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J C Nicolas

Publications and source records attributed to J C Nicolas.

At least 145 records · Page 8Linked to original sources
PubMed1983

Host range temperature-conditional mutants in the adenovirus DNA binding protein are defective in the assembly of infectious virus.

R(ts107)202 is a host range temperature-conditional mutant of adenovirus type 5. This mutant is temperature sensitive for replication and plaquing in 293 cells but is temperature independent for growth and plaquing in HeLa cells (J. C. Nicolas, F. Suarez, A. J. Levine, and M. Girard (1981) Virology 108, 521-524). The mutant was isolated in HeLa cells as a temperature-independent revertant of the H5ts107 temperature-sensitive mutant that maps in the adenovirus DNA binding protein (DBP). The reasons for the temperature conditional phenotype of this mutant in 293 cells were investigated. The mutant synthesized an unstable DBP in both HeLa and 293 cells at 39 degrees. In 293 cells at 39 degrees, about two- to threefold less viral DNA was synthesized by r(ts107)202 as compared to Ad5wt. R(ts107)202 infected cells at 39 degrees produced normal (wild-type) amounts of all detectable late viral structural proteins. The mutant failed, however, to produce infectious virus or assemble virus particles in 293 cells at 39 degrees. The altered DBP may therefore play a role in the assembly of virus particles, either directly or indirectly via an altered DNA structure. The failure of r(ts107)202 to assemble virion particles in 293 cells at 39 degrees furthermore suggests that virus assembly is dependent upon cellular factors that differ in HeLa and 293 cell.

Adenoviruses, Human↗
PubMed1983

Structure and function of DNA binding proteins from revertants of adenovirus type 5 mutants with a temperature-sensitive DNA replication.

H5ts107 and H5ts125 are two adenoviruses type 5 (Ad5) mutants with a temperature-sensitive DNA replication. Both mutants contain an altered gene encoding the DNA binding protein (DBP). We have established by nucleotide sequence analysis that both mutants carry exactly the same mutation in the DBP gene resulting in the substitution of a proline residue at position 413 in the wild-type DBP amino acid sequence (529 amino acid residues long) by a serine residue. Revertants of H5ts107 and H5ts125, which are temperature independent in plaque efficiency and growth in HeLa cells at 32 degrees and 39 degrees, were characterized by nucleotide sequence analysis of their DBP genes. Four types of revertants could be distinguished: revertants with the wild-type DBP amino acid sequence (type I) and, revertants carrying, in addition to the original H5ts107/H5ts125 mutation at position 413, intragenic second site mutations at position 508 histidine leads to tyrosine (type II), at position 352 glycine leads to aspartic acid (type III), and at position 347 alanine leads to proline (type IV), respectively. All intragenic second site mutations are located, together with the H5ts107/H5ts125 mutation, in the C-terminal 45-kD fragment of the adenovirus DBP molecule. This provides further evidence that this part of the DBP molecule plays an important role in viral DNA replication. Phenotypic characterization of the revertants (J.C. Nicolas, F. Suarez, A.J. Levine, and M. Girard (1981), Virology 108, 521-524; (J.C. Nicolas, D. Ingrand, P. Sarnow, and A.J. Levine (1982), Virology 122, 481-485) has shown that the second site mutations reveal additional functional domains in the DBP molecule.

Adenoviruses, Human↗
PubMed1983

Isolation of a human pararotavirus.

During a survey of 350 different isolates of human rotaviruses, a single isolate with unique properties was obtained. Unlike all rotaviruses tested this unique isolate failed to react in an ELISA test with antibody directed against the group-specific rotavirus antigen. The morphology of this unusual virus isolate was identical in electron micrographs with other rotaviruses and the virus also contained 11 double-stranded RNA segments characteristic of this virus group. The electrophoretic migration of these RNAs (electrophoretype) in polyacrylamide gels did not coincide with the typical pattern of distinct size classes observed with all human rotaviruses. The unique isolate thus had two distinct properties that distinguish it from human rotaviruses, lack of a group-specific antigen and an atypical electrophoretype of the RNA segments. Based upon this, the new human isolate is given the name pararotavirus to distinguish this new type of virus group. Porcine and chicken pararotaviruses have recently been described with similar properties.

Antigens, Viral↗
PubMed1983

Bioluminescent assay of femtomole levels of estrone and estradiol.

A rapid and very sensitive enzymatic assay of estrogens was developed using the transhydrogenase reaction of the human estradiol-17 beta dehydrogenase and bioluminescence for the quantification of NADH accumulated during the first reaction. The assay requires two steps: first, the addition of transhydrogenase buffer, and a few minutes later the addition of the bioluminescence reagent. The method can determine 0.1 to 50 pg of estrogens in the assay, in 1 h, with a coefficient of variation of 5%.

17-Hydroxysteroid Dehydrogenases↗
PubMed1983

Enzymatic determination of dehydroepiandrosterone sulfate in biological fluids.

A simple method is described for the determination of androgens, particularly dehydroepiandrosterone sulfate (DHEAS), in human plasma and urine. The method does not involve extraction or chromatography. An internal standard is used as reference. The androgens are enzymatically converted to estrogens and these latter compounds can be measured by the enzymatic method previously described [1]. The range of accuracy was between 99 and 103%. The precision of the method was 2.5%. The sensitivity was 0.1 pM per sample. The method is suitable for routine use, and one worker can easily perform twenty assays in one day.

Aromatase↗
PubMed1983

Active site-directed irreversible inhibition of 3 beta-hydroxysteroid dehydrogenase from human placenta.

