| Co-Investigator(Kenkyū-buntansha) |
高木 敦子 国立循環器病センター研究所, 病因部, 室長 (90179416)
IKEDA Yasuyuki NCVC, Dept. Etiology/Pathogenesis, Staff, 病因部, 室長 (90176107)
TAJIMA Shoji NCVC, Dept. Etiology/Pathogenesis, Chief, 病因部, 室長 (50132931)
MIYAKE Yasuko NCVC, Dept. Etiology/Pathogenesis, Chief, 病因部, 室長 (00132936)
YAMAMURA Taku NCVC, Dept. Etiology/Pathogenesis, Chief, 病因部, 室長 (20132938)
YAKOYAMA Shinji NCVC, Dept. Etiology/Pathogenesis, Chief (10142192)
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| Research Abstract |
Hyperlipoproteinemia is one of the most important risk factors for atherosclerotic vascular diseases. Hyperchylomicronemia often results in fatal pancreatitis. Although the plasma lipid level is strongly influenced by dietary conditions,most cases of hyperlipoproteinemia are based upon the genetic of abnormalities in plasms lipoprotein metabolism. Depending upon such hereditary predispositions,the response to dietary management is different. The present study is aimed at elucidating the etiology and pathogenesis of hyperlipoproteinemia on the molecular basis and establishing the criteria for discriminating of trait of hyperlipoproteinemia.
1) Analysis of the LDL-receptor defect in familial hypercholesterolemia (FH): Fibrobasts were cultured from skin biopsy samples from 23 cases pf FH homozygotes belonging to 17 different families. Functional activities of LDL-receptor proteins were estimated by measuring ^<125>I-LDL binding and uptake. LDL- receptor proteins were analyzed by ^<35>S-met
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hionine pulse labeling followed by the immunoprocipitation and SDS electophoresis. Ten families were diagnosed as a receptor-negative type. Among them, receptor proteins were completely lacking in 5 families and markedly reduced in 2 families. Processing of the precursor to mature protein was disturbed in 2 families and a receptor protein 5kd smaller in size was formed in one family. Among 5 families with receptor-defective type, two showed a reduction in number of the receptors and three showed a delay in processing from precursor to the mature form. In one family, a peculiar mutation was found. The EGF precursor homology domain was completely lacking due to the Alu-Alu recombination. Although the receptor protein appeared on the cell surface, it could not bind LDL. The receptor could bind beta-VLDL but recycling was impaired. Our results show that a variety of mutants exist in LDL receptors of PH among Japanese population. [Ref.1-3]
2) Purification and Characterization of lipoprotein lipase(LpL) and the analysis of its deficiency in type I hyperlipoproteinemia: LpL and hepatic triglyceride lipase(H-TGL) were purified from human postheparin plasma(PHP) and specific antibodies (polyclonal and monoclonal) were prepared. Assay kits for LPL and H-TGL with selective immunoinactivation method (for enzyme activities) and sandwich EIA method (for mass) were developed. Using THP-1 cells, a human monocytic leukemia cell line, the synthesis and secretion of LpL were studied. It was synthesized as a precursor with 55kd molecular weight and processed by the addition of sugar moiety into 61kd mature protein. In one of the patient with type I hyperlipoproteinemia, no LpL nor MRNA was detected in monocute-macrophages. In another patient a small amount of LPL with normal molecular weight was detected in monocute-macrophages, while there was no mass nor activity of the enzyme was found in his PHP. [Ref.4, 5]
3) Apolipoprotein E (apo E) polymorphism and its relation to hyperlipoproteinemia: Two new mutants of apo E were found and the site of mutation was determined; apo E5 with the change of the 3rd amino acid residue from Glu to Lys and apo E7 with the change at 244 and 245 both from Glu to Lys. The substitution occurred by the same base exchange G->A. The frequency distribution of the major apo E isoforms and these new mutants was investigated among 199 patients with ischemic heart diseases (mainly myocardial infarction) administered to CCU of our hospital. The frequency of E4,5, and 7 was significantly higher in relatively young male patients aged below 60 years and in female patients compared to the healthy population. [Ref.6-8]
4) Purification and characterization of lipid transfer protein (cholesterol ester transfer protein CETP): A rapid purification procedure using 3 steps gel chromatography was established and CETP purified 43,000 fold with a yield of 30% from human plasma. CETP-catalyzed cholesterol ester transfer (trig ly-ceride transfer into the reverse direction) was estimated by using LDL, HDL and artificial triolein particle covered by phosphatidylcholine monolayer and stabilized by apolipoprotein A-I,A-II, C-II, C-III, or E. The kinetics was consistent with the model that the reaction is bidirectional in equilibrium, taking both non-polar lipids as substrate in a single pool. In one patient with strong hyperalphalipoproteinemia with HDL-cholesterol 239 mg/dl, a reduction in CETP activity was observed by using patient's HDL as a substrate suggesting the existence of an inhibitor. [Ref.9, 10] Less
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