Biomedical subjects
Publications and source records attributed to C F Clarke.
A premenarcheal girl aged 12 years presented with an abdominopelvic mass and virilisation. A large ovarian cyst was removed at laparotomy. A histological diagnosis of polycystic ovarian syndrome was made, with no evidence of an associated masculinising tumour.
Oligomeric protein complexes containing the nuclear oncogene p53 and the simian virus 40 large tumor antigen (D. I. H. Linzer and A. J. Levine, Cell 17:43-51, 1979), the adenovirus E1B 55-kilodalton (kDa) tumor antigen, and the heat shock protein hsc70 (P. Hinds, C. Finlay, A. Frey, and A. J. Levine, Mol. Cell. Biol. 7:2863-2869, 1987) have all been previously described. To begin isolating, purifying, and testing these complexes for functional activities, we have developed a rapid immunoaffinity column purification. p53-protein complexes are eluted from the immunoaffinity column by using a molar excess of a peptide comprising the epitope recognized by the p53 monoclonal antibody. This mild and specific elution condition allows p53-protein interactions to be maintained. The hsc70-p53 complex from rat cells is heterogeneous in size, with some forms of this complex associated with a 110-kDa protein. The maximum apparent molecular mass of such complexes is 660,000 daltons. Incubation with micromolar levels of ATP dissociates this complex in vitro into p53 and hsc70 110-kDa components. Nonhydrolyzable substrates of ATP fail to promote this dissociation of the complex. Murine p53 synthesized in Escherichia coli has been purified 660-fold on the same antibody affinity column and was found to be associated with an E. coli protein of 70 kDa. Immunoblot analysis with specific antisera demonstrated that this E. coli protein was the heat shock protein dnaK, which has extensive sequence homology with the rat hsc70 protein. Incubation of the immunopurified p53-dnaK complex with ATP resulted in the dissociation of the p53-dnaK complex as it did with the p53-hsc70 complex. This remarkable conservation of p53-heat shock protein interactions and the specificity of dissociation reactions suggest a functionally important role for heat shock proteins in their interactions with oncogene proteins.
A retrospective study of 90 adolescents admitted to a district general hospital after deliberate drug overdoses was carried out. Underlying risk factors, the inpatient assessment, and the initial management offered to each patient were recorded. The longer term outcomes were assessed, with particular emphasis on psychiatric and related disorders. Many had underlying family problems; the parents of nearly half the patients were separated or divorced, and over half the families had already been seen by the social services or at the child guidance clinic. Three quarters of the patients had psychiatric assessments during admission, and of these 59 (66%) were referred for further psychiatric treatment. Of these over half withdrew from treatment. Eleven (12%) of the children took a further drug overdose. This study emphasises the need for psychiatric assessment and treatment in these children, but the results suggest that the success of management is limited by poor patient compliance.
Differential hybridization and molecular cloning have been used to isolate CR39, a cDNA which hybridizes to a 1.2-kilobase (kb) mRNA in rat liver. The level of CR39 mRNA was increased seven- to ninefold over normal levels by dietary cholestyramine and mevinolin and decreased about fourfold compared with normal levels by cholesterol feeding or administration of mevalonate. Similar changes in the mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase were observed under the various conditions. In vitro translation of either CR39 hybrid selected RNA or 1.2-kb CR39 RNA generated by an SP6 in vitro transcription system produced a polypeptide of 39,000 daltons. As deduced from the nucleotide sequence of a full-length CR39 cDNA, the rat CR39 polypeptide contained 344 amino acids and had a molecular weight of 39,615. The predicted amino acid composition and submit molecular weight of the rat CR39 were very similar to those of prenyltransferases isolated from chicken, pig, and human. The sequence of amino acid residues 173 through 203 in the rat CR39 polypeptide showed that 17 out of 30 matched an active-site peptide of avian liver prenyltransferase. Thus, alterations in the rate of cholesterogenesis resulted in the coordinate regulation of three mRNAs encoding HMG-CoA reductase, HMG-CoA synthase, and CR39, the latter being tentatively identified as prenyltransferase.
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was purified to homogeneity from rat liver cytoplasm. The active enzyme is a dimer composed of identical subunits of Mr = 53,000. The amino acid composition and the NH2-terminal sequence are presented. Partial cDNA clones for the enzyme were isolated by screening of a rat liver lambda gt11 expression library with antibodies raised against the purified protein. The identity of the clones was confirmed by hybrid selection and translation. When rats were fed diets supplemented with cholesterol, cholestyramine, or cholestyramine plus mevinolin, the hepatic protein mass of cytoplasmic synthase, as determined by immunoblotting, was 25, 160, and 1100%, respectively, of the mass observed in rats fed normal chow. Comparable changes in enzyme activity were observed. Approximately 9-fold increases in both HMG-CoA synthase mRNA mass and synthase mRNA activity were observed when control diets were supplemented with cholestyramine and mevinolin. When rats were fed these two drugs and then given mevalonolactone by stomach intubation, there was a 5-fold decrease of synthase mRNA within 3 h. These results indicate that cytoplasmic synthase regulation occurs primarily at the level of mRNA. This regulation is rapid and coordinate with that observed for HMG-CoA reductase. The chromosomal localization of human HMG-CoA synthase was determined by examining a panel of human-mouse somatic cell hybrids with the rat cDNA probe. Interestingly, the synthase gene resides on human chromosome 5, which has previously been shown to contain the gene for HMG-CoA reductase. Regional mapping, performed by examination of a series of chromosome 5 deletion mutants and by in situ hybridization to human chromosomes indicates that the two genes are not tightly clustered.
