| Project/Area Number |
13480297
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biomedical engineering/Biological material science
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| Research Institution | Ehime University |
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Principal Investigator |
TAMURA Minoru Ehime University, Fac.Engineer., Assoc.Prof., 工学部, 助教授 (00128349)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAMURA Yoichi Osaka Prefectural University, Fac.Agriculture, Prof., 農学部, 教授 (90180413)
YOSHIDA Satoshi Gifu University, Fac.Engineer., Prof., 工学部, 教授 (50158440)
MIURA Satoshi Yokohama City University, Fac.Medicine, Assoc.Prof., 医学部, 助教授 (60157427)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥12,400,000 (Direct Cost: ¥12,400,000)
Fiscal Year 2002: ¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 2001: ¥6,500,000 (Direct Cost: ¥6,500,000)
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| Keywords | Superoxide / Reactive oxygen / NADPH oxidase / Cytochrome b_<558> / Noxl Duox family / Neutrophils / Fusion Proteins / Small GTPase / 低分子量Gタンパク / Superoxide / Reactive oxygen / Cytochrome b_<558> / duox family / Neutrophilts / Fusion protein / Small GTPase / Reactive oxygen species / Phox / Nox family / Neutrophils / Small G-protein |
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| Research Abstract |
1) Establishment of a new O_2^- generating device
We created a new-type O_2^- generating nano-machine by improving the regulatory components of neutrophil NADPH oxidase. A fusion protein between p47N and p67N was genetically engineered and used in a cell-free reconstitution in combination with RacQ61L, a constitutively active form of Rac, and found that this method produced a extremely stabilized enzyme. Furthermore, we succeeded to activate the oxidase without a stimulator SDS by optimizing the lipid composition for relipidation of cytochrome b_<558>. When the enzyme was activated with high concentrations of protein components and frozen quickly, the formed complex was maintained in storage and showed a full activity after thawing.
2) Application of the new device to cell experiments
When the device was added to HEK cells which has often been used for experiments of amyloid formation, a characteristic of Alzheimer's disease, proliferation of the cells stopped at 1 : 5000 dilution of the device solution and cell death took place at 1 : 500dilution of the device. Such phenomena was not observed when superoxide dismutase was added, indicating that they were caused by O_2^-. On the other hand, a neuron cell line was more resistant to O_2^-, but when added at a higher concentration of the device cells started to die.
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