LIVER/INTESTINAL METABOLISM ON BILE ACIDS/CHOLESTEROL

胆固醇和胆汁酸被认为是重要的 在“西方社会”的几种主要疾病中扮演的角色，包括： 动脉硬化、胆固醇结石形成、胆汁淤积性肝 疾病和结肠癌的发生。此次更新的总体目标是 应用目的是为了更详细地了解胆汁的作用 酸在调节胆固醇和胆汁酸的动态平衡中发挥作用， 肝脏/肠道生理学和胆固醇结石疾病。这个 总体目标将通过以下具体措施实现 目的：[1]a)确定蛋白激酶C的哪个亚型(S) 参与胆固醇7α-羟基酶的调节；b) 确定蛋白激酶C亚型激活的机制 通过胆汁酸；c)定义蛋白激酶C和胆汁酸反应 胆固醇7α-羟基酶启动子元件；d) 测定胆汁酸在体内是否激活蛋白激酶C亚型 肝脏和回肠(Vlahcevic、Stravitz、Heuman、Hylemon)； 量化胆盐对模型膜的吸附；b)开发 并验证了胆汁盐膜的一般定量模型。 吸附；c)确定卵磷脂和胆固醇对 胆盐对膜的毒性；d)确定 胆汁脂类成分对胆盐中毒动物模型的影响 胆盐诱导的肝损伤；e)确定蛋白激酶C 胆盐的活化是胆汁蓄积的后果。 膜表面的盐分可以用定量的方法预测 胆盐-膜吸附模型(Heuman，Stravitz， Valhcevic；[3]a)选择性过表达胆固醇7α- 羟基酶、甾醇27-羟基酶、胆固醇酯水解酶和 乙酰辅酶A：胆固醇酰基转移酶(Hep G2和中国人) 仓鼠卵巢细胞)；b)评估每种酶在 维持细胞和全身胆固醇动态平衡 体内重组腺病毒载体(仓鼠)；c)研究 胆固醇酯水解酶和酰辅酶A：胆固醇的调节 大鼠肝细胞原代培养和体内胆汁中的酰基转移酶 酸、胆固醇和激素(Pandak，Vlahcevic)； 完成一个大分子胆汁酸的克隆、测序和分析 肠道真细菌的可诱导操纵子(BAI)。VPI 12708；b)确定每个基因产物编码的功能 该操纵子发挥熊胆酸7α/β-脱羟化作用；c)分离， 胆汁酸7α-脱羟基细菌的鉴定 来自胆固醇结石患者具有高水平(&gt；30%) 脱氧胆酸，并确定这些患者是否被 独特的7种α-脱羟基细菌(Hylemon，Berr)。

Cholesterol and bile acids have been implicated in playing important roles in several major diseases of "Western Society" including: arteriosclerosis, cholesterol gallstone formation, cholestatic liver disease and colon carcinogenesis. The overall goal of this renewal application is aimed at a more detailed understanding of the role bile acids play in the regulation of cholesterol and bile acid homeostasis, liver/intestinal physiology, and cholesterol gallstone disease. The overall goal will be accomplished through the following specific aims: [1] a) Determine which isoform(s) of protein kinase C is involved in the regulation of cholesterol 7 alpha-hydroxylase; b) Determine the mechanism of activation of protein kinase C isoforms by bile acids; c) Define protein kinase C and the bile acid responsive element of the cholesterol 7 alpha-hydroxylase promoter; d) Determine if bile acids activate protein kinase C isoforms in vivo in liver and ileum (Vlahcevic, Stravitz, Heuman, Hylemon); [2] a) Quantify adsorption of bile salts to model membranes; b) Develop and validate a general quantitative model of bile salt-membrane adsorption; c) Determine the effects of lecithin and cholesterol on toxicity of bile salts toward membranes; d) Determine the effect of biliary lipid composition on biliary bile salt toxicity in animal models of bile salt induced liver injury; e) Determine if protein kinase C activation by bile salts are consequences of the accumulation of bile salts on the membrane surface that can be predicted by quantitative modeling of bile salt-membrane adsorption (Heuman, Stravitz, Valhcevic ; [3] a) Selective overexpression of cholesterol 7 alpha- hydroxylase, sterol 27-hydroxylase, cholesterol ester hydrolase and acyl CoA:cholesterol acyltransferase in vitro (Hep G2 and Chinese hamster ovary cells); b) Assess the role each enzyme plays in maintaining cellular and whole body cholesterol homeostasis using recombinant adenovirus vectors in vivo (hamsters); c) Investigate the regulation of cholesterol ester hydrolase and acyl CoA:cholesterol acyltransferase in primary rat hepatocyte cultures and in vivo by bile acids, cholesterol and hormones (Pandak, Vlahcevic); [4] a) Complete the cloning, sequencing and analysis of a large bile acid inducible operon (bai) from the intestinal Eubacterium sp. VPI 12708; b) Determine the function that each gene product encoded by this operon plays inbile acid 7 alpha/beta-dehydroxylation; c) Isolate, characterize and identify bile acid 7 alpha-dehydroxylating bacteria from cholesterol gallstone patients having high (>30%) levels of deoxycholic acid and determine if these patients are colonized by unique 7 alpha-dehydroxylating bacterial species (Hylemon, Berr).