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C L Thompson

Publications and source records attributed to C L Thompson.

At least 37 records · Page 2Linked to original sources
PubMed1995

Quantitative characterization of alpha 6 and alpha 1 alpha 6 subunit-containing native gamma-aminobutyric acidA receptors of adult rat cerebellum demonstrates two alpha subunits per receptor oligomer.

gamma-Aminobutyric acidA (GABAA) receptors were purified from adult rat cerebella by anti-alpha 6(1-16 Cys) antibody affinity chromatography. Immunoblots of the alpha 6 subunit-containing receptors showed the copurification of the alpha 1, beta 2/3, gamma 2, delta but not alpha 2 and alpha 3 GABAA receptor polypeptides. Further fractionation of this receptor subpopulation by anti-GABAA receptor subunit alpha 6(1-16 Cys) and anti-alpha 1(413-429) antibody affinity columns in series substantiated the coassociation of the alpha 1 and alpha 6 polypeptides. The percentage of coexistence of the two subunits was determined by quantitative immunoblotting, which found that 41 +/- 12% of alpha 6 subunit immunoreactivity is associated with the alpha 1 subunit. The ratios of the alpha 1:alpha 6 subunits in the double purified receptor preparations was found to be 1:1, thus determining directly for the first time subunit ratios within native GABAA receptors. The benzodiazepine pharmacology of the alpha 1 alpha 6 subunit-containing receptors was shown to be predominantly benzodiazepine-insensitive by quantitative immunoprecipitation assays. These results are the first direct quantitative studies of subunit ratios within a population of native GABAA receptors.

Amino Acid Sequence↗
PubMed1995

Impact of age and environment on somatic mutation at the hprt gene of T lymphocytes in humans.

Analysis of two human populations for dependence of somatic mutation on age has revealed both similarities and differences. The studies performed employed peripheral blood lymphocytes and measured the efficiency with which these cells form clones in vitro (cloning efficiency, CE) and the frequency of cells with inactivating mutations of the hypoxanthine phosphoribosyltransferase gene (mutant frequency, MF). The people studied were between 19 and 64 years of age. In one population, composed of 78 never smokers and 140 current smokers from the United States (US), both CE and MF were dependent on age: CE declined with age (p = 0.005); MF increased 0.15 per 10(6) cells per year of age for nonsmokers (p < 0.001) and at 1.3 times that rate for smokers (p = 0.01). In the second population, 80 people of unknown smoking status from Russia, the increase in MF per year was even greater, 2.5 times that of the US nonsmokers (p = 0.001) but the dependence of CE on age was the same as for the US population (p = 0.043). Because the increase of MF of the Russians with age is 2-fold greater than that of the US smokers, the intensity of smoking and/or other environmental exposures, or the susceptibility to these exposures, must account for the difference in age dependent MF increase, not the proportion of Russians that are smokers. Differences in the lymphocyte subpopulations that survived the longer transit from Russia may have contributed to the observed differences in MF. However, overall, the mutant frequency results suggest that the Russians were chronically exposed to higher levels of agents that induce somatic mutation and that, on an age adjusted basis, the Russia population studied is at increased risk for health consequences from accumulated genetic damage.

Adult↗
PubMed1995

Critical care-acquired pneumonia.

Nosocomial infections are the second most common hospital-acquired infections. The Centers for Disease Control and Prevention recently published an updated Guidelines for the Prevention of Nosocomial Pneumonia. A summary of these recommendations and general preventive strategies are presented in this article.

Critical Care↗
PubMed1994

GABAA receptor subtypes expressed in cerebellar granule cells: a developmental study.

The developmental properties of primary rat cerebellar granule cells have been characterised with respect to their expression of GABAA receptor subtypes using both an immunological approach and radioligand binding assays. At day 1 in culture, the GABAA receptor alpha 1 subunit was detectable in immunoblots and increased in level up to day 9. The GABAA receptor alpha 6 subunit was not detectable at day 1; however, at days 3-5, a specific M(r) 58,000 anti-alpha 6 1-16 Cys immunoreactive species was present which further increased in level up to 9 days in culture. Similar qualitative results were obtained for the expression of the GABAA receptor alpha 6 subunit in age-matched rat cerebellar membranes. In parallel studies, it was found that although there was an overall increase in [3H]Ro 15-4513 binding sites with days in culture, the relative contributions of diazepam-sensitive and diazepam-insensitive [3H]Ro 15-4513 binding changed. A time-dependent enrichment of the diazepam-insensitive binding site up to a maximum of 74% of total [3H]Ro 15-4513 sites was found. This was concomitant with the appearance of the GABAA receptor alpha 6 subunit. These results are in agreement with the pharmacology described for alpha 6 beta gamma 2 cloned receptors. They suggest a developmentally regulated expression of the GABAA receptor alpha 6 subunit gene at a time that is correlated in vivo with establishment of neuronal connections.

