| Project/Area Number |
10557230
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
医薬分子機能学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
SUGIYAMA Yuichi Graduate School of Pharm. Sci. The Univ. of Tokyo, Professor, 大学院・薬学系研究科, 教授 (80090471)
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| Co-Investigator(Kenkyū-buntansha) |
TAKIKAWA Hajime School of Medicine Teikyo Univ., Professor, 医学部, 助教授 (70197226)
KATO Yukio Graduate School of Pharm. Sci. The Univ. of Tokyo, Research Associate, 大学院・薬学系研究科, 助手 (30251440)
SUZUKI Hiroshi Graduate School of Pharm. Sci. The Univ. of Tokyo, Assistant Professor, 大学院・薬学系研究科, 助教授 (80206523)
KATO Motohiro Drug Metabolism & Pharmacokinetics Lab. Chugai Pharm. Co., Ltd., Researcher, 薬物動態研究所, 研究主査(研究職)
NAKAMUARA Toshikazu School of Medicine Osaka Univ., Professor, 医学部, 教授 (00049397)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥12,600,000 (Direct Cost: ¥12,600,000)
Fiscal Year 1999: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1998: ¥9,800,000 (Direct Cost: ¥9,800,000)
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| Keywords | Intracellular sorting / Drug Delivery System / iologically Active Proteins / Receptor / Endocytosis / ドラッグデリバリーシステム |
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| Research Abstract |
Biologically active proteins usually exhibit the short plasma half-life, repeated and high doses being necessary to obtain their pharmacological activity in vivo. Since their clearance mechanism is the receptor-mediated endocytosis and subsequent lysosomal degradation in target organs, the aim of this study is to regulate their intracellular sorting in order to avoid such degradation pathway. Hepatocyte growth factor (HGF) was fused with endoplasmic reticulum retention signal and nuclear localization signal by gene recombinant techniques. HGF genes obtained in this way were inserted into PUC-SRaipha vector and transfected into COS-7 cells. HGF produced in the medium was dialyzed and purified by heparin-affinity chromatography and reverse phase HPLC. HGF was iodinated by the chloramine-T method, its molecular weight being checked by SDS-PAGE. The biological activity of each HGF derivative was checked by assessing the stimulatory effect on DNA synthesis in primary cultured rat hepatocytes. After incubating with cell lines which highly express HGF receptor, the trichloroacetic acid-soluble radioactivity in the medium gradually increased after a lag-time. However, the increase in TCA-soluble radioactivity during the incubation period with radiolabeled HGF mutant was much lower, indicating that its intracellular degradation is much slower than the wild-type HGF. Thus, the regulation of the intracellular fate of the endocytosed ligand can be, at least partially, regulated by the insertion of a certain type of signal sequences. However, the stability of such a mutant in the medium was not so much increased probably because of the low efficiency in the recycle of the ligand. Therrefore, further improvement has to be considered to change the sorting after avoiding lysosomal degradation. The dissociation kinetics in the endosomal compartment may be the other target to improve the efficiency of ligand recycle.
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