| Project/Area Number |
09672285
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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 |
医薬分子機能学
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| Research Institution | Josai University |
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Principal Investigator |
SHIRAHATA Akira Josai University, Faculty of Pharmaceutical Sciences, Department of Biochemistry, Associate Professor, 薬学部, 助教授 (50150107)
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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 |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1998: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1997: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | polyamine / transglutaminase / photo- affinity labeling / biotin / arginyltransferase / cell death / histon / NMDA receptor / スペルミジン / スペルミン / 遊離型濃度 / 蛍光標識 / TFO / 細胞容積 / アルギニルトランスフェラーゼ / NMDA / アジド / メランスグルタミナーゼ / フォトアフィニティーラベル / ポリアミントランスポーター |
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| Research Abstract |
Synthesis of polyamine analogs having groups and detection of proteins as targets of cellular polymine actions: Biotinylated polyamines were systematically synthesized. When they were administered to rat hepatoma HTC cells, their structure specific labeling of cellular proteins were found by Western blot analysis. Microtiter plate assay method of tissue transglutaminase was newly developed using biotinylated polyamine. From the substrate specifisity of the enzyme elucidated by using the method, its active site structure could be proposed. A biotinylated and azidophenylated analog was usefull for detecting cell membrane protein having affinity to physiological polyamine. Artificial substrate of arginyltransferase were synthesized by using fluorescence labeled polyamines, and the assay method of the enzyme was established with the substrate.
Analysis cell function which are affected by depletion and accumulation of polyamine : Polyamine-depleted HTC cells, treated with inhibitor of polyam
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ine biosynthesis for relatively long period, easily accumulate administered spermidine and the abnormal accumulation of spermidine in the cells leads to abnormal cell death like apoptosis. In order to investigate the mechanism of the cell death, the protein whose cellular level changed during the death process was searched. It was observed that the level of cellular histon decreased during the process and its degradation preceded the cell death. The degradation and the cell death were partially suppressed by the administration of serine protease inhibitor, suggesting the histon degradation would be involved in the cell death accompanied by the accumulation of spermidine. Ultrafiltration method for cytosol fraction in corn oil was proposed to determine the free concentration of cellular polyamine, and applied to HTC cell culture system. It was found that the method was useful for the estimation of free cellular polyamine level, and that cell volume of HTC cells was affected by the depletion or accumulation of polyamine. A new determination method of polyamine which does not have separation steps was developed by using mass- spectrometry.
Syntheis of polyamine analogs and examination of their action : Polyamine analogs including ones having long chain lenghth were synthesized and their effect on NMDA receptor, DNA triple helix formation and so on were examined. Less
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