This study addresses whether transcriptional control of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene in rat liver plays a role in determining the level of reductase mRNA. Isolated rat liver nuclei were allowed to elongate nascent RNA transcripts in the presence of [alpha-32P]CTP, and radiolabeled nuclear reductase RNA was quantitated by filter hybridization. Rats fed a diet supplemented with the drugs cholestyramine and mevinolin and having 20-60-fold induced levels of reductase mRNA exhibited levels of reductase transcription which were 20-fold higher than in rats fed an unsupplemented diet. Over 90% of the transcription of the reductase gene was inhibited by concentrations of alpha-amanitin which selectively inhibit RNA polymerase II. Administration of mevalonolactone (the end product of the reaction catalyzed by reductase) to rats fed cholestyramine and mevinolin caused an 80% decrease in the rate of reductase transcription by approximately 1 h. We conclude that under these conditions changes in reductase transcription are primarily responsible for the regulation of reductase mRNA levels.
H4-II-E-C3 hepatoma cells in culture respond to lipid-depleted media and to mevinolin with increased sterol synthesis from [14C]acetate and rise of 3-hydroxy-3-methylglutaryl coenzyme A reductase levels. Mevalonate at 4 mM concentration represses sterol synthesis and the reductase, and completely abolishes the effects of mevinolin. Mevalonate has little or no effect on sterol synthesis or reductase in enucleated hepatoma cells (cytoplasts) or on reductase in cytoplasts of cultured Chinese hamster ovary (CHO) cells. The sterol-synthesizing system of hepatoma cell cytoplasts and the reductase in the cytoplasts of CHO cells were completely stable for at least 4 hr. While reductase levels and sterol synthesis from acetate followed parallel courses, the effects on sterol synthesis--both increases and decreases--exceeded those on reductase. In vitro translation of hepatoma cell poly(A)+RNAs under various culture conditions gave an immunoprecipitable polypeptide with a mass of 97,000 daltons. The poly(A)+RNA from cells exposed for 24 hr to lipid-depleted media plus mevinolin (1 microgram/ml) contained 2.8 to 3.6 times more reductase-specific mRNA than that of cells kept in full-growth medium, or cells exposed to lipid-depleted media plus mevinolin plus mevalonate. Northern blot hybridization of H4 cell poly(A)+RNAs with [32P]cDNA to the reductase of CHO cells gave two 32P-labeled bands of 4.6 and 4.2 K-bases of relative intensities 1.0, 0.61-1.1, 2.56, and 1.79 from cells kept, respectively, in full-growth medium, lipid-depleted medium plus mevinolin plus mevalonate, lipid-depleted medium plus mevinolin, and lipid-depleted medium. These values approximate the reductase levels of these cells. We conclude that mevalonate suppresses cholesterol biosynthesis in part by being a source of a product that decreases the level of reductase-specific mRNA.
Rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase exhibits a diurnal rhythm of activity which coincides with a diurnal rhythm of reductase protein and reductase mRNA levels. This diurnal rhythm of reductase activity, polypeptide mass, and mRNA exists in rats fed a normal diet (unsupplemented rat chow) and in rats fed a diet supplemented with cholestyramine plus or minus mevinolin. Levels of reductase protein were determined by 8 M urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Reductase mRNA was measured by in vitro translation or blot hybridization of liver RNA. Functional reductase mRNA levels in rats fed a normal diet were approximately 10-fold higher during the middle of the dark cycle than during the middle of the light cycle. Maximum induction of functional reductase mRNA was observed in rats fed cholestyramine and mevinolin. This latter level was 157-fold higher than the level measured at the diurnal low point in rats fed a normal diet. Blot hybridization of liver RNA showed two predominant mRNAs of 4.6 and 4.2 kilobase pairs and a minor species at 6.9 kilobase pairs. These mRNAs exhibited a diurnal rhythm for rats on all three diets and reached peak levels during the 12-h dark period. These data indicate that the diurnal rhythm of reductase mass and activity is closely paralleled by the level of its mRNA.
Addition of cholestyramine or cholestyramine plus mevinolin to the diet has been reported to increase the activity and mass of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase. The present data show that these same dietary manipulations cause an induction of functional reductase mRNA. RNA was isolated from rat livers and added to an in vitro translation system, and the reductase was immunoprecipitated and analyzed by polyacrylamide gel electrophoresis under denaturing conditions. One protein was specifically immunoprecipitated and was found to have a Mr of 90,000 on 0.5 M urea/sodium dodecyl sulfate/polyacrylamide gels and a Mr of 94,000 on 8 M urea/sodium dodecyl sulfate/polyacrylamide gels. In animals fed rat chow supplemented with 5% cholestyramine and 0.1% mevinolin, reductase mRNA levels were 5.7-fold higher than in animals fed rat chow with 5% cholestyramine and were 16-fold higher than in animals fed rat chow with 5% cholestyramine and given mevalonolactone by stomach intubation. RNA isolated from animals fed a normal diet and killed at the nadir of the diurnal cycle of enzyme activity contained no detectable amounts of reductase mRNA as determined by this assay.
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