| Project/Area Number |
16590643
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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 |
Gastroenterology
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| Research Institution | KANAZAWA MEDICAL UNIVERSITY |
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Principal Investigator |
DATE Takayasu KANAZAWA MEDICAL UNIVERSITY, Department of Medicine, Professor, 医学部, 教授 (50019676)
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| Co-Investigator(Kenkyū-buntansha) |
KAWAHARA Hiroshi KANAZAWA MEDICAL UNIVERSITY, Department of Medicine, Associate Professor, 医学部, 助教授 (10177727)
MATSUI Tadashi KANAZAWA MEDICAL UNIVERSITY, Department of Medicine, Assistant Researcher, 医学部, 助手 (60288272)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | RNA interference (RNAi) / double-stranded RNA (dsRNA) / dsRNA-activated protein kinase(PKR) / eIF2alpha / Hepatitis Cvirus (HCV) / Japanese encephalitis virus (JEV) / core-protein / HepG2 / dsRNA依存性プロテインキナーゼ / cIF2-α / HCV / リン酸化 / Coreタンパク質 / RNA干渉(siRNA) / RNA / アポトーシス / NS3 / タンパク質合成 |
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| Research Abstract |
1)Recombinant His-tagged PKR was prepared as a non-phosphorylated form and characterized. PKR bound double-stranded (ds) RNA with less than 26 nucleotides in length. However, PER was not autophosphorylated (activated) by such short dsRNA in vitro. Autophosphorylation of PKR requires dsRNA with longer than 31 nucleotides in length in vitro. Activation of PKR by long dsRNA was inhibited by short dsRNA (21.23 nucleotides in length)
2)Partial activation of cellular PKR in HepG2 cells was observed by Western blot analysis using antibody raised to phosphothreonine at 446. It may be induced by intrinsic dsRNA. When shRNA-expression plasmid was introduced, PKR was strongly suppressed, thereby, resulting in the inhibition of phosphorylation of the alpha subunit of eukaryotic translation factor. (eIF2α). The sequence of short dsRNA generated from shRNA had a random sequence and no target for cellular mRNA.The rate of protein synthesis was increased several times by luciferase reporter assay.
3)In order to investigate the effect of short dsRNA on RNA virus production, we examined whether Japanese encephalitis virus (JEV), a model HCV virus, was suppressed in shRNA-expressing cells. Activation of PKR was observed in shRNA expressing cells in the same extent as observed in control cells. Levels of phosphorylation of eIF2α and expression of viral envelope protein were almost the same as that in control cells. Therefore, inactivation of PKR by short dsRNA was not found in the virulent conditions.
4)The effect of the hepatitis C virus (HCV)-core protein on RNA interference was examined in HepG2 cells using luciferase-expression plasmid and its siRNAs. The core protein slightly enhanced the RNAi effect compared with other HCV RNA-binding proteins, NS3 and NS5A proteins. However, the effect was very small and was independent of RNA binding activity of the core protein.
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