Aging↗
PubMed1993

Characterization of in vivo somatic mutations at the hypoxanthine phosphoribosyltransferase gene of a human control population.

The ability to recognize a change in mutation spectrum after an exposure to a toxic substance and then relate that exposure to health risk depends on the knowledge of mutations that occur in the absence of exposure. Toward this end, we have been studying both the frequency and molecular nature of mutations of the hypoxanthine phosphoribosyltransferase (hprt) gene in peripheral blood lymphocytes as surrogate reporters of genetic damage. We have analyzed mutants, one per donor to ensure independence, from a control population in which the quantitative effects of smoking and age on mutant frequency have been well defined. Analyses of cDNA and genomic DNA by polymerase chain reaction and sequencing have identified the mutations in 63 mutants, 45 from males and 18 from females, of which 34 were smokers and 29 were nonsmokers. Slightly less than half of the mutations were base substitutions; they were predominantly at GC base pairs. Different mutations at the same site indicated that there are features of the hprt polypeptide that affect the mutation spectrum. Two pairs of identical mutations indicated that there may also be hot spots. Mutations not previously reported have been detected, indicating that the mutation spectrum is only partly defined. The remainder of the mutations were deletions or insertions/duplications; deletions ranged from one base pair to complete loss of the locus. Despite a small average increase in mutant frequency for smokers, an increased proportion of base substitutions at AT base pairs in smokers (p = 0.2) hinted at a smoking-associated shift in the mutation spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

CYP1A1 mRNA levels as a human exposure biomarker: use of quantitative polymerase chain reaction to measure CYP1A1 expression in human peripheral blood lymphocytes.

Accurate human risk assessment requires sensitive methods to evaluate dose-response relationships, especially following low level exposures. We have developed a reverse transcriptase polymerase chain reaction (RT-PCR) method to quantitative cytochrome P450-1A1 (CYP1A1) mRNA levels in human blood lymphocytes. Many polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene, and chlorinated PAH such as polychlorinated dibenzodioxins, dibenzofurans and biphenyls induce CYP1A1 expression through activation of an endogenous protein, the Ah receptor. Using a quantitative competitive RT-PCR method that included a synthetic internal standard we determined copy numbers of CYP1A1 mRNA in resting as well as mitogen-stimulated human blood lymphocytes. In mitogen-stimulated human blood lymphocytes assay variation was approximately 10% for measurement of this low expression gene and mRNA levels correlated well with ethoxyresorufin-O-deethylase (EROD) activity. The expression of mRNA was induced 20-fold upon culturing human lymphocytes with 10 nM TCDD. In nonstimulated, uninduced lymphocytes CYP1A1 levels are extremely low (1000 copies mRNA/10(4) cells) and cannot be measured by EROD activity. Studies of CYP1A1 mRNA expression in chemically-exposed populations are in progress.

Base Sequence↗
PubMed1992

Mapping of GABAA receptor alpha 5 and alpha 6 subunit-like immunoreactivity in rat brain.

The distribution of the alpha 5 and alpha 6 subunits of the GABAA receptor has been mapped in rat brain using affinity-purified antibodies generated against peptide sequences unique to the respective polypeptides. alpha 5 Subunit-like immunoreactivity was of low density but was distributed across several cell groups including cortical interneurones, hippocampal CA3 pyramidal neurones, the anterior thalamic reticular nucleus and cerebellar Purkinje neurones. alpha 6 Subunit-like immunoreactivity was observed in high density in cerebellar granule cells. These patterns are compatible with in situ hybridisation studies and provide a further anatomical substrate for GABAA receptor heterogeneity in the CNS.

Amino Acid Sequence↗
PubMed1992

Genetic monitoring of human polymorphic cancer susceptibility genes by polymerase chain reaction: application to glutathione transferase mu.

