『徳島大学 教育・研究者情報データベース (EDB)』---[学外] /
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種別 (必須): 学術論文 (審査論文) [継承]
言語 (必須): 英語 [継承]
招待 (推奨):
審査 (推奨):
カテゴリ (推奨):
共著種別 (推奨):
学究種別 (推奨):
組織 (推奨):
著者 (必須): 1. (英) Naganawa, Yasunori (日) (読)
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[継承]
2.伊藤 孝司 ([徳島大学.大学院医歯薬学研究部.薬学域.薬科学部門.統合医薬創製科学系.創薬生命工学])
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[継承]
3. (英) Shimmoto, Michie (日) (読)
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[継承]
4. (英) Kamei, Sachiko (日) (読)
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学籍番号 (推奨):
[継承]
5. (英) Takiguchi, Kyoko (日) (読)
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貢献度 (任意):
学籍番号 (推奨):
[継承]
6. (英) Doi, Hirofumi (日) (読)
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貢献度 (任意):
学籍番号 (推奨):
[継承]
7. (英) Sakuraba, Hitoshi (日) (読)
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貢献度 (任意):
学籍番号 (推奨):
[継承]
題名 (必須): (英) Stable expression of protective protein/cathepsin A-green fluorescent protein fusion genes in a fibroblastic cell line from a galactosialidosis patient.  (日)    [継承]
副題 (任意):
要約 (任意): (英) Fibroblastic cell lines derived from a galactosialidosis patient, stably expressing the chimaeric green fluorescent protein variant (EGFP) gene fused to the wild-type and mutant human lysosomal protective protein/cathepsin A (PPCA) cDNA, were first established as a model system for revealing the sorting and processing of lysosomal enzymes and for investigating the molecular bases of their deficiencies. In the cell line expressing the wild-type PPCA-EGFP chimaera gene (EGFP-PPwild), an 81 kDa form (27 kDa EGFP fused to the C-terminus of the 54 kDa PPCA precursor) was produced, then processed into the mature 32/20 kDa two-chain form free of the EGFP domain. The intracellular cathepsin A, alpha-N-acetylneuraminidase and beta-galactosidase activities, which are deficient in the parent fibroblastic cells, could also be significantly restored in the cells. In contrast with the uniform and strong fluorescence throughout the cytoplasm and nucleus in the mock-cell line expressing only EGFP cDNA, weak reticular and punctate fluorescence was distributed throughout the EGFP-PPwild cell line. Bafilomycin A1, a potent inhibitor of vacuolar ATPase and intracellular acidification, induced the distribution of Golgi-like perinuclear fluorescence throughout the living and fixed cells, in which only the 81 kDa product was detected. After removal of the agent, time-dependent transport of the chimaeric protein from the Golgi apparatus to the prelysosomal structure in living cells was monitored with a confocal laser scanning microscope system. Leupeptin caused the distribution of lysosome-like granular fluorescence throughout the cytoplasm in the fixed cells, although it was hardly observed in living cells. The latter agent also dose-dependently induced an increase in the intracellular amount of the 81 kDa product containing the EGFP domain and inhibited the restoration of cathepsin A activity in the EGFP-PPwild cells after the removal of bafilomycin A1. In parallel, both the mature two-chain form and PPCA function disappeared. These results suggested that the chimaera gene product was transported to acidic compartments (endosomes/lysosomes), where proteolytic processing of the PPCA precursor/zymogen, quenching of the fluorescence, and random degradation of the EGFP portion occurred. A cell line stably expressing a chimaeric gene with a mutant PPCA cDNA containing an A1184-->G (Y395C) mutation, commonly detected in Japanese severe early-infantile type of galactosialidosis patients, showed an endoplasmic reticulum (ER)-like reticular fluorescence pattern. The PPCA-immunoreactive gene product was hardly detected in this cell line. The mutant chimaeric product was suggested to be degraded rapidly in the ER before transport to post-ER compartments. A cell line expressing the chimaeric gene with a T746-->A (Y249N) PPCA mutation exhibited both ER-like reticular and granular fluorescence on the reticular structure that was stronger than that in the EGFP-PPwild cells. Some of them contained large fluorescent inclusion-body-like structures. The ineffectiveness of transport inhibitors in the distribution changes in the two mutant chimaeric proteins suggested that they were not delivered to acidic compartments. Therefore this expression system can possibly be applied to the direct analysis of the sorting defects of mutant gene products in living cells and will be useful for the molecular investigation of lysosomal diseases, including galactosialidosis.  (日)    [継承]
キーワード (推奨): 1. (英) Amino Acid Sequence (日) (読) [継承]
2. (英) Anti-Bacterial Agents (日) (読) [継承]
3. (英) Base Sequence (日) (読) [継承]
4. (英) Biological Transport (日) (読) [継承]
5. (英) Carboxypeptidases (日) (読) [継承]
6. (英) Cathepsin A (日) (読) [継承]
7. (英) Cell Line (日) (読) [継承]
8. (英) DNA, Complementary (日) (読) [継承]
9. (英) Fibroblasts (日) (読) [継承]
10. (英) Green Fluorescent Proteins (日) (読) [継承]
11. (英) Humans (日) (読) [継承]
12. (英) Leupeptins (日) (読) [継承]
13. (英) Luminescent Proteins (日) (読) [継承]
14. (英) Lysosomal Storage Diseases (日) (読) [継承]
15. (英) Lysosomes (日) (読) [継承]
16. (英) Macrolides (日) (読) [継承]
17. (英) Microscopy, Fluorescence (日) (読) [継承]
18. (英) Models, Biological (日) (読) [継承]
19. (英) Molecular Sequence Data (日) (読) [継承]
20. (英) Mutation (日) (読) [継承]
21. (英) Protein Processing, Post-Translational (日) (読) [継承]
22. (英) Recombinant Fusion Proteins (日) (読) [継承]
発行所 (推奨):
誌名 (必須): The Biochemical Journal ([The Biochemical Society])
(pISSN: 0264-6021, eISSN: 1470-8728)