To obtain a placental microsome preparation able to convert androstenedione and testosterone specifically to estrogens, 3 beta-hydroxysteroid dehydrogenase must be eliminated. After solubilisation by Triton X-100, the remaining 3 beta-hydroxysteroid dehydrogenase activity is inhibited by 11 alpha-bromoacetoxyprogesterone, an alkylating analogue of progesterone, which behaves as an irreversible active site-directed inhibitor. The enzyme is protected against inactivation and alkylation by steroid and coenzyme substrates. The inhibition is specific to 3 beta-hydroxysteroid dehydrogenase and the estrogen synthetase (aromatase) activity contained in the preparation is not affected by this inhibitor.

3-Hydroxysteroid Dehydrogenases↗
PubMed1983

Detection, rescue, and mapping of mutations in the adenovirus DNA binding protein gene.

r(ts107)202 was isolated in HeLa cells as a temperature-independent revertant of H5ts107, an adenovirus mutant that maps in the structural gene of the viral DNA-binding protein. r(ts107)202 is a host-range temperature-conditional mutant: it is temperature independent for growth in HeLa cells but temperature sensitive for growth in 293 cells, a type 5 adenovirus-transformed human cell line. Marker rescue experiments using H5ts107 DNA and restriction enzyme fragments from r(ts107)202 DNA demonstrated that the mutations causing the r(ts107)202 phenotype were localized in HindIII fragment A containing the entire DNA-binding protein gene. To obtain a fine structure map of the r(ts107)202 mutations, overlap recombination between EcoRI fragment A (0-75.9 map units) from either Ad5wt or r(ts107)202 and BamHI fragment B (59.5-100 map units) from either Ad5wt or r(ts107)202 was performed. Segregation of the H5ts107 primary-site mutation away from the accompanying reversion mutation could be demonstrated in 5 of 200 plaques when r(ts107)202 EcoRI fragment A was crossed with the Ad5wt BamHI fragment. In the reciprocal cross, none of 200 plaques contained the H5ts107 mutant. These results permitted a determination of the order of the primary-site mutation (H5ts107) and secondary-site mutation in r(ts107)202, and the frequency of recombination predicted a distance of 50-340 base pairs between these two mutations. This experimental result agrees with the nucleotide sequence of the r(ts107)202 mutant which shows that 182 base pairs separate the primary-site and secondary-site mutations in r(ts107)202.

Adenoviruses, Human↗
PubMed1983

Centrifugal analyzer used for enzyme immunoassay of progesterone and choriomammotropin.

Recently we developed an enzyme immunoassay involving the use of steroid delta-isomerase (EC 5.3.3.1) as enzyme label and exclusion-affinity chromatography for rapid separation of free antigen-enzyme conjugate that bound to antibodies (J. Immunol. Methods 35: 267-284, 1980). Here we describe an automated version of this procedure, for immunoassay of progesterone and choriomammotropin (human placental lactogen) in serum with the use of a centrifugal analyzer. After incubation, suitable dilutions of sera or extract plus antiserum, conjugate, and double antibody were filtered on an estradiol affinity gel-filtration column; the enzyme activity of the filtrates was determined with the centrifugal analyzer. Results correlate well with those obtained by radioimmunoassay: r (progesterone) = 0.980, r (choriomammotropin) = 0.940. The within-run and between-run precision, specificity, sensitivity, accuracy, and speed of this system make it a useful tool for immunoassay.

Autoanalysis↗
PubMed1983

[Detection of rubella-specific IgM by an immunoenzyme technic].

An enzyme-linked immunosorbent assay (ELISA) was compared to sucrose gradient centrifugation technique for rubella specific IgM detection of 114 sera. The 28 sera which were rubella specific IgM positive, were also positive by ELISA. Among the 86 sera which were rubella IgM negative by centrifugation, 6 were equivocal and 2 were rubella IgM positive by the ELISA test, without clinical or epidemiological connection. These false results make a necessity of confirming by an immunoglobulin separation technique the ELISA IgM positive sera.

Adult↗
PubMed1983

[Effect of the hormonal environment on the human oocyte and its later development in vitro].

The biosynthesis of progesterone by the granulosa cells that come from follicles from which the oocyte has been fertilized and has divided in vitro is far superior to that from the cells coming from follicles in which the oocyte has failed to develop. This result is in accordance with the raised levels of progesterone and the influence of androgens on lower levels of oestrogens in follicular fluid that evolved favourably. In the same follicles the ratio of concentrations of prostaglandins PGE2 to PGF2 alpha is twice as high as in the liquid from other follicles.

Androgens↗
PubMed1982

A one-year virological survey of acute intussusception in childhood.

A prospective study was carried out during 1 year in order to correlate intussusceptions in childhood with a viral infection. A virus was incriminated in 50% of the 64 patients in this survey. Adenovirus infections remain predominant as compared to rotavirus infections.

Adenoviridae Infections↗
PubMed1982

[Enzymatic determination of urinary estrone and estradiol: correlations with plasma estradiol in the menstrual cycle in women].

Enzymatic determination of urine estrone and estradiol concentrations were done daily during a menstrual cycle in six female volunteers. The results were compared with plasma estradiol determined by radioimmunoassay in the same patients. The pattern of estrone and estradiol excretion in morning urine sample fairly reproduced plasma estradiol variations and a good correlation was observed (r = 0.78). The urine pattern of estrone and estradiol variation determined by the enzyme assay can be considered as a good index of oestrogen secretion. The preovulatory peak of estrone and estradiol was observed in urine the day before or the same day as the LH peak. Estrone and estradiol concentration in urine doubled the mean value of the concentration of the follicular phase several days before the LH peak giving a useful indication for prediction of ovulation.

Estradiol↗