Several genes involved in the metabolism of carcinogens have been found to be polymorphic in human populations and are associated with increased risk of cancer at some sites. This study focuses on the polymorphic enzyme glutathione transferase mu (GT mu). Smokers with low lymphocyte GT mu activity are at an approximately 2-fold higher risk for lung cancer and an approximately 3-fold higher risk for stomach and colon adenocarcinomas. Recent cloning and sequencing of the GST1 gene has allowed the development of convenient genotyping methods based on restriction fragment length polymorphisms (RFLP) or the polymerase chain reaction (PCR). The GST1 polymorphism has been shown to be a deletion of the gene locus. To detect the presence or absence of the gene we amplified exons 4-5 and/or exons 6-7 of the GST1 gene by PCR. PCR amplification produced bands of 215-bp or 273-bp from individuals with one or two copies of the GST1 allele and no band if the individual was homozygously deleted (0/0). In the exon 6-7 PCR, we co-amplified a 268-bp portion of the beta-globin gene as an internal reference standard for quantitative analysis of product yield. This allowed homozygote individuals (+/+) to be distinguished from heterozygotes (+/0). We have compared the GST1 genotype to lymphocyte GT mu activity measured on trans-stilbene oxide (TSO) in the lymphocytes of 45 individuals. Low GT mu activity (< 67 pmole/min/10(7) cells) was strongly associated (24/24) with the GST1 0/0 genotype. With the exception of one individual, activities greater than 67 pmole/min/10(7) were associated with the presence of the GST1 allele (20/21). Individuals with the highest GT-TSO activity were found to be homozygous for GST1. (+/+), while heterozygotes (+/0) generally had lower activity, suggesting a gene dosage effect in lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene Deletion↗
PubMed1991

Glutathione S-transferase mu in human lymphocyte and liver: role in modulating formation of carcinogen-derived DNA adducts.

Glutathione transferase (GT) activity towards trans-stilbene oxide (tSBO), benzo[a]pyrene-4,5-oxide (B[a]PO) and 1-chloro-2,4-dinitrobenzene (CDNB) was measured in human liver and lymphocytes. GT-tSBO activity is catalyzed by GT mu which has polymorphic expression in human lymphocytes. Our results show that activity of GT-tSBO in lymphocytes correlates with its activity in liver (r = 0.7, P less than 0.001). GT activity towards BPO (GT-BPO) also correlated with GT-tSBO in lymphocytes and liver. However, interindividual variation of GT-BPO is less than that of GT-tSBO, suggesting that BPO may not be as specific a substrate for GT mu and therefore other GT isozymes may contribute to BPO conjugation. Conjugation of CDNB by GT was not different using cytosols from either high or low GT mu individuals. The functional significance of the GT-mu polymorphism was evaluated by measuring its effect on benzo[a]pyrene (B[a]P)- and aflatoxin B1 (AFB1)-DNA adduct formation in vitro. Human liver cytosols prepared from persons having low or high GT-tSBO activity were incubated with human liver microsomes, calf thymus DNA and B[a]P or AFB1. HPLC analysis revealed that the major B[a]P adduct was dG(N2)-7 beta, 8 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-dG). BPDE-dG adducts were decreased equally by cytosols from either low or high conjugators. In contrast, AFB1-DNA binding was inhibited to a greater extent in high conjugators than low conjugators. HPLC analysis demonstrates that adducts formed were AFB1-FAPyr and AFB1-N7-Gua. The correlation between AFB1-DNA adduct concentrations and GT mu activity was highly significant with a correlation coefficient of r = 0.88 at P less than 0.001. These results suggest that GT mu plays an important role in detoxifying DNA reactive metabolites of AFB1 and this enzyme may be a susceptibility marker for AFB1 related liver cancer. Moreover, our data demonstrate that lymphocytes are a reliable surrogate tissue for detecting liver GT mu polymorphisms.

Adolescent↗
PubMed1990

Multiple DNA adducts in lymphocytes of smokers and nonsmokers determined by 32P-postlabeling analysis.

