| Project/Area Number |
11672278
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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 |
応用薬理学・医療系薬学
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| Research Institution | Showa Pharmaceutical University |
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Principal Investigator |
UTSUNOMIYA Iku Showa Pharmaceutical Universit, Department of Neuroscience, Assistant Professor, 薬学部, 講師 (70168722)
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| Co-Investigator(Kenkyū-buntansha) |
MIYATAKE Tadashi Showa Pharmaceutical Universit, University, Department of Neuroscience, Professor, 薬学部, 教授 (50048998)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Neurotrophic factor / Motor neuron / Acetylcholine receptor / Hybridoma / Gangliosides / CNTF / GM2 / IL-6 receptor |
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| Research Abstract |
To develop pharmacotherapy for motor neuron disease, we examined whether ciliary neurotrophic factor (CNTF) can alter serum-free cell survival of immortalized motor neuron-1ike cells, which were established by fusing mouse neuroblastoma N18TG2 with mouse motor neurons. One of the cell lines, NSC-34 exhibited cell survival in the presence of CNTF. NSC-34 preserves the most characteristics of motor neurons, such as the formation of neuromuscular junctions on co-cultured myotube. GM2 ganglioside is characteristic of motor neurons, and expressed highly in NSC-34. When NSC-34 was cultured with exogenous GM2 and CNTF, GM2 facilitated the cell survival effect of CNTF. In addition, GM2 synthase activity was enhanced up to 3.9-fold by culture in the presence of CNTF. Then we examined whether CNTF-induced cell survival is associated with the collaboration between GM2 and the CNTF receptor (CNTF-R). Despite the presence of CNTF (50 ng/ml), anti-CNTF-R antibody caused cell death and prevented the up-regulation of GM2 synthase expression. The addition of GM2 (1 to 20 μM) abrogated the anti-CNTF-R antibody effect which shortened cell survival and blocked GM2 synthase activation. Use of [<125>^I] CNTF showed the specificity of CNTF binding in NSC-34 cells in situ. GM2 produced a 5-fold increase in the CNTF binding affinity per cell but did not change the binding site number. The study by metabolic labeling with [1-<14>^C]N-acetyl-D-galactosamine ([<14>^C]GalNAc) showed that biosynthesized GM2 was involved in the immunoprecipitation of CNTF-R. These findings indicate that up-regulated GM2 synthesis induces functional conversion of CNTF-R to the activated state, in which it has high affinity for CNTF. We conclude that GM2 is a bio-regulating molecule of CNTF-R in motor neurons.
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