Search PubMed⌕ Search

Biomedical subjects

L H Thompson

Publications and source records attributed to L H Thompson.

At least 91 records · Page 5Linked to original sources
PubMed1991

Metabolic activation and cytogenetic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) in Chinese hamster ovary cells expressing murine cytochrome P450 IA2.

We have utilized Chinese hamster ovary cell lines which stably express a murine cytochrome P450IA2 (P(3)450) cDNA to characterize more fully the mechanisms of genotoxicity of heterocyclic amines derived from cooked meats. To verify that these cell lines were capable of converting promutagens into active metabolites, we studied the microsomal metabolism and cytogenetic effects of 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pridine (PhIP). Microsomal preparations derived from excision repair-deficient Chinese hamster ovary cells expressing the mouse cytochrome P(3)450 cDNA (UV5P3) converted PhIP to the genotoxic N-hydroxy-PhIP metabolite. Cytotoxic activity in UV5P3 was observed at concentrations of PhIP as low as 1 microM. Cytotoxicity of PhIP was an order of magnitude lower in a matched repair-proficient cell line (5P3R2) expressing the P(3)450 cDNA. PhIP produced a concentration-dependent increase in sister chromatid exchange (SCE) in UV5P3. N-Hydroxy-PhIP, at concentrations as low as 0.1 microM, produced an increase in SCE in both UV5P3 and in UV5 cells which lack the P(3)450 cDNA. Incubation of PhIP with UV5P3 cells increased the frequency of micronuclei (MN) in cytokinesis-blocked cells. Chromatid gaps, but not aberrations also were induced by treatment with PhIP. N-Hydroxy-PhIP produced increases in MN and chromatid gaps in both UV5 and UV5P3 cell lines; chromosomal aberrations were induced in UV5P3 cells. These results suggested that UV5P3 cells metabolize sufficient quantities of PhIP to produce cytogenetic damage and further indicated that N-hydroxylation of PhIP was requisite for mammalian genotoxicity.

Animals↗
PubMed1990

ERCC2: cDNA cloning and molecular characterization of a human nucleotide excision repair gene with high homology to yeast RAD3.

Human ERCC2 genomic clones give efficient, stable correction of the nucleotide excision repair defect in UV5 Chinese hamster ovary cells. One clone having a breakpoint just 5' of classical promoter elements corrects only transiently, implicating further flanking sequences in stable gene expression. The nucleotide sequences of a cDNA clone and genomic flanking regions were determined. The ERCC2 translated amino acid sequence has 52% identity (73% homology) with the yeast nucleotide excision repair protein RAD3. RAD3 is essential for cell viability and encodes a protein that is a single-stranded DNA dependent ATPase and an ATP dependent helicase. The similarity of ERCC2 and RAD3 suggests a role for ERCC2 in both cell viability and DNA repair and provides the first insight into the biochemical function of a mammalian nucleotide excision repair gene.

Amino Acid Sequence↗
PubMed1990

Myogenin is in an evolutionarily conserved linkage group on human chromosome 1q31-q41 and unlinked to other mapped muscle regulatory factor genes.

Myogenin is a member of a family of muscle-specific regulatory factors which includes MyoD1, Myf-5, and Myf-6 (also called MRF4 and herculin). Extensive regions of sequence homology in genes for these three factors suggest duplication events associated with their evolution. In the present study, the chromosomal location of the myogenin gene in humans (MYOG), mice (Myog), and Chinese hamsters (MYOG) was determined using in situ hybridization to human metaphase chromosomes as well as segregation analysis among interspecific somatic cell hybrid panels and interspecific backcrossed mice. We localize the gene encoding myogenin to human chromosome 1q31-q41 within a linkage group homologous with a region on mouse chromosome 1 and Chinese hamster chromosome 5. The results verify the nonlinkage of MYOG to MYOD1, MYF5, and MYF6 genes and indicate that events associated with the duplication of MYOG with respect to MYOD1, MYF5, or MYF6 loci were not chromosome-wide.

Animals↗
PubMed1990

Cyclobutane-pyrimidine dimer excision in UV-sensitive CHO mutants and the effect of the human ERCC2 repair gene.

