| Project/Area Number |
05660141
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
食品科学・栄養科学
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| Research Institution | Faculty of Agriculture, Shimane University |
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Principal Investigator |
YOKOTA Kazushige Shimane University, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (90158361)
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| Co-Investigator(Kenkyū-buntansha) |
JISAKA Mitsuo Shimane University, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (60243424)
TAKINAMI Koichi Shimane University, Faculty of Agriculture, Professor, 農学部, 教授 (40243422)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1994: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1993: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | essential fatty acid / Madin-Darby canine kidney cells / arachidonic acid / prostanoid / prostaglandin / phorbol diester / prostaglandin endoperoxide synthase / phospholipase D / arachidonate cascades / 細胞情報伝達機構 / 膜リン脂質 / ホスファチジルエタノールアミン |
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| Research Abstract |
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
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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
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