| Project/Area Number |
58870052
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
Neurology
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| Research Institution | The University of Tokyo |
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Principal Investigator |
INOUE Kiyoharu (1985) 東京大学, 医, 講師 (80107664)
豊倉 康夫 (1983-1984) 東京大学, 医学部, 教授
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| Project Period (FY) |
1983 – 1985
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| Project Status |
Completed (Fiscal Year 1985)
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| Budget Amount *help |
¥13,000,000 (Direct Cost: ¥13,000,000)
Fiscal Year 1985: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1984: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1983: ¥10,000,000 (Direct Cost: ¥10,000,000)
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| Keywords | Alzheimer's disease / Paired helical filament / monoclonal antibody / amino acid sequence / イソペプチド結合 |
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| Research Abstract |
Paired helical filaments (PHF) are unusual neuronal fibers which accumulate progressivily in Alzheimer's disease (AD) brain. The insolubility of PHF in various kinds of solvents enabled us to obtain highly purified PHF, but prevented the application of conventional analytical methods to identify their components. Here we report immunochemical identification of a possible precursor of PHF, using a monoclonal antibody. We obtained one hybridoma secreting antibody to PHF (DF-1). DF-1 is considered to bind to PHF themselves from following observations. First, DF-1 stained neurofibrillary tangles isolated by extensive extraction in SDS. Second, electron microscopy showed that immunogold-conjugated DF-1 specifically decorated PHF. By immunoblotting, DF-1 was found to label a lowmolecular weight polypeptide at <M_r> [5000 (p5) in soluble fractions of both normal and AD brains. This indicates that a certain portion of PHF and p5 share a common antigenic determinant, possibly the same or homologous aminoacid sequence. To investigate further the molecular properties of p5 and so PHF, we purified p5 from soluble fractions of normal brains. Purified p5 was analyzed by a gas-phase protein sequencer. As a result amino acid sequence was determined from N terminal to position 34. A computer search of this sequence revealed that it is identical to that of ubiquitin. Ubiquitin is known to have an important role in cytoplasmic protein degradation. Once ubiquitin is covalently linked to a target protein at the expense of ATP, this marked protein is rapidly degraded with an unidentified protease also in a ATP dependent manner. Currently we do not know the reason why ubiquitin is concentrated on and tightly bound to PHF. Cytoskeletal proteins in degenerating neurons might be ubiquitinated and linked to the framework of PHF.
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