Yoshio Tsujita

WHHL-rabbit: a low density lipoprotein receptor-deficient animal model for familial hypercholesterolemia.

Skin fibroblasts from normal human subjects possess a specific receptor for low density lipoprotein (LDL), but cells from subjects with homozygous form of familial hypercholesterolemia (FH) lack it [l--3]. Although LDL receptor-deficiency was eludicated to be a primary metabolic defect of FH, many unknown processes responsible for its pathogenesis have not yet been clarified. Thus an animal model for FH is required. A method for producing LDL receptordeficient mice using mutagenized cultures of teratocarcinoma stem cells appeared in [4]. The lipid metabolism of rhesus monkeys with spontaneous hypercholesterolemia was analyzed [ 51. In spite of these efforts, the presence of LDL receptor-deficient animal has not been reported. WHHL-rabbit (Watanabe-heritable hyperlipidemic rabbit), characterized by its abnormally high level of serum lipids, is a strain developed by inbreeding from a mutant of spontaneous hyperlipidemia [6,7]. Here, we describe the biochemical characteristics of WHHLrabbit including LDL receptor-deficiency in its skin fibroblasts.

Hypolipidemic effects in dogs of ML-236B, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

ML-236B, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, significantly reduced both serum cholesterol and phospholipid levels in dogs, when used at a dosage higher than 10 mg/kg per day. Triglyceride levels were not consistently changed, but beta- and pre-beta-lipoproteins were preferentially reduced. Serum cholesterol levels were reduced by 44--45% at the higher dosage of 100--400 mg/kg per day (for 5 weeks) but ML-236B caused no significant changes in the cholesterol content of the liver and aorta and in the activities of serum GOT, GPT, CPK and lecithin : cholesterol acyltransferase. Fecal excretion of neutral sterols was unaffected but that of bile acids was markedly elevated by the drug. Under these conditions, hepatic cholesterol 7alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, showed no detectable changes.

Mechanism for elevation of hepatic cholesterol synthesis and serum cholesterol levels in triton WR-1339-induced hyperlipidemia.

Abstract 1. 1. During the first 3 h after the intravenous injection of Triton (WR-1339) to rats at a dose of 400 mg/kg (first phase), a 2-fold increase in serum cholesterol levels was accompanied by a 17–35% decrease in hepatic cholesterol levels, but the liver plus serum cholesterol levels (mg/100 g body weight) were essentially unchanged. No increases were seen in the activity of either hepatic 3-hydroxy-3-methylglutaryl-CoA reductase or sterol synthesis during this period. 2. 2. At 6–9 h after Triton injection (second phase), a 3–4-fold increase in serum cholesterol was accompanied by a 2–3-fold increase in hepatic sterol synthesis and a 4–5-fold elevation of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity. During this phase, liver cholesterol levels increased to control level, and the liver plus serum cholesterol levels increased by 14–40%. 3. 3. Treatment with ML-236B, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, had no effect on changes in the first phase but reduced both the liver and serum cholesterol levels in the second phase. 4. 4. These results strongly suggest that the increased serum cholesterol levels in the first phase was mainly caused by removing cholesterol from liver into the serum compartment, and that in the second phase the increased sterol synthesis in liver resulted from the elevation of 3-hydroxy-3-methylglutaryl-CoA reductase may explain the rise of serum cholesterol levels.

Purification and characterization of the two molecular forms of Aspergillus oryzae acid protease.

The isolation and partial characterization of the acid proteases A1 and A2 (EC3.4.23.6) from Aspergillus oryzae grown on solid bran culture are described. The purified preparations were essentially homogeneous by several criteria including sedimentation analysis and polyacrylamide gel electrophoresis. The physiochemical properties of the proteases A1 and A2 were as follows (in the order: A1, A2): molecular weight: 63 000 & 32 000; sedimentation coefficient s20, w: 3.93 and 3.16 S; diffusion constant D20, w, 5.63 - 10(-7) and 8.61 - 10(-7) CM2/S, partial specific volume, v: 0.73 ml/g for both; nitrogen content: 16.30 and 13.42%; E1% 1 cm at 280 nm: 5.9 and 11.1. The two enzymes had the same pH optima in the acid pH range, and both activated bovine pancreatic trypsinogen. The enzymes were essentially of the same amino acid composition and immunologically cross-reacted with each other. The protease A2 contained little or no carbohydrate, whereas the protease A1 was glycoprotein, containing 49% carbohydrate comprising glucose, mannose, and galactose. These results suggest that the protein portion of acid protease A1 is the same as that of acid protease A2.

Mechanism of action of probucol on cholesteryl ester transfer protein (CETP) mRNA in a Chinese hamster ovary cell line that had been stably transfected with a human CETP gene

Probucol, a widely used lipid-lowering agent, is associated with a significant reduction of plasma high density lipoprotein (HDL)-cholesterol levels. To examine the mechanism of probucol HDL-lowering and probucols effects on cholesteryl ester transfer protein (CETP) and cholesterol metabolism in cells, we used a Chinese hamster ovary (CHO) cell line that had been stably transfected with a human CETP gene (hCETP-CHO). After this cell line was incubated with various concentrations of probucol (5, 10 and 50 microM) for 24 h, mean intracellular probucol concentrations reached 0.47, 0.67, and 1.52 microg/mg cell protein, respectively. Northern blot analysis showed that cellular CETP mRNA was increased by probucol in a dose-dependent manner (137%, 162%, and 221% of the control, respectively). The specific CET activity in the culture medium, measured as the percentage of [3H]cholesterol oleate transferred from discoidal bilayer particles (which mimic HDL) to LDL, also increased in a dose-dependent manner. Intracellular total cholesterol levels were decreased to 87.5%, 74.9%, and 52.5% of the control, respectively. Probucol had no effects on HMG-CoA reductase activity or cholesterol synthesis from [14C]acetate in hCETP-CHO. However, 14C-incorporated cholesterol secretion into the culture medium from hCETP-CHO was increased to 181%, 256% and 354% of the control by 5, 10 and 50 microM probucol, respectively. We concluded that (1) treatment with probucol increased the CETP mRNA level and specific CET activity in the hCETP-CHO cell line, and (2) probucol promoted cholesterol efflux from hCETP-CHO, which resulted in a decrease in intracellular cholesterol levels.

Schizostatin, a potent squalene synthase inhibitor from Schizophyllum commune: Isolation, structure elucidation, and total synthesis

Abstract The novel schizostatin ( 1 ) has been isolated as a potent inhibitor of squalene synthase. Its structure elucidation and total synthesis are described. Synthesis of the Z-isomer 12 and its biological activity are also reported.

Evidence for the presence of membrane-bound forms of acid protease in Aspergillus oryzae

Abstract While approximately 85% of the cell-bound acid protease of Aspergillus oryzae were recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be present tightly associated with the membranes. Two forms of membrane-bound enzyme, which were solubilized with Triton X-100, were similar to the external acid protease found in culture medium in that they had an optimum pH at 3.2, activated trypsinogen at pH 3 and lost their activity upon treatment with 5.1 mM sodium dodecylsulfonate. However, they differed in their hydrophobic properties (i.e. aggregation in the absence of Triton X-100 and activation by the detergent) from both the cell-bound, soluble form and the one excreted into culture medium.