2003 Fiscal Year Final Research Report Summary
Analysis of autophagy-related gene products during pexophagy in hepatocytes
| Project/Area Number |
14580693
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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 |
Cell biology
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| Research Institution | University of Yamanashi (2003)
山梨医科大学 (2002) |
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Principal Investigator |
YOKOTA Sadaki University of Yamanashi, Interdisciplinary Graduate School of Medicine and Engineering, Professor, 大学院・医学工学総合研究部, 教授 (40020755)
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| Project Period (FY) |
2002 – 2003
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| Keywords | Pexophagy / Apg proteins / autophagy / proliferated peroxisomes / hepatocytes / immunocytochemistry |
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| Research Abstract |
In previous studies, we suggested that excess peroxisomes are removed by autophagy specific for the proliferated peroxisomes which were isolated by endoplasmic reticulum (ER) membrane. Then, in this study, we investigated the early event in autophagic process, in which the proliferated peroxisomes were isolated and enclosed, using immunocytochemical and immunoblotting techniques. We prepared peptide antibodies against Apg 5, LC3 and Apg 12. Rat liver peroxisomes were proliferated with-di-(2-ethylhexyl) phthalate (DEHP) and the animals were treated with leupeptin for 30 min to 120 min. By immunofluorescence microscopy, these three antigens showed similar behavior. They were detected in small vesicles located around bile canaliculi 30 min after leupeptin treatment. Afterwards the positive granules increased gradually in the number and size. Double staining with catalase and Apg proteins showed close association of Apg proteins and peroxisomes. No ER staining was observed. Primary cultured-hepatocytes isolated from peroxisome-proliferated rats were cultured for 2 -4 days in presence of absence of 3-methyladenine and then the cells were doubly stained for catalase and Apg proteins. The close association of Apg proteins with peroxisomes were observed but no ER staining was noted. The results suggest that Apg 5, LC3 and Apg 12 are not concerned with the ER membrane, but with other membranes which form the isolation membrane.
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