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The regulation of the gene expression of the enzymes in arachidonate cascades by dietary factors

饮食因素对花生四烯酸级联酶基因表达的调节

基本信息

批准号：
05660141
负责人：
YOKOTA Kazushige
金额：
$ 1.41万
依托单位：
Faculty of Agriculture, Shimane University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

To determine the nutritional regulation of arachidonate cascades, Madin-Darby canine kidney (MDCK) cells were modified by supplementation with n-6 and n-3 essential fatty acids. The acyl moiety of membrane phospholipids was analyzed for obtaining the insight into the metabolism of essential fatty acids. Moreover, the activation mechanism of arachidonate cascades in cell signaling in the presence of both phorbol diesters and calcium ionophore. The analysis of the cellular interaction of n-6 and n-3 essential fatty acids revealed that n-3 fatty acids failed to inhibit the incorporation of arachidonic acid into membrane phospholipids. In contrast, n-6 linoleic acid significantly blocked the conversion of linoleic acid to arachidonic acid. Additional study demonstrated that the predominant inhibitory site by n-3 essential fatty acid was the chain elongation of gamma-linolenic acid to dohomo-gamma-linolenic acid. Although prostanoid synthesis activated in cell response changed depending on … More the level of arachidonic acid in membrane phospholipids, the relationship between them was not necessarily straightforward. The cultured cells modified with n-6 essential fatty acids showed the more reduced prostaglandin (PG) endoperoxide synthase activity than those with n-3 essential fatty acids. These findings implicated the feedback inhibition of prostanoid synthesis caused by n-6 essential fatty acids.On stimulation with 50 nM PMA and 0.1 muM A23187, the synthesized PGE_2 and prostanoid synthetic activities reached to a maximal level after 24h of the stimulation. On the other hand, the expressed protein level was higher at 24h than at 3h as determined by Western blotting and immunostaining. cDNAs specific for PG endoperoxide synthase isoforms were amplified by polymerase chain reaction (PCR). In response to phorbol diesters, mitogen-responsive type II increased and reached to a maximum level around 3h rather than 24h. We also studied and characterize the mammalian phospholipase D involved in cell signaling using MDCK cells. Less

为了研究花生四烯酸级联反应对细胞的营养调节作用，用n-6和n-3必需脂肪酸对MDCK细胞进行了修饰。为了深入了解必需脂肪酸的代谢，对膜磷脂的酰基进行了分析。此外，在佛波醇二酯和钙离子载体存在的情况下，花生四烯酸的激活机制在细胞信号转导中级联。N-6和n-3必需脂肪酸的细胞相互作用分析表明，n-3脂肪酸不能抑制花生四烯酸掺入膜磷脂。相反，n-6亚油酸显著抑制了亚油酸向花生四烯酸的转化。进一步的研究表明，n-3必需脂肪酸的主要抑制部位是γ-亚麻酸向DOHO-γ-亚麻酸的链延长。尽管激活的前列腺素合成在细胞反应中的变化取决于…膜磷脂中花生四烯酸的水平越高，它们之间的关系就不一定是直接的。经n-6必需脂肪酸修饰的培养细胞的前列腺素(PG)过氧化物酶活性较经n-3必需脂肪酸修饰的细胞更低。在50 nM的PMA和0.1um的A23187刺激下，PGE_2和前列腺素合成活性在刺激后24小时达到最高水平。另一方面，免疫印迹和免疫染色显示，24小时的蛋白表达水平高于3小时。聚合酶链式反应(PCR)扩增PG内源性过氧化物合成酶亚型特异的cDNAs。对佛波醇二酯的反应，丝裂原反应型II增加，并在3h左右达到最大水平，而不是24小时。我们还利用MDCK细胞研究和表征了参与细胞信号转导的哺乳动物磷脂酶D。较少