| Project/Area Number |
15570125
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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 |
Functional biochemistry
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| Research Institution | Niigata University of Pharmacy and Applied life Sciences |
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Principal Investigator |
ICHIKAWA Shinichi Niigata University of Pharmacy and Applied Life Sciences, Department of Applied life Sciences, Associate Professor, 応用生命科学部, 助教授 (10223083)
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| Co-Investigator(Kenkyū-buntansha) |
NAGANO Michiyo Niigata University of Pharmacy and Applied Life Sciences, Department of Applied life Sciences, Assistant Professor, 応用生命科学部, 助手 (80350718)
HIRABAYASHI Yoshio The Institute of Chemical and Physical Research (RIKEN) Brain Research Institute, Unit Leader, 脳科学総合研究センター, ユニットリーダー (90106435)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | ceramide / glucosylceramide / apoptosis / hydrogen peroxide / プロモーター / アポートシス |
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| Research Abstract |
1. The promoter analysis of ceramide glucosyltransferase
In several cell lines, ceramide are believed to serves as second messenger. In the case of stress or DNA damage, intracellular ceramide level elevates and induces apoptosis. In the other hand, ceramide glucosyltransferase, GlcT-1 inactivates ceramide and inhibits apoptosis. Thus, we constructed the chimera gene of the promoter and luciferase and introduced into several animal cell lines. Using these cell lines we examined a variety of genotoxic agents. In B16 cells 2μM camptothcine increase the activity twice and 10μM etoposide increased 3,2 times. We also examined 40 kinds of plant extracts and identified two kinds of plant extract that suppress the promoter activity.
2. The functional analysis of GlcT-1 molecule by genetic engineering
GlcT-1 localizes in the Golgi membrane as well as its substrate ceramide. The enzyme is believed to binds to the membrane by the signal anchor sequence. To prove this hypothesis, we constructed cDNA lacking the sequence and introduced it to GlcT-1 deficient GM95 cells. The GlcT-1 lacking the signal anchor sequence did not show the activity, indicating the importance of the sequence. Localization of the mutant enzyme is now under examination.
3. Expression cloning of proteins which inhibit apoptosis
We tried to isolate genes that can inhibit ceramide induced apotosis using retroviral-mediate expression cloning. In the course of the study, we happened to isolate genes that inhibit hydrogen peroxide induced apoptosis. We infected mouse 7-day cDNA library to NIH/3T3 cells and selected with hydrogen peroxide. The cDNAs were recovered by PCR from the genomic DNA of the survived cells. We have isolated 11 genes and two were functionally unknown.
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