Using a radiochromatographic assay, we have examined cis-syn cyclobutane-pyrimidine dimer removal after ultraviolet irradiation in cell lines representative of the first 6 complementation groups of Chinese hamster ovary DNA nucleotide excision repair mutants. AA8, the CHO cell line from which these mutants were derived, consistently showed normal dimer excision for a rodent cell. The mutants uniformly exhibited no significant dimer excision within the limits of determination. Additionally, V-H1, a mutant belonging to complementation group 2 and derived from V79 hamster cells, exhibited no dimer excision. Two UV5 derived transformants that carry the complementing human ERCC2 repair gene showed a capacity for dimer excision comparable to the AA8 wild-type cells.

Animals↗
PubMed1990

Biochemical and genetic analysis of the Chinese hamster mutants irs1 and irs2 and their comparison to cultured ataxia telangiectasia cells.

Two mutants of the Chinese hamster cell line V79-4 (irs1 and irs2) were previously isolated on the basis of their hypersensitivity (2- to 3-fold) to cell inactivation by ionizing radiation. One of these mutants, irs1, displays an unusual phenotype of cross-sensitivity to other varied genotoxic agents including UV light (2- to 3-fold), ethyl methanesulphonate (approximately 10-fold) and mitomycin C (approximately 60-fold). The possibility that these sensitivities might be due to more than one gene mutation in irs1 was investigated. Hybrids formed between irs1 and human lymphocytes were isolated in which the mitomycin C (MMC) sensitivity of irs1 was corrected by complementing human chromosomal material. These MMC-resistant hybrids and their subclones also showed concordant correction of the gamma-ray, UV and EMS sensitivities of irs1, suggesting that a single gene defect is most likely responsible for the phenotype of irs1. In addition it was shown that the MMC-sensitivity of irs1 is complemented by four CHO cell mutants (UV20, UV41, UV-1 and irs1SF), which also display extreme sensitivity to MMC. Mutants irs1, irs1SF and UV-1 define three new complementation groups for MMC sensitivity. The biochemical nature of the ionizing radiation sensitivity of irs1 and irs2 was also investigated. The production and repair of DNA single- and double-strand breaks were studied using the techniques of alkaline and neutral elution, respectively. Irs1 and irs2 both showed repair kinetics for each lesion that are indistinguishable from wild-type. Analysis of the rate of DNA synthesis following gamma-irradiation showed irs1 to have a dose-dependent inhibition similar to that of wild-type.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Molecular cloning of the human DNA excision repair gene ERCC-6.

The UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to 5. It harbors a deficiency in the repair of UV-induced cyclobutane pyrimidine dimers but permits apparently normal repair of (6-4) photoproducts. Genomic (HeLa) DNA transfections of UV61 resulted, with a very low efficiency, in six primary and four secondary UV-resistant transformants having regained wild-type UV survival. Southern blot analysis revealed that five primary and only one secondary transformant retained human sequences. The latter line was used to clone the entire 115-kb human insert. Coinheritance analysis demonstrated that five of the other transformants harbored a 100-kb segment of the cloned human insert. Since it is extremely unlikely that six transformants all retain the same stretch of human DNA by coincidence, we conclude that the ERCC-6 gene resides within this region and probably covers most of it. The large size of the gene explains the extremely low transfection frequency and makes the gene one of the largest cloned by genomic DNA transfection. Four transformants did not retain the correcting ERCC-6 gene and presumably have reverted to the UV-resistant phenotype. One of these appeared to have amplified an endogenous, mutated CHO ERCC-6 allele, indicating that the UV61 mutation is leaky and can be overcome by gene amplification.

Animals↗
PubMed1990

Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange.

We describe the cloning and function of the human XRCC1 gene, which is the first mammalian gene isolated that affects cellular sensitivity to ionizing radiation. The CHO mutant EM9 has 10-fold-higher sensitivity to ethyl methanesulfonate, 1.8-fold-higher sensitivity to ionizing radiation, a reduced capacity to rejoin single-strand DNA breaks, and a 10-fold-elevated level of sister chromatid exchange compared with the CHO parental cells. The complementing human gene was cloned from a cosmid library of a tertiary transformant. Two cosmid clones produced transformants that showed approximately 100% correction of the repair defect in EM9 cells, as determined by the kinetics of strand break repair, cell survival, and the level of sister chromatid exchange. A nearly full-length clone obtained from the pcD2 human cDNA expression library gave approximately 80% correction of EM9, as determined by the level of sister chromatid exchange. Based on an analysis of the nucleotide sequence of the cDNA insert compared with that of the 5' end of the gene from a cosmid clone, the cDNA clone appeared to be missing approximately 100 bp of transcribed sequence, including 26 nucleotides of coding sequence. The cDNA probe detected a single transcript of approximately 2.2 kb in HeLa polyadenylated RNA by Northern (RNA) blot hybridization. From the open reading frame and the positions of likely start sites for transcription and translation, the size of the putative XRCC1 protein is 633 amino acids (69.5 kDa). The size of the XRCC1 gene is 33 kb, as determined by localizing the endpoints on a restriction endonuclease site map of one cosmid clone. The deduced amino acid sequence did not show significant homology with any protein in the protein sequence data bases examined.

