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Biomedical subjects

M R James

Publications and source records attributed to M R James.

86 records · Page 5Linked to original sources
PubMed1987

Strategies to analyse mutagenesis in mammalian cells using simian virus 40 or shuttle vectors.

The use of exogeneous DNA probes, which replicate extrachromosomally, is proposed in order to study spontaneous and induced mutagenesis in mammalian cells. Simian virus 40 has already proved to be very useful, since it has provided much important information in this field. Recently, several shuttle vectors have been designed for this purpose; however, it seems that these molecules have high spontaneous mutation frequencies when replicating in mammalian cells. We have developed new alternative systems, such as Epstein-Barr virus-based shuttle vectors that can be episomally maintained in human cells. Furthermore, we have constructed packageable shuttle vectors, which appear to be stable in the host cell and thus suitable for analysis of mutagenesis.

Animals↗
PubMed1987

A gamma-ray-resistant derivative of an ataxia telangiectasia cell line obtained following DNA-mediated gene transfer.

Genomic DNA from normal human or mouse cells was transfected together with the selectable marker gpt into the simian virus 40-transformed ataxia telangiectasia fibroblast line, AT5BIVA. From a series of experiments involving over 400,000 clones selected for the gpt marker, one unambiguously radiation-resistant clone (clone 67) was recovered following selection with repeated cycles of gamma irradiation. The normal level of radiation resistance of clone 67 has been maintained for at least 11 months in the absence of further selection by radiation. The resistant clone contains one copy of the gpt gene. Its DNA synthesis following gamma-irradiation is inhibited to an extent intermediate between that of ataxia telangiectasia and normal cells. Three out of four thioguanine-resistant derivatives of clone 67 have either lost or do not express the gpt sequence and show almost the same sensitivity to gamma irradiation as the original AT5BIVA line. This suggests that the radiation resistance of clone 67 may be linked to the gpt sequence and may have arisen as a consequence of the transfection, rather than as the result of an independent mutation to radiation resistance.

Animals↗
PubMed1986

Efficient immortalization and morphological transformation of human fibroblasts by transfection with SV40 DNA linked to a dominant marker.

Immortal cell lines are essential for genetic and biochemical studies. Unlike rodent cells, which will form continuously growing cultures either spontaneously or after infection with an oncogenic virus (e.g., Simian Virus 40 (SV40)), human cells fail to form continuous cell lines spontaneously and in only rare cases from cell lines after oncogenic virus infection. We have used a plasmid (pSV3gpt) containing both the SV40 early region encoding T antigen and the bacterial gene xanthine-guanine phosphoribosyl transferase (gpt) to achieve high efficiency morphological transformation and immortalization of primary human skin fibroblasts. Transfection of this plasmid into primary human skin fibroblasts derived from a normal individual, two Cockayne's syndrome patients, and an immuno-deficient patient and selection for the gpt gene resulted in an altered cell morphology and growth properties characteristic of previously described SV40-transformed cells. Transfected cultures subsequently senesced, entered crisis and in each case formed a rapidly growing culture. The high efficiency of immunortalization described here (four out of four cell strains) is in contrast to previously described procedures utilizing focal overgrowth. We suggest that the use of a dominant selectable marker linked to the SV40 early region increases the probability of establishing an immortal human cell line.

Antigens, Viral, Tumor↗
PubMed1986

A derivative of an ataxia-telangiectasia (A-T) cell line with normal radiosensitivity but A-T-like inhibition of DNA synthesis.

Ataxia-telangiectasia (A-T) cells are hypersensitive to the lethal effects of ionizing radiation and fail to inhibit DNA synthesis following radiation exposure. A cell line derived from an A-T line following DNA-mediated gene transfer has normal radiation sensitivity, but the kinetics of DNA synthesis after gamma-irradiation are similar to those of A-T cells.

Ataxia Telangiectasia↗
PubMed1985

NAD and the synthesis of (ADP-ribose)n in a human cell strain (46BR) hypersensitive to the lethal effects of 3-aminobenzamide.

The cell strain 46BR, derived from an immunodeficient individual, is hypersensitive to the lethal effects of DNA-damaging agents, and of 3-aminobenzamide (3AB), the latter being an inhibitor of the enzyme ADP-ribosyltransferase (ADPRT). This hypersensitivity is not found with the noninhibitory analogue, 3-aminobenzoate. The NAD content of 46BR cells is similar to that of fibroblasts from normal human donors, as is the decrease in NAD content following treatment with dimethylsulphate. Both the activity of ADP-ribosyltransferase and its inhibition by 3AB in permeabilized cells are similar in 46BR and in normal cell strains. High concentrations of 3AB interfere with purine metabolism in cultured cells. Again this effect is similar in 46BR and normal cells. Thus there is no apparent anomaly either in the activity of ADPRT or in the gross effects of 3AB in 46BR. The sensitivity to 3AB may be caused by a defect in a specific acceptor for the ADP-ribose synthesized by ADPRT, or in some as yet undiscovered action of the inhibitor.