Identification of DNA adducts in peripheral lymphocytes could serve as a means of monitoring human exposure to potential genotoxic agents. In this study, DNA from peripheral lymphocytes of smokers and nonsmokers was examined for adducts by the P1 nuclease 32P-postlabeling technique. Thin layer chromatography (TLC) maps from both groups revealed multiple DNA adducts which ranged from no adducts for one individual to six adducts for a different individual. The total DNA adduct concentrations were approximately one adduct in 10(8)-10(10) normal nucleotides. Comparison of the adduct TLC profiles revealed individual variation in both pattern and level of DNA adducts. The type and amount of adduct was not influenced by smoking history and remained unchanged in four out of six subjects who were resampled after a 1 month interval. The capacity of lymphocytes to form BaP-derived DNA adducts after a 72 h incubation with 10(-6) M [3H]BaP was measured by both high-performance liquid chromatography (HPLC) and 32P-postlabeling analysis. The in vitro adduct values detected by [3H]nucleoside concentrations on HPLC ranged from 1 to 7 fmol adduct per micrograms DNA (3.3-23.3 adducts per 10(7) nucleotides). The [3H]nucleoside values were consistent with values obtained by 32P-postlabeling of the same sample (correlation coefficient of 0.88). No relationship was apparent between the capacity of lymphocytes to form a [3H]BaP-derived adduct in vitro and the concentration of any adduct, or total adducts present in untreated lymphocytes. These results suggest that multiple DNA adducts are present in lymphocytes from nonsmokers as well as smokers, although the profile and extent of these adducts can vary among individuals. The relationship of the lymphocyte DNA adducts detected in this study to human cancer susceptibility remains to be determined.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1990

Teaching home care for ventilator-dependent patients: the patients' perception.

An increasing number of ventilator-dependent patients are being transferred from hospitals to their homes. Discharge teaching has been based on health professionals' perception of what patients need to know and how they can learn best. In this study we asked ventilator-dependent patients who were living at home (N = 13) to complete a questionnaire concerning their perceptions of ideal discharge teaching. Most respondents believed that teaching should occur (1) at the bedside, (2) with one or two family members, (3) with only the team member doing the teaching, (4) for a 30-minute session, (5) using demonstration technique. Subjects thought that teaching regarding the lung disease process was less important than other content. The patients' criteria for learning mostly agree with those identified by health care professionals, and can be met easily in most settings.

Adolescent↗
PubMed1990

Involvement of cytochrome P-450c in alpha-naphthoflavone metabolism by rat liver microsomes.

Metabolism of alpha-naphthoflavone (ANF) is increased markedly in rat liver microsomes by 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), two inducers of cytochromes P-450c and P-450d (P-450c and P-450d). Although several indirect lines of evidence in the literature suggest that ANF is metabolized by P-450c, Vyas et al. [J. Biol. Chem. 258:5649-5659 (1983)] reported that ANF metabolism by 3-MC-induced rat liver microsomes was only partially inhibited by antibodies against P-450c. Our laboratory has previously reported clastogenic effects of metabolites of ANF, and in the present study we reexamined the role of P-450c in ANF metabolism by both uninduced and TCDD-induced rat liver microsomes, using monospecific polyclonal antibodies to P-450c and P-450d. ANF metabolism was inhibited to different extents in TCDD-induced microsomes by different preparations of anti-P-450c. One lot of anti-P-450c produced only 50% inhibition of ANF metabolism in TCDD-induced microsomes, whereas another lot of anti-P-450c inhibited ANF metabolism by 80%. Anti-P-450d had no effect on ANF metabolism. Neither anti-P-450c nor anti-P-450d inhibited ANF metabolism in uninduced rat liver microsomes. In a reconstituted enzyme system, purified P-450c metabolized ANF 47 and 510 times more rapidly than P-450d and P-450b, respectively. Metabolites resulting from oxidation at 7,8- or 5,6-positions (7,8-dihydro-7,8-dihydroxy-ANF, 5,6-dihydro-5,6-dihydroxy-ANF, 5,6-oxide-ANF, and 6-hydroxy-ANF) were formed by all preparations of microsomes. An unknown toxic ANF metabolite was formed only with a reconstituted P-450c system and with 3-MC- or TCDD-induced microsomes. Our results indicate that P-450c is responsible for the majority of the metabolism of ANF in TCDD-induced microsomes, whereas other constitutive isozymes are responsible for the metabolism seen in uninduced liver microsomes. The variable inhibition of ANF metabolism with different lots of anti-P-450c probably reflects the differences in the proportion of antibodies to different epitopes important in the binding or metabolism of this substrate.

