujeszky's disease virus).

Purified DNA from 45 isolates of suis herpes virus 1 (SHV1) collected between 1980 and 1987 from clinical outbreaks of Aujeszky's disease on French farms was compared by restriction fragment pattern (RFP) analysis. The BamHI generated RFPs were found to be distinguishable, confirming RFP analysis as a potential epidemiological tool. The RFP could be assigned to two established major electrophoretic types and different subtypes. The RFP analysis indicated that the majority of outbreaks were caused by ADV with a central European genome type. The heterogeneity of RFPs among PRV isolates recovered from the central nervous system, lung, and foetus was not restricted specifically to one clinical entity. Variations in the virulence of the 45 isolates studied in mice, chicks, or piglets were unrelated to the RFP subtypes. One unusual RFP has been described for one strain of low virulence.

Analysis of Variance↗
PubMed1990

Cytomegalovirus-specific B cell activation as a potential marker for the diagnosis of cytomegalovirus infection.

In vitro secretion of antibodies to cytomegalovirus was investigated by analysis of peripheral blood mononuclear cell culture supernatants from subjects infected with cytomegalovirus. Patients with primary or recurrent cytomegalovirus infection showed transient in vitro cytomegalovirus-specific antibody secretion. A high proportion of patients infected with human immunodeficiency virus and sero-positive for cytomegalovirus showed in vitro cytomegalovirus-specific antibody secretion. All peripheral blood mononuclear cell cultures from patients with symptomatic human immunodeficiency virus infection were found to secrete in vitro antibodies to cytomegalovirus, despite the fact that isolation of cytomegalovirus from some of these patients was not achieved. In human immunodeficiency virus-infected patients, in vitro secretion of anti-cytomegalovirus antibodies appeared to be persistent. In vitro cytomegalovirus-specific antibody secretion by peripheral blood lymphocytes probably reflects an in vitro cytomegalovirus-specific B cell activation. This new assay could be considered an interesting method for detecting both acute or chronic cytomegalovirus infection, with potential use in routine laboratory practice.

Adult↗
PubMed1990

Silver staining of DNA restriction fragments for the ultrarapid identification of pseudorabies virus strains.

Restriction endonuclease analysis of pseudorabies virus DNA has been used to study various virus strains. To make use of a rapid technique for the identification of viral strains, studies have been undertaken to facilitate purification of the DNA from viral particles present in cytoplasmic fractions. The ultrasensitive photochemical silver staining of nucleic acid, described by Beidler et al. (Analytic Biochemistry 1982: 126) has been adapted and applied to the detection of pseudorabies virus restriction fragments. In a period of 5 h more than 1 microgram of DNA can be extracted from a 25 cm2 plastic flask containing infected cells and purified without ultra centrifugation. Low molecular weight DNA was separated from high molecular weight DNA by polyacrylamide gel electrophoresis. The restriction fragments were selectively visualized by silver staining which can detect 0.025 micrograms of total pseudorabies virus DNA. The electrophoretic DNA pattern of vaccine and wild strains has been studied using these techniques and the results agree with previously published data.

DNA Restriction Enzymes↗
PubMed1990

Nucleotide sequence of SHV-2 beta-lactamase gene.

The nucleotide sequence of plasmid-mediated beta-lactamase SHV-2 from Salmonella typhimurium (SHV-2pHT1) was determined. The gene was very similar to chromosomally encoded beta-lactamase LEN-1 of Klebsiella pneumoniae. Compared with the sequence of the Escherichia coli SHV-2 enzyme (SHV-2E.coli) obtained by protein sequencing, the deduced amino acid sequence of SHV-2pHT1 differed by three amino acid substitutions.

Amino Acid Sequence↗