| 研究課題/領域番号 |
18K14520
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| 研究機関 | 愛媛大学 |
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研究代表者 |
Mohapatra Sipra 愛媛大学, 南予水産研究センター, 助教(特定教員) (80715441)
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| 研究期間 (年度) |
2018-04-01 – 2021-03-31
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| キーワード | autophagy / Hexokinase / Red Sea bream |
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| 研究実績の概要 |
I have scouted the Red Sea bream genome and identified the probable sequences of four hexokinases, i.e. hexokinase (HK) I, HKII, HKIII and HKIV. All these gene share a common core sequences representing mammalian and other fish. I have isolated the complete HK1 sequence and partial sequences of remaining three hexokinases and importantly found one alternatively spliced sequence of HK1 (hereafter named as HK1b). Tissue distribution analysis shows highest expression of HK1a and HK1b in liver followed by brain and gonad. Both these HK1 isoforms localizes in the liver hepatocytes. In vitro and ex vivo experiments using Red Sea bream liver cells shows that glucose and serum starvation induces HK1 expression and further enhances autophagy. The conditions were further aggravated by HK1 over expression suggests HK1, at least, is very important for autophagy initiation in Red Sea bream.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
The project is slightly delayed due to child birth. Since November, 2018. I went to maternity and child care holiday. I will rejoin from June, 2019 and restart my experiments.
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| 今後の研究の推進方策 |
I will isolate the remaining HKII, HKIII and HKIV and check their tissue distribution using PCR. I will analyse the cellular localization of all Hexokinase isoforms in liver by in situ hybridization and immunohistochemistry (using commercially available antibody).
Meanwhile I will develop a primary cell culture of red sea bream hepatocytes and analyze the Hexokinases modulation under both glucose and serum starved condition. The data will be gathered using live microscopy and realtime PCR. I will also check the autophagic status of various treatment groups and corelate with hexokinase modulation.Later I will overexpress and knockdown candidate hexokinase and check the autophagy modulation invitro, using realtime PCR, microscopy and immunohistochemistry and decipher the hexokinase associated molecular cascade in red sea bream. If necessary other fish cell lines or NGS will be performed to fast track the experiment.
The promoter sequence of candidate hexokinase will be isolated and its effects on autophagy induction will be ascertain invitro using mammalian cell lines, luciferase assay and realtime PCR. Later the promoter overexpressed red sea bream hepatocytes will be analyzed using next generation sequencing and Chromatin immunoprecipitation and compared with control to further characterized the autophagy cascade. In separate invivo experiments, I will perform a fasting and E. tarda challenge experiment using both medaka and red sea bream, and compare the autophagy induction and hexokinase association to determine the common autophagy pathway in fish.
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| 次年度使用額が生じた理由 |
I am currently in maternity and child care leave since November 2018. So I stopped the research and did not use the money. I planned to finish the planed work after restart the work from June 2019.
Briefly, I will isolate the remaining HKII, HKIII and HKIV and check their tissue distribution using PCR. I will also analyse the cellular localization of all Hexokinase isoforms in liver by in situ hybridization and immunohistochemistry. I will overexpress and knockdown candidate hexokinase and check the autophagy modulation in vitro and with the help of NGS and other analysis I will determine the autophagy pathway. In separate invivo experiments, I will perform a fasting and E. tarda challenge experiment using both medaka and red sea bream, and compare the autophagy induction and hexokinase association to determine the common autophagy pathway in fish.
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