Adenosine Diphosphate Ribose↗
PubMed1985

An attempt to transfer radiation resistance to an ataxia-telangiectasia cell line.

Fibroblasts from ataxia-telangiectasia (AT) are hypersensitive to the lethal effects of ionizing radiation. Genomic DNA from normal human cells was transfected together with the selectable bacterial marker, gpt, on plasmid pSV2 into an SV40-transformed AT line, AT5BIVA. One radiation resistant clone (67) was recovered following repeated cycles of gamma-irradiation from a population of 90,000 clones. The normal level of radiation resistance has been maintained for at least 11 months in the absence of further selection by radiation. The resistant clone is not a contaminant as determined by isoenzyme analysis, carries one copy of the gpt gene, and its DNA synthesis is inhibited after radiation to an extent intermediate between that of AT and normal cells. It is not yet established whether clone 67 is a bona fide transformant or arose as a consequence of mutation of the parent AT line.

Ataxia Telangiectasia↗
PubMed1983

A biochemical defect in the repair of alkylated DNA in cells from an immunodeficient patient (46BR).

The fibroblast cell strain 46BR, derived from an immunodeficient individual, is hypersensitive to the lethal effects of a variety of DNA-damaging agents, this effect being particularly marked for monofunctional methylating agents. After u.v. irradiation 46BR cells show normal unscheduled DNA synthesis, daughter strand repair, and recovery of DNA and RNA synthesis. The inhibition of DNA replicative synthesis by u.v. is slightly less than that of normal cells. After gamma-irradiation the rejoining of strand breaks is normal as are the kinetics of replicative DNA synthesis. Following treatment with dimethylsulphate, replicative DNA synthesis is affected in a similar way to normal cells, unscheduled DNA synthesis may be increased relative to normal cells, but more strand breaks persist in 46BR than in normal cells. In addition 46BR cells are hypersensitive to the toxic effects of 3-aminobenzamide, an inhibitor of ADP-ribosyl transferase. This enzyme is involved in the ligation step of repair of alkylation damage. A hypothesis is presented suggesting that 46BR may be defective in DNA ligase I.

Benzamides↗
PubMed1982

Role of poly(adenosine diphosphate ribose) in deoxyribonucleic acid repair in human fibroblasts.

We have investigated the role of poly(adenosine diphosphate ribose) in DNA repair in human fibroblasts by observing the effects of 3-aminobenzamide (3AB), a specific inhibitor of poly(ADP-ribose) synthesis, on various aspects of DNA repair. After treatment of human fibroblasts with dimethyl sulfate (DMS), 3AB retarded the joining of strand breaks; unscheduled DNA synthesis was unaffected after low doses of DMS but was stimulated after high doses. 3AB also enhanced the cytotoxicity of DMS. After gamma irradiation there was a slight inhibition by 3AB of the rejoining of single-strand breaks but no effect on the rejoining of double-strand breaks, unscheduled DNA synthesis, DNA replicative synthesis, or cytotoxicity. There were no effects of 3AB on the repair of UV damage. On the basis of the different kinetics of the various steps of excision repair processes after different treatments of fibroblasts, our results are interpreted as evidence that the synthesis of poly(ADP-ribose) is involved in the ligation step of excision repair.

Benzamides↗
PubMed1977

The effect of ouabain on the guinea pig ileum longitudinal smooth muscle: 1. ATPase activities in a sarcolemma-enriched fraction prepared with the aid of divalent cation depletion of the intact muscle.

Depletion of divalent cations before fractionation of the longitudinal muscle of the guinea pig ileum yielded a sarcolemma-enriched microsomal fraction free of mitochondria. A major portion of the ATPase activity in the presence of Mg, Na, and K was due to stimulation by Na alone. A further small stimulation by K was demonstrated only in the presence of an activating factor from the 105 000 X g supernatant. Ouabain inhibited only the K activation and had no effect on the Na-stimulated Mg-ATPase.

Adenosine Triphosphatases↗
PubMed1977

The effect of ouabain on the guinea pig ileum longitudinal smooth muscle: 2. Intracellular levels of Ca, Na, K, and Mg during the ouabain response and the dependence of the response on extracellular Ca.