Animals↗
PubMed1989

Relationships among benzo(a)pyrene metabolism, benzo(a)pyrene-diol-epoxide:DNA adduct formation, and sister chromatid exchanges in human lymphocytes from smokers and nonsmokers.

In the present study, benzo(a)pyrene (BP) metabolism, DNA adduct formation, ethoxyresorufin-O-deethylase activity, and sister chromatid exchange induction by BP were compared in human lymphocytes prepared from whole blood of smokers and nonsmokers following an in vitro incubation with BP. There was an approximate 7- to 10-fold variation in all parameters measured. To determine the source of this variation, participants were resampled, the assays were repeated, and all the data were analyzed to assess (a) smoking-related effects, (b) differences in multiple samples from the same individual, and (c) intraindividual, experimental, and interindividual variation. No smoking-related effects were observed except for baseline sister chromatid exchange frequency. The variation observed for BP-related DNA adducts and ethoxyresorufin-O-deethylase activity was primarily due to interindividual variation. For example, in vitro formation of DNA adducts did not change when samples were obtained at different times from the same individual and were not influenced significantly by culture conditions. No significant correlation existed between DNA adduct formation and BP metabolism [correlation coefficient (r) = 0.27] for either the total population or when segregated based on smoking status. Furthermore, no correlation was seen between DNA adducts and sister chromatid exchange induction by BP. Our studies have compared a number of commonly used lymphocyte markers and conclude that it is difficult to predict changes in one marker based on changes in another. However, in vitro formation, of PB-derived DNA adducts is consistent over time for individuals.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1989

Fine structure mapping of five temperature-sensitive mutants in the 22- and 147-kilodalton subunits of vaccinia virus DNA-dependent RNA polymerase.

We have mapped the temperature-sensitive (ts) lesions of three mutants, ts51, ts53, and ts65, and two other mutants, ts7 and ts20, to regions on the vaccinia virus genome that encode the 147- and 22-kilodalton subunits of the viral DNA-dependent RNA polymerase, respectively. Plasmid and bacteriophage clones from the HindIII J region and the region spanning the HindIII J-H junction were used in marker rescue experiments to map the mutations. Sequence analysis of the region encoding the 22-kilodalton subunit in the wild-type, ts7, and ts20 viruses revealed a single base change in the mutants compared with that in the wild-type virus. The identification of these RNA polymerase mutants provides us with tools to understand transcription and its regulation in vaccinia virus.

Amino Acid Sequence↗
PubMed1989

Detailed phenotypic characterization of five temperature-sensitive mutants in the 22- and 147-kilodalton subunits of vaccinia virus DNA-dependent RNA polymerase.

We have carried out detailed phenotypic characterization of five temperature-sensitive (ts) mutants of vaccinia virus, the ts lesions of which have previously been mapped to two different subunits of the viral RNA polymerase. We have also attempted to determine the mechanism of temperature sensitivity in these mutants. Phenotypic characterization of each of the mutants showed that at the nonpermissive temperature, all five mutants exhibited normal levels of early viral mRNA and protein synthesis, but for an extended period of time, all mutants accumulated normal levels of DNA in abnormally large pools in the cell cytoplasm; all mutants were defective in the synthesis of late viral mRNA and proteins and in viral morphogenesis. In an attempt to address the mechanism of temperature sensitivity in these mutants, we measured the effect of a temperature shift on the ability of the mutants to direct late viral protein synthesis. If infected cells were shifted down from a nonpermissive temperature late during infection, late protein synthesis was initiated after a lag period of 1 to 2 h. If infected cells were shifted up from a permissive temperature early during infection, late protein synthesis continued to be defective. If infected cells were shifted up to the nonpermissive temperature after late protein synthesis had commenced, late protein synthesis was maintained at the nonpermissive temperature at the level observed when the temperature was shifted up. We interpret these results to mean that once a functional RNA polymerase has been assembled at the permissive temperature during a mutant infection, it remains functional at the nonpermissive temperature, but that the ts mutants are defective in the assembly of a newly synthesized RNA polymerase at the nonpermissive temperature. This interpretation implies that the virion RNA polymerase is responsible for early viral transcription and that a newly synthesized RNA polymerase transcribes late viral genes.

Blotting, Northern↗