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， 抑制类花生酸关键酶的表达 生物合成这些研究的结果将提供重要的信息 在理解膳食氮的某些生物学作用机制方面， 3不能根据改变解释的PUFA 类花生酸生物合成的底物可用性。此外，结果 从这些研究可以打开令人兴奋的可能性，修改 膳食脂肪酸的组成（也许还有其他营养素）可以是 用作改善某些慢性疾病的补充手段， 其改变了关键酶（即环氧合酶（考克斯））的表达 参与合成炎症介质是有益的。的 具体目标是：1）确定膳食n-3 PUFA是否抑制 前列腺素合成中限速酶（考克斯）的表达， 和5-脂氧合酶（5-LO）在人类血液单核细胞消耗的饮食 n-3或n-6 PUFA。2)方法的抑制表达式是否 通过n-3 PUFA介导的酶的活性和/或表达的改变， 蛋白激酶C（PKC）在培养的细胞中。(The假设是 n-3 PUFA介导的这些酶表达的降低是由 抑制PKC的活性和/或表达，这反过来又导致 降低花生四烯酸（PKC激活剂）释放的水平， 信号转导）。3)为了确定是否改变了 考克斯，5-LO或PKC的饮食n-3多不饱和脂肪酸的结果，从特定的变化， 相应基因的转录水平，而不是改变 mRNA或酶蛋白的降解。两者的表达 将在刺激的细胞中评估组成型和诱导型考克斯和5-LO。 人单核细胞或巨噬细胞和平滑肌细胞（SMC）（来源于 从喂食n-3 PUFA的大鼠）通过测定：i）通过逆转录聚合酶链反应测定mRNA水平， 转录和聚合酶链反应ii）酶蛋白的速率 通过代谢标记和免疫沉淀使用特异性 抗体iii）酶活性。 在动物研究中， 酶的表达和PKC易位的课程将是 在巨噬细胞和SMC中进行，所述巨噬细胞和SMC在含有 同源血清核径流转录测定和脉冲追踪 将进行实验以评估转录水平， 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.