Date of Award

1997

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

First Advisor

Adeli, K.

Keywords

Biology, Molecular.

Rights

CC BY-NC-ND 4.0

Abstract

The objectives of my research program were: (i) to develop a mRNA-dependent cell-free system capable of translation of apoB mRNA, since common in vitro translation systems are unable to synthesize apoB 100 because of its unusually large size; (ii) to study the effect of hypolipidemic drugs including a newly developed HMG-CoA reductase inhibitor, atorvastatin, on intracellular degradation of apoB in HepG2 cells; and (iii) to study degradation of different intracellular apoB pools in HepG2 cells in order to elucidate factors regulating apoB degradation. An mRNA-dependent cell-free system was developed from HepG2 cells which can accommodate in vitro translation of apoB mRNA. Upon addition of cytoplasmic RNA extracted from HepG2 cells, the HepG2 cell-free system was capable of synthesis of the full length apoB100, with a size of 550 kDa. The HepG2 cell-free system also proved to be sensitive to heterologous mRNAs and actively translated Brome Mosaic Virus RNA. Studies on apoB degradation in HepG2 cells were also performed by using a permeabilized HepG2 system developed in our laboratory. Degradation of apoB was studied under the influence of a battery of hypolipidemic drugs including the fibrate derivatives, nicotinic acid, probucol, as well as the HMG-CoA reductase inhibitors lovastatin and atorvastatin. The fibrates, nicotinic acid, probucol, and lovastatin did not influence apoB degradation. However atorvastatin appeared to stimulate apoB degradation by approximately 25%. Atorvastatin showed this stimulatory effect under different conditions in which the HepG2 cells were provided with oleate or low density lipoprotein (LDL) as a source of lipid. Fractionation of different apoB pools in HepG2 cells showed that atorvastatin enhanced degradation of apoB bound to the ER membrane as well as apoB present in the lumen of the ER. Subsequent fractionation of luminal apoB into dense (HDL) and light (LDL-VLDL like) particles revealed that apoB in the HDL-like particles is more sensitive to the degradation under the influence of atorvastatin. Characterization of degradation of different intracellular apoB pools in permeabilized HepG2 cells showed that degradation of membrane bound apoB occurs at a faster rate and to a larger extent compared to the luminal apoB. Degradation of apoB in the membrane pool was also not sensitive to N-acetylleucylleucylnorleucinal (ALLN), a cysteine protease inhibitor which repressed luminal apoB degradation. (Abstract shortened by UMI.)Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1996 .M635. Source: Dissertation Abstracts International, Volume: 59-08, Section: B, page: 3909. Adviser: K. Adeli. Thesis (Ph.D.)--University of Windsor (Canada), 1997.

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