Amino Acid Sequence↗
PubMed1990

Epidemiologic study of bluetongue viruses in Central America and the Caribbean: 1986-1988. Regional Bluetongue Team.

Results of a prospective serologic and virologic study of ruminant livestock in Central America and the Caribbean islands revealed bluetongue virus (BTV) to be enzootic in the 9 countries participating in the study. Bluetongue virus serotypes 1, 3, 6, and 12 were isolated from sentinel animals. To the authors' knowledge, these are the first isolations of BTV from the region studied and the first isolations of these serotypes in the Western Hemisphere. Clinical disease attributable to BTV infection was not observed in sentinel animals. The incidence pattern, with respect to age and geographic location, was determined. The need to evaluate the epizootiologic features or arthropod-borne viruses (arboviruses) on a regional ecologic basis is stressed.

Animals↗
PubMed1989

Complementation of repair gene mutations on the hemizygous chromosome 9 in CHO: a third repair gene on human chromosome 19.

A human DNA repair gene, ERCC2 (Excision Repair Cross Complementing 2), was assigned to human chromosome 19 using hybrid clone panels in two different procedures. One set of cell hybrids was constructed by selecting for functional complementation of the DNA repair defect in mutant CHO UV5 after fusion with human lymphocytes. In the second analysis, DNAs from an independent hybrid panel were digested with restriction enzymes and analyzed by Southern blot hybridization using DNA probes for the three DNA repair genes that are located on human chromosome 19: ERCC1, ERCC2, and X-Ray Repair Cross Complementing 1 (XRCC1). The results from hybrids retaining different portions of this chromosome showed that ERCC2 is distal to XRCC1 and in the same region of the chromosome 19 long arm (q13.2-q13.3) as ERCC1, but on different MluI macrorestriction fragments. Similar experiments using a hybrid clone panel containing segregating Chinese hamster chromosomes revealed the hamster homologs of the three repair genes to be part of a highly conserved linkage group on Chinese hamster chromosome number 9. The known hemizygosity of hamster chromosome 9 in CHO cells can account for the high frequency at which genetically recessive mutations are recovered in these three genes in CHO cells. Thus, the conservation of linkage of the repair genes explains the seemingly disproportionate number of repair genes identified on human chromosome 19.

Animals↗
PubMed1989

UV mutagenesis, cytotoxicity and split-dose recovery in a human-CHO cell hybrid having intermediate (6-4) photoproduct repair.

Somatic cell hybrids constructed between UV-hypersensitive Chinese hamster ovary cell line UV20 and human lymphocytes were used to examine the influence of a human DNA repair gene, ERCC1, on UV photoproduct repair, mutability at several drug-resistance loci, UV cytotoxicity and UV split-dose recovery. In hybrid cell line 20HL21-4, which contains human chromosome 19, UV-induced mutagenesis at the APRT, HPRT and Na+/K+-ATPase loci was comparable to that in repair-proficient CHO AA8 cells, whereas cell line 20HL21-7, a reduced human-CHO hybrid not containing human chromosome 19, exhibited a hypermutable phenotype at all 3 loci indistinguishable from that of UV20 cells. The response of 20HL21-4 cells to UV cytotoxicity reflected substantial but incomplete restoration of wild-type UV cytotoxic response, whereas responses of UV20 and 20HL21-7 cell lines to UV cytotoxicity were essentially the same, reflecting several-fold UV hypersensitivity. Repair of UV-induced (5-6) cyclobutane dimers and (6-4) photoproducts was examined by radioimmunoassay; (6-4) photoproduct repair was deficient in UV20 and 20HL21-7 cell lines, and intermediate in 20HL21-4 cells relative to wild-type CHO AA8 cells. UV split-dose recovery in 20HL21-4 cells was also intermediate relative to AA8 cells. These results show that the human ERCC1 gene on chromosome 19 is responsible for substantial restoration of UV survival and mutation responses in repair-deficient UV20 cells, but only partially restores (6-4) UV photoproduct repair and UV split-dose recovery.