ISSN (任意): 0264-6021
ISSN: 0264-6021 (pISSN: 0264-6021, eISSN: 1470-8728)
Title: The Biochemical journal
Title(ISO): Biochem J
Publisher: Portland Press, Ltd.
 (NLM Catalog  (Scopus  (CrossRef (Scopus information is found. [need login])
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(必須): 340 [継承]
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(必須): 467 474 [継承]
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年月日 (必須): 西暦 1999年 6月 1日 (平成 11年 6月 1日) [継承]
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PMID (任意): 10333491    (→Scopusで検索) [継承]
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備考 (任意): 1.(英) Article.Affiliation: Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113-8613, Japan.  (日)    [継承]
2.(英) Article.PublicationTypeList.PublicationType: Journal Article  (日)    [継承]
3.(英) Article.PublicationTypeList.PublicationType: Research Support, Non-U.S. Gov't  (日)    [継承]
4.(英) OtherID: PMC1220273  (日)    [継承]

標準的な表示

和文冊子 ● Yasunori Naganawa, Kouji Itou, Michie Shimmoto, Sachiko Kamei, Kyoko Takiguchi, Hirofumi Doi and Hitoshi Sakuraba : Stable expression of protective protein/cathepsin A-green fluorescent protein fusion genes in a fibroblastic cell line from a galactosialidosis patient., The Biochemical Journal, Vol.340, 467-474, 1999.
欧文冊子 ● Yasunori Naganawa, Kouji Itou, Michie Shimmoto, Sachiko Kamei, Kyoko Takiguchi, Hirofumi Doi and Hitoshi Sakuraba : Stable expression of protective protein/cathepsin A-green fluorescent protein fusion genes in a fibroblastic cell line from a galactosialidosis patient., The Biochemical Journal, Vol.340, 467-474, 1999.

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