| Project/Area Number |
07458201
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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 | 一般 |
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| Research Field |
Nerve anatomy/Neuropathology
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| Research Institution | Osaka University |
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Principal Investigator |
UCHIYAMA Yasuo Osaka University Medical School, Professor, 医学部, 教授 (10049091)
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| Co-Investigator(Kenkyū-buntansha) |
OHSAWA Yoshiyuki Osaka Univ.Med.School, Assistant Prof., 医学部, 助手 (30273642)
GOTOW Takahiro Osaka Univ.Med.School, Associate Prof., 医学部, 助教授 (20135693)
WATANABE Tsuyoshi Osaka Univ.Medical School, Associate Prof., 医学部, 助教授 (80220903)
和栗 聡 大阪大学, 医学部, 助手 (30244908)
似鳥 徹 岩手医科大学, 医学部, 講師 (90128934)
佐藤 昇 大阪大学, 医学部, 助手 (00254756)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥7,500,000 (Direct Cost: ¥7,500,000)
Fiscal Year 1997: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1996: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1995: ¥5,300,000 (Direct Cost: ¥5,300,000)
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| Keywords | Apoptosis / PC12 Cells / Bcl-2 gene / Neurotrophic factor / Cathepsin B / Cathepsin D / Lysosomes / Dorsal roof ganglion / bcl-2遺伝子 / bcl-2 / 遺伝子導入 / cDNAクローニング / nedd-2 / 脳虚血 / オートファジ- / リソゾーム / CAl錐体細胞 |
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| Research Abstract |
1) PC12 cells undergo apoptosis when cultured under serum deprivation. In this situation, the activity of caspase-3-like proteinases was elevated, and the survival rate could be maintained by treatment with acetyl (AC)-DEVD-CHO,a specific inhibitor of caspase-3. In a culture of PC12 cells with AC-DEVD-CHO,where caspase-3-like proteinases are not activated, CA074, a specific inhibitor of cathepsin B induced apoptosis of the cells. This ability of CA074 was also replaced by cathepsin B antisense oligonucleotides. By double staining of TUNEL and activated caspase-3, the dying cells treated with CA074 were positive for TUNEL staining but negative for caspase-3. Ultrastructures of the cells were relatively large and had nuclei with chromatin condensation, suggesting that they died by apoptosis. Thus cell death effect by CA074 or the cathepsin B antisense were inhibited by the addition of pepstatin A,a lysosomal aspartic proteinase inhibitor, or cathepsin D antisense. The results suggest tha
… More
t a novel pathway of apoptosis exists, which is regulated by lysosomal cathepsins, and in which cathepsin D acts as a death factor, but that this death-inducing activity is usually suppressed by cathepsin B.
2) PC12 cells transfected with the human bcl-2 gene can survive when cultured under serum deprivation. In this situation, the transfected cells extended neurite-like processes. Culture media of the transfected cells contained factor (s) which rescued wild-type PC12 cells following serum withdrawal. We therefore partially purified the factor and separated it by native SDSPAGE.From this, the N-terminal amino acid sequence of the factor was decided and the protein was found to be novel. Following the routine method of cDNA cloning, the cDNA clones of the factor protein were isolated, consisting of 2332 bp of total sequence having an ORF of 768 bp, encoding a protein of 256 amino acids. The predicted amino acid sequence has a signal sequence of 23 amino acid and an active form sequence of 233 amino acid. Antibodies against synthesized peptides corresponding to several parts of the protein indicated that the molecular weight of the protein is approximately 35 kD by SDSPAGE and Western blotting. Transfection study of the cloned cDNA into PC12 cells demonstrated that the cells can survive following serum withdrawal. We, therefore, named the factor as PCTF35. Less
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