| Project/Area Number |
17591946
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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 basic dentistry
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| Research Institution | Health Sciences University of Hokkaido, |
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Principal Investigator |
TOJYO Yosuke Health Sciences University of Hokkaido, School of Dentistry, Professor, 歯学部, 教授 (90111731)
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| Co-Investigator(Kenkyū-buntansha) |
TANIMURA Akihiko Health Sciences University of Hokkaido, School of Dentistry, Associate Professor, 歯学部, 准教授 (70217149)
MORITA Takao Health Sciences University of Hokkaido, School of Dentistry, Instructor, 歯学部, 助教 (20326549)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | salivary glands / calcium ion / inositol 1, 4, 5-trisphosphate / calcium signal / fluorescent imaging / GFP / FRET / LIBRA |
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| Research Abstract |
1. The Ca^<2+> signaling in salivary ductal cells was visualized by multi-photon microscopy. The results indicated that ducts contain a certain subpopulation of cells, which exhibits particularly high sensitivity to these Ca^<2+>-mobilizing agonists. Also, low concentrations of phenylephrine, carbachol and ATP activated different subpopulations of ductal cells.
2. To analyze IP_3 production in salivary cells, variants of the FRET-based biosensor LIBRA were constructed. To obtain the pH-stable LIBRA, we replaced the FRET acceptor EYFP to Venus, a more bright and pH-stable mutant. Next, we optimized the linkers between ECFP and IP_3-binding domain and between Venus and IP_3-binding domain. Furthermore, we replaced Venus and ECFP to their circularly permuted types.
3. We expressed the variants of LIBRA to cultured cells and measured the changes in LIBRA fluorescence in saponin- permeabilized cells. Using the improved LIBRA, change in the ratio increased to about 140%.
4. Dispersed rat parotid acinar cells were prepared by enzymatic digestion and cultured in DMEM-F12 medium. At 6 h after dispersion, the cultured acinar cells showed a similar morphology to that of the cells immediately after dispersion. The polarity seemed to maintain at least for 6 h. At 48 h after dispersion, the polarity was lost in most of the cells, but some cells maintained it. Intracellular Ca^<2+> concentrations ([Ca^<2+>]i) were measured in fura-2-loaded cells. Stimulation with agonists induced significant increases in [Ca^<2+>]i even in the cells cultured for 48 h.
5. Dispersed rat parotid acinar cells were transfected with cDNA for GFP and PKC-GFP. The optimal condition for tranfection was examined using various reagents and medium. The used of Lipofectoamine 2000 and Opti MEM was most effective for tranfection. Stimulation with ionomycin induced translocation of PKC-GFP from cytoplasm to plasma membrane.
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