| Project/Area Number |
09660139
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
食品科学・栄養科学
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| Research Institution | Shimane University |
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Principal Investigator |
YOKOTA Kazushige Shimane University Faculty of Life and Environmental Science Professor, 生物資源科学部, 教授 (90158361)
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| Co-Investigator(Kenkyū-buntansha) |
JISAKA Mitsuo Shimane University Faculty of Life and Environmental Science Associate Professor, 生物資源科学部, 助教授 (60243424)
滝波 弘一 島根大学, 生物資源科学部, 教授 (40243422)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1999: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1998: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1997: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | arachidonic acid / cyclooxygenase / reverse transcriptase-polymerase chain reaction / lipoxygenase / 8-lipoxygenase / 15-lipoxygenase / chimera / Madin-Darby canine kidney cell line / エイコサノイド / RT-PCR / アポトーシス / アラキドン酸カスケード / 不飽和化反応 / 必須脂肪酸 / エイコサペンタエン酸 / プロスタグランジン / プロスタノイド / アラキド酸 / アイソフォーム / 逆転写・ポリメラーゼ連鎖反応 / Maclin-Darbyイヌ腎臓上皮性細胞株 / リノール酸 |
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| Research Abstract |
Arachidonate metabolites function as informational lipid molecules involved in the signal transduction of mammalian cells. We studied the regulatory mechanism of the arachidonate cascade by food-derived functional agents. As an interacting point of the food agents, we focused the regulation of gene expression of two isoforms of cyclooxygenase (COX), an rate-limiting enzyme in the biosynthetic pathway of prostanoids. To determine their levels specifically with higher sensitivity, the method using reverse transcriptase-polymerase chain reaction was developed with cRNA as internal standards. This method enabled us to determine the change in the levels of 2-type isoform in Madin-Darby canine kidney cells and mouse Swiss 3T3 cells. Tumor-promoting phorbol diesters caused an increase in the level of COX-2. The modification of the membrane phospholipids with n-6 and n-3 essential fatty acids resulted in the distinct regulation of the gene expression of COX isoforms depending on the types of unsaturated fatty acids.
The structures and function of phorbol diester-inducible novel lipoxygenase (LOX)s were studied by the recombinant DNA technology and protein engineering. The exchange of the C-terminal one-third of the 8S-LOX for the corresponding human homologue, 15S-LOX-2 produced a chimeric enzyme with exclusive 15S-lipoxygenase activity. The stereospecificity was found to be S configuration. These findings suggested that the C-terminal regions were determinant for the positional specificity of the lipoxygenases. By using the in situ chimeragenesis in Escherichia coli followed by the site-directed mutagenesis, a pair of amino acids, Tyrosine-603 and Histidine-604 was identified as the positional determinant. The mutation of those in 8S-LOX to the corresponding 15S-LOX-2 residues converted to the enzyme expressing almost 15S-lipoxygenase activity.
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