Isotonic Tris-HCl containing 10 mM LaCl3 at 4 degrees C effectively removed extracellular ions in 30 min while preventing loss of intracellular ions. Intracellular Ca and Na increased during the contraction in the presence of 10 mM ouabain and then decreased during relaxation. Intracellular Na increased again during the latter part of the relaxation phase when K loss became apparent. Mg levels remained essentially constant. Ouabain responses were rapidly lost in Ca-free medium indicating that they were dependent on extracellular Ca. A 5.5-fold increase in the normal levels of extracellular K did not reduce the contraction to a submaximal dose of ouabain. A full phasic response to high K (60 mM) was observed after a 10-min exposure of the tissue to ouabain, at which time the ouabain response had returned to basal tension. The contraction to ouabain appears to be dissociated from inhibition of the Na,K-ATPase at the K site. The changes in intracellular ions indicated that ouabain contracted the muscle by increasing the plasma membrane permeability to Ca and Na and later decreased the K and Na concentration gradients, probably by inhibition of the Na,K-ATPase.

Adenosine Triphosphatases↗
PubMed1976

Numerosity discrimination of tactile stimuli.

The ability of 40 college students to discriminate the number of tactile stimuli presented simultaneously was measured in two experiments. Simulation was provided by 12 solenoids fixed to points on the arms and legs. Exp. I showed a mean correct discrimination level of between 1.55 and 2.10 solenoids. Exp II compared discrimination of numerosity by a group who received immediate feedback with a no-feedback control group. Feedback produced a small increase in this ability. However, the most stimulation points correctly discriminated did not exceed three. The data are discussed in terms of the possible role of cutaneous masking.

Aptitude↗
PubMedDate not supplied

Comparative study of Epstein-Barr virus and SV40-based shuttle-expression vectors in human repair-deficient cells.

Shuttle vectors and expression vectors have been used in human cells to examine various aspects of DNA repair including effects of DNA damage on mutagenesis, transcription, replication and recombination. A combined shuttle-expression system should provide further advantages for the stable expression of and perhaps selection/rescue strategies for DNA repair genes. We describe 2 such systems. The first is a simian virus 40 (SV40) shuttle system which allows a quasi-stable episomal vector/host relationship in which the shuttle vector may be recovered in extrachromosomal DNA preparations many months after transfection and selection but in which a high proportion of the plasmids rescued in bacteria are heavily mutated and rearranged. Secondly, we describe Epstein-Barr virus-based shuttle-expression vectors which exist as stable, multicopy episomes in human cells. Using a reporter gene and a metal-inducible promoter we have obtained low basal and very high induced expression from episomal vectors in a variety of human cells including xeroderma pigmentosum and ataxia telangiectasia cell lines. This should facilitate many molecular genetic experiments in human cells and may have particular application to molecular cloning, expression and analysis of DNA repair genes.

Animals↗
PubMedDate not supplied

Steroid metabolism in the hormone dependent MCF-7 human breast carcinoma cell line and its two hormone resistant subpopulations MCF-7/LCC1 and MCF-7/LCC2.

Androgen and estrogen metabolism was investigated in the hormone-dependent human breast cancer cell line MCF-7 and its two hormone-resistant sublines MCF-7/LCC1 and MCF-7/LCC2. Using the product isolation method, the activity of aromatase, 5alpha-reductase, 3alpha/beta-hydroxysteroid oxidoreductase and 17beta-hydroxysteroid oxidoreductase were investigated isolating the following steroids: estriol (E3), estradiol (E2), estrone (E1), 3alpha/beta-androstanediol (A-diol), testosterone (T), dihydrotestosterone (DHT), androsterone (AND), androstenedion (4-AD) and androstanedione (A-dion). For all experiments, cells were preincubated with cortisol and subsequently incubated with [14C]T or [14C]4-AD as the substrate in medium without phenol red and with serum charcoal stripped of steroids. The results showed no aromatase activity in any of the cell lines under the experimental conditions used, and preincubation with cortisol had no effect on the enzyme activity. With [14C]T as the substrate, the metabolized level of DHT was very similar in the three cell lines, though MCF-7/LCC1 and MCF-7/LCC2 utilized the substrate to a much lesser extent. The amount of DHT and 4-AD produced were comparable in the two hormone-resistant cell lines, while the amount of 4-AD was significantly higher in MCF-7 cells. No differences in enzyme activity were found in the three cell lines when [14C]4-AD was used as the substrate. This study showed an altered androgen metabolism in the MCF-7/LCC1 and MCF-7/LCC2 sublines compared to the parent MCF-7. However, since treatment with DHT and T inhibited cell growth equally well in all three tumor cell lines, it is unlikely that the found differences in steroid metabolism was involved in the acquisition of the endocrine resistance of the two MCF-7 sublines.

Aminoglutethimide↗