Adenine Phosphoribosyltransferase↗
PubMed1989

Hypoxia-selective antitumor agents. 2. Electronic effects of 4-substituents on the mechanisms of cytotoxicity and metabolic stability of nitracrine derivatives.

The mechanism of cytotoxicity of a series of 4-substituted derivatives of 9-[[3-(dimethylamino)propyl]amino]-1-nitroacridine (nitracrine) has been studied, using a panel of DNA repair-defective mutants of the Chinese hamster ovary cell line AA8. Cell lines UV-4 and UV-5 were hypersensitive to nitracrine, with sensitivities approximately 10-fold greater than that of AA8, while EM-9 showed a hypersensitivity factor (HF) of about 2-fold. This pattern suggests the major cytotoxic lesions induced by nitracrine are bulky DNA monoadducts, rather than DNA interstrand cross-links as previously suggested. The desnitro analogue of nitracrine, which retains the intercalative potential of the latter but cannot be metabolically activated by nitro reduction, showed no hypersensitivity, indicating the specificity with which this panel of cell lines can discriminate different types of DNA damage. Several of the highly cytotoxic 4-substituted nitracrine derivatives showed HFs similar to that of the parent, but the less potent 4-dialkylamino and 4-COOMe derivatives showed much lower HFs for UV-4, suggesting that different mechanisms of cytotoxicity contribute. All compounds showed similar HFs under both aerobic and hypoxic conditions, indicating that hypoxia-selective toxicity in this series is due to a quantitative rather than qualitative change in the presence of oxygen. Rates of metabolic consumption of the compounds were measured under both aerobic and hypoxic conditions by bioassay against the sensitive UV-4 cell line. The results agreed well with previous inferences on metabolic stability derived from cell-killing kinetics and showed that electron-donating 4-substituents can be used to increase metabolic stability in vitro. Such stabilization may enhance the therapeutic utility of the nitroacridines in cancer therapy since rapid metabolism of nitracrine appears to prevent its activity against hypoxic cells in solid tumors.

Aminoacridines↗
PubMed1989

CHO mutant UV61 removes (6-4) photoproducts but not cyclobutane dimers.

The CHO mutant UV61 was previously assigned to complementation group 6 of UV-sensitive rodent cell mutants. UV61 is less sensitive to killing by UV radiation than mutants such as UV5, which is highly defective in the incision process that acts on UV-induced lesions. The D37 for cell survival is approximately 4 J/m2 for UV61, compared with 10 J/m2 for the parental AA8 line and approximately 2 J/m2 for UV5. Similarly, mutation induction at the hprt and aprt loci shows an intermediate response to UV61. In a post-replication recovery assay, the kinetics of maturation of pulse-labelled nascent DNA were normal after UV irradiation in UV61. Data from alkaline elution and alkaline unwinding assays showed that the rates of break accumulation and resealing, measured 0-120 min after irradiation, were also normal in the mutant. This repair incision correlated with the rapid, normal removal of pyrimidine(6-4)pyrimidone photoproducts in UV61 measured using a radioimmunoassay that is specific for this class of damage. In contrast, after exposure to 10 or 15 J/m2, no detectable removal of cyclobutane dimers from DNA was found in UV61 while AA8 cells removed 32% by 24 h. We suggest that the mutation in UV61 specifically lowers the affinity of a repair protein for cyclobutane dimers, which are also inefficiently removed from the bulk DNA of normal CHO cells. The resistance of UV61 to killing by the direct acting chemical 7-bromomethylbenz[a]anthracene was only slightly greater than that of UV5, indicating defective repair of bulky chemical adducts in addition to cyclobutane dimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Bilateral removal of the cauda epididymides in the neonatal pig as a technique for creating teaser boars.

