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Biomedical subjects

M A Flanagan

Publications and source records attributed to M A Flanagan.

8 recordsLinked to original sources
PubMed1992

Inhibition of cholesterol synthesis by cyclopropylamine derivatives of squalene in human hepatoblastoma cells in culture.

Two squalene derivatives, trisnorsqualene cyclopropylamine and trisnorsqualene N-methylcyclopropylamine, were synthesized and tested for inhibition of lanosterol and squalene epoxide formation from squalene in rat hepatic microsomes, and for the inhibition of cholesterol synthesis in human cultured hepatoblastoma (HepG2) cells. Trisnorsqualene cyclopropylamine inhibited [3H]-squalene conversion to [3H]squalene epoxide in microsomes (IC50 = 5.0 microM), indicating that this derivative inhibited squalene mono-oxygenase. Trisnorsqualene N-methylcyclopropylamine inhibited [3H]squalene conversion to [3H]lanosterol (IC50 = 12.0 microM) and caused [3H]-squalene epoxide to accumulate in microsomes, indicating that this derivative inhibited 2,3-oxidosqualene cyclase. Cholesterol biosynthesis from [14C]acetate in HepG2 cells was inhibited by both derivatives (IC50 = 1.0 microM for trisnorsqualene cyclopropylamine; IC50 = 0.5 microM for trisnorsqualene N-methylcyclopropylamine). Cells incubated with trisnorsqualene cyclopropylamine accumulated [14C]squalene, while cells incubated with trisnorsqualene N-methylcyclopropylamine accumulated [14C]squalene epoxide and [14C]squalene diepoxide. The concentration range of inhibitor which caused these intermediates to accumulate coincided with that which inhibited cholesterol synthesis. The results indicate that cyclopropylamine derivatives of squalene are effective inhibitors of cholesterol synthesis, and that substitutions at the nitrogen affect enzyme selectivity and thus the mechanism of action of the compounds.

Acetates↗
PubMed1990

Differential regulation of hepatic triglyceride lipase and 3-hydroxy-3-methylglutaryl-CoA reductase gene expression in a human hepatoma cell line, HepG2.

The level of hepatic triglyceride lipase (H-TGL) synthesis and secretion was examined in response to changes in cholesterol biosynthesis in the human hepatoma cell line HepG2. Cells were first fed a lipoprotein-deficient serum-supplemented medium to eliminate exogenous cholesterol. Mevinolin, a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, was then added at a concentration (37 microM) which inhibited cholesterol biosynthesis by greater than 85% and decreased total cell cholesterol from 36.1 to 27.4 micrograms/ml of cell protein. Mevinolin treatment caused a 4.9 +/- 0.8-fold increase in the amount of H-TGL activity secreted into the medium, a 1.8 +/- 0.4-fold rise in H-TGL-specific mRNA, and a concurrent 14-fold increase in HMG-CoA reductase mRNA. Addition of 1 mM mevalonic acid to normal or mevinolin-treated cells raised the cellular cholesterol content and decreased the amount of secreted H-TGL activity to levels below control values. Mevalonic acid also prevented mevinolin-induction of H-TGL and HMG-CoA reductase mRNA, suggesting a common regulatory step for H-TGL and HMG-CoA reductase. Exposure of cells to mevinolin and 25-hydroxycholesterol together resulted in a marked repression of HMG-CoA reductase mRNA levels, whereas these conditions further enhanced the secretion of H-TGL activity and the expression of H-TGL mRNA. These results demonstrate a differential role for 25-hydroxycholesterol in the regulation of H-TGL and HMG-CoA reductase expression.

Carcinoma, Hepatocellular↗
PubMed1989

Characterization and sequence analysis of the human ornithine decarboxylase gene.

This report describes the characterization and complete sequence of the human ornithine decarboxylase (ODC) gene. Genomic Southern blot analysis shows only a single gene hybridizing at high stringency, in contrast to the murine multigene family. A Pst I restriction fragment length polymorphism was identified and an allele of the human ODC gene containing the polymorphic Pst I site was cloned and sequenced. The ODC gene is divided into 12 exons and spans 8 kb. Comparison of the human, rat, and mouse ODC genes shows striking conservation of genomic organization, as well as 82% identity in the first 148 bp of the 5'-flanking region. This region contains a TATA box, cAMP-responsive element, CCAAT box, and AP-2 binding site and is consistent with induction of ODC gene expression by both the cAMP and protein kinase C-mediated signaling pathways. The first intron of the human gene is 2,849 bp in length, and contains two putative Sp1 binding sites, as well as an Ap1 binding site, suggesting a role for the first intron in transcriptional regulation. The 5' noncoding region of the predicted mRNA contains regions of virtual identity with that of mouse and rat ODC mRNA, suggesting sequences involved in translational regulation. In addition, it was found that the exon segments corresponding to the amino and carboxyl termini of Saccharomyces cerevisiae and Trypanosoma b. brucei are unrelated to their mammalian counterparts, whereas the middle segments of the protein are conserved. These differences may influence the difference in protein half-life seen between T. b. brucei and mammalian ODC.

Amino Acid Sequence↗
PubMed1985

pH-dependent processes in proteins.

Recent improvements in the understanding of electrostatic interactions in proteins serve as a focus for the general topic of pH-dependent processes in proteins. The general importance of pH-dependent processes is first set out in terms of hydrogen ion equilibria, stability, ligand interactions, assembly, dynamics, and events in related molecular systems. The development of various theoretical treatments includes various formalisms in addition to the solvent interface model developed by Shire et al. as an extension of the Tanford-Kirkwood treatment. A number of detailed applications of the model are presented and future potentialities are sketched.

Animals↗
PubMed1983

Contributions of individual amino acid residues to the structural stability of cetacean myoglobins.

The acid-denaturation behavior of eleven cetacean myoglobins has been studied at two ionic strengths, 0.01 and 0.10 M, at 25.0 degrees C. The myoglobulins studied fall into four phylogenetic suborders, representing the sperm whales, dolphins, baleen whales, and beaked whales. The differences in response to acid denaturation among these closely related myoglobins are small but statistically significant. In three cases, free-energy differences between myoglobins can be ascribed to one amino acid difference and in three others to two differences. The differences in response were analyzed in terms of the changes in noncovalent interactions occurring in the native structure. The effects of changes in electrostatic interactions over the whole charge array were calculated for each myoglobin species by using the modified Tanford-Kirkwood theory. The predicted changes in stability correlated well with the experimental observations in most cases. When differences in hydrogen-bonding capability were considered at a first approximation, substantial effects were predicted. When these effects were taken in conjunction with the electrostatic interactions, the correlation with experiment was improved. Additionally, restrictions in motional freedom and packing constraints appeared to be significant in the single-site analysis. The detectable differences in stability due to single amino acid substitutions along with the small differences in stability between the cetacean suborders indicate that compensatory interactions provide the mechanism for the conservation of stability among the myoglobins studied.

Amino Acid Sequence↗