DIETARY N-3 FATTY ACIDS AND EXPRESSION OF CYCLOOXYGENASE

长期目标是确定饮食中的n-3 多不饱和脂肪酸(PUFA)调节关键酶的表达 参与受体介导的信号转导，进而 抑制二十烷类化合物中的关键酶的表达 生物合成。这些研究的结果将提供至关重要的信息 在了解饮食中某些生物作用的机制中，n- 3不能以变更为依据解释的PUFA 二十烷类化合物生物合成的底物利用率。此外，结果 从这些研究中可以打开令人兴奋的可能性，修改 膳食脂肪酸(也许还有其他营养素)的组成可以是 作为一种补充手段来改善某些慢性病 改变关键酶(即环氧合酶(COX))的表达 参与合成炎症介质是有益的。这个 具体目标是：1)确定膳食n-3多不饱和脂肪酸是否抑制 前列腺素合成过程中限速酶COX的表达 和5-脂氧合酶(5-LO)在饮食人群血单核细胞中的表达 N-3或n-6多不饱和脂肪酸。2)确定是否抑制了 N-3PUFA介导的酶活性和/或表达的改变 培养细胞中的蛋白激酶C(PKC)。(假设是 N-3多不饱和脂肪酸降低这些酶的表达是通过 抑制PKC的活性和/或表达，进而导致 降低花生四烯酸(PKC激活剂)水平 信号转导)。3)确定是否改变了 膳食n-3多不饱和脂肪酸引起的COX，5-LO或PKC的特异性变化 相应基因的转录水平而不是改变 信使核糖核酸或酶蛋白的降解。两者的表达 结构性和诱导性COX和5-LO将在刺激中被评估 人单核细胞或巨噬细胞和平滑肌细胞(SMC)(来源 来自饲喂n-3多不饱和脂肪酸的大鼠)通过测定：i)反向的mRNA水平 转录和聚合酶链式反应II)酶蛋白比率 代谢标记和免疫沉淀法合成 抗体III)酶活性。在动物研究中，综合时间 这些酶的表达和PKC的转位的课程将是 巨噬细胞和SMC在含有 同源血清。核径流转录分析与脉冲追逐 将进行实验以评估转录水平和 从头合成酶的半衰期。

The long term objective is to determine whether dietary n-3 polyunsaturated fatty acids (PUFA) modulate the expression of key enzymes involved in the receptor mediated signal transduction, and in turn suppress the expression of key enzymes involved in eicosanoid biosynthesis. Results from these studies will provide crucial information in understanding the mechanism of some of biological actions of dietary n- 3 PUFA that can not be explained on the basis of the alteration of substrate availability for eicosanoid biosynthesis. Furthermore, results from these studies can open exciting possibility that modifying the composition of dietary fatty acids (and perhaps other nutrients) can be used as a supplemental means to ameliorate certain chronic diseases for which altered expression of key enzymes (i.e. cyclooxygenase (COX)) involved in synthesizing inflammatory mediators is beneficial. The specific aims are: 1) to determine whether dietary n-3 PUFA suppress the expression of the rate limiting enzyme (COX) in prostaglandin synthesis, and 5-lipoxygenase (5-LO) in blood monocytes of humans consuming dietary n-3 or n-6 PUFA. 2) To determine whether suppressed expression of the enzymes by n-3 PUFA is mediated by altered activity and/or expression of protein kinase C (PKC) in cells in culture. (The hypothesis is that decreased expression of these enzymes by n-3 PUFA is mediated by suppressed activity and/or expression of PKC, which in turn results from reduced level of arachidonic acid (PKC activator) released during the signal transduction). 3) To determine whether or not altered expression of COX, 5-LO or PKC by dietary n-3 PUFA results from specific change in the level of transcription of corresponding gene instead of altered degradation of mRNA or enzyme proteins. The expression of both constitutive and inducible COX and 5-LO will be assessed in stimulated human monocytes or in macrophages and smooth muscle cells (SMC) (derived from rats fed n-3 PUFA) by determining: i) levels of mRNA by reverse transcription and polymerase chain reaction ii) the rate of enzyme protein synthesis by metabolic labeling and immunoprecipitation using specific antibodies iii) enzyme activities. In animal studies, comprehensive time courses for the expression of the enzymes and translocation of PKC will be performed in macrophages and SMC cultured in the media containing homologous serum. Nuclear runoff transcription assay and pulse chase experiments will be carried out to assess the level of transcription and the half lives of de novo synthesized enzyme.