Five pairs of crossbred littermate boars were used to assess the efficacy of bilateral removal of the cauda epididymides at an early age as a technique for creating teaser boars. The cauda epididymides were surgically removed in one of each litter pair; the other of the pair served as an intact control. Boars subjected to removal of the epididymides (Epid) were rendered sterile by the technique. The Epid-treated and control untreated littermate boars had similar levels of sexual aggression and libido, as measured by behavioral characteristics at semen collection. The Epid-treated boars showed a slight, but not significant, reduction in ejaculate volume. Upon slaughter at 273 d of age, Epid and control boars had similar weights for the accessory sexual organs and penis and similar penile lengths. The Epid-treated boars displayed enlarged caput epididymides and granulomata. It is suggested that bilateral removal of the cauda epididymides in the neonatal pig may prove a worthwhile alternative to the traditional vasectomy procedure to create teaser boars.

Animals↗
PubMed1989

Cross-protective immunity between equine encephalomyelitis viruses in equids.

Eighteen equids were inoculated with eastern equine encephalomyelitis (EEE) and 18 equids with western equine encephalomyelitis (WEE) viruses to produce EEE virus- and WEE virus-immunized equids. Twelve surviving EEE virus-seropositive equids, 15 surviving WEE virus-seropositive equids, and 10 nonimmunized, seronegative equids (controls) were subsequently inoculated with an equine pathogenic (epizootic) strain of Venezuelan equine encephalomyelitis (VEE) virus to determine cross-protective immunity. Challenge infection produced 90% mortality in control (nonimmunized) equids, and 40% mortality in WEE virus-seropositive equids; all EEE virus-seropositive equids survived. Postchallenge exposure VEE viremia levels in EEE virus- or WEE virus-seropositive equids were lower than those in the 10 nonimmunized VEE virus-inoculated control equids. Plaque-neutralizing antibody responses to VEE virus in the EEE virus- and WEE virus-seropositive equids were similar in time of onset and titer to the antibody responses of nonimmunized equids. Neutralizing antibody to the third equine encephalomyelitis virus (either EEE virus or WEE virus) was detectable in 19 of 27 equids after inoculation with the challenge virus, VEE. Demonstration of cross-protective immunity between EEE or WEE virus and VEE virus in equids confirmed field observations made during the VEE epizootic in Texas in 1971.

Alphavirus↗
PubMed1988

A yeast DNA repair gene partially complements defective excision repair in mammalian cells.

The RAD10 gene of Saccharomyces cerevisiae is required for nucleotide excision repair of DNA. Expression of RAD10 mRNA and Rad10 protein was demonstrated in Chinese hamster ovary (CHO) cells containing amplified copies of the gene, and RAD10 mRNA was also detected in stable transfectants without gene amplification. Following transfection with the RAD10 gene, three independently isolated excision repair-defective CHO cell lines from the same genetic complementation group (complementation group 2) showed partial complementation of sensitivity to killing by UV radiation and to the DNA cross-linking agent mitomycin C. These results were not observed when RAD10 was introduced into excision repair-defective CHO cell lines from other genetic complementation groups, nor when the yeast RAD3 gene was expressed in cells from genetic complementation group 2. Enhanced UV resistance in cells carrying the RAD10 gene was accompanied by partial reactivation of the plasmid-borne chloramphenicol acetyltransferase (cat) gene following its inactivation by UV radiation. The phenotype of CHO cells from genetic complementation group 2 is also specifically complemented by the human ERCC1 gene, and the ERCC1 and RAD10 genes have similar amino acid sequences. The present experiments therefore indicate that the structural homology between the yeast Rad10 and human Ercc1 polypeptides is reflected at a functional level, and suggest that nucleotide excision repair proteins are conserved in eukaryotes.

Animals↗
PubMed1988

An eighth complementation group of rodent cells hypersensitive to ultraviolet radiation.

Two mutant lines (US31, US46) of mouse lymphoma cells that are hypersensitive to ultraviolet (UV) radiation were previously found to belong to different complementation groups. The mutants were tested for their ability to complement the six known complementation groups of UV-sensitive Chinese hamster ovary (CHO) cells, which are defective in nucleotide excision repair, as well as a seventh group represented by a V79 mutant. Hybrid cells were produced by fusion with polyethylene glycol and tested in situ for UV resistance. The mouse mutant US46 complemented all CHO mutants except UV61. Therefore, US46 is assigned to the same complementation group as UV61, and it is probably defective in the same locus. The mouse mutant US31 produced UV-resistant hybrid cells in each of the seven crosses, indicating that it forms an eighth complementation group among the rodent mutants. Thus, at least eight genes are likely required to repair UV damage in rodent cells.

Animals↗