| Project/Area Number |
14370309
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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 |
Hematology
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| Research Institution | Saitama Medical School |
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Principal Investigator |
HIGASHIO Kanji Saitama Medical School, Associate professor, 医学部, 助教授 (20337603)
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| Co-Investigator(Kenkyū-buntansha) |
GOTO Masaaki Chugai Pharmaceutical Co. Ltd., Researcher, 創薬企画推進部, 研究員
SHIMA Nobuyuki Saitama Medical School, Assistant Professor, 医学部, 講師 (30337604)
SUDA Tatsuo Saitama Medical School, Professor, 医学部, 教授 (90014034)
TOMOYASU Akihiro Chugai Pharmaceutical Co. Ltd., Researcher, 創薬企画推進部, 研究員
KODAIRA Kunihiko Chugai Pharmaceutical Co. Ltd., Researcher, 創薬企画推進部, 研究員
後藤 雅行 中外製薬株式会社, 創薬企画推進部, 研究員
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥12,800,000 (Direct Cost: ¥12,800,000)
Fiscal Year 2003: ¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 2002: ¥6,800,000 (Direct Cost: ¥6,800,000)
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| Keywords | megakaryocytes / proplatelet_formation / PPF commitment factor, PCF / human fibroblasts / platelet / ceramic culture system / ubiquitin / ユビキチン / 胞体突起形成 / proplatelet formation (PPF) |
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| Research Abstract |
Proplatelet formation (PPF) of megakaryocytes (MKs) is an essential process to release platelets from MKs. To investigate the existence of PPF commitment factor(s), PCF, that is involved in differentiation from MKs to MKs PPF, we constructed a screening system composed of two assay steps, and searched such factor(s). UT-7/TPO cell line was preincubated in 96-well plates in the presence of 5 ng/ml TPO and test sample for 48 hr (1^<st> step). The cell suspension (10 μL) was added to 100 υ1 of DMEM containing 10% human plasma and 10 ng/ml TPO in 96 well plates coated with type I collagen. After incubation for 2hr. the numbers of MKs PPF were counted (2^<nd> step). Three PCF-like active fractions (PCF-I, II, III) were detected in heparin chromatography of the conditioned medium of human fibroblast, IMR-90 cells, cultivated by ceramic culture system. These active fractions were shifted into one component, PCF-III further purification. PCF-III protease-sensitive small molecule, and was effectively purified from dialytic fraction (<MW. 8,000) of the eluate from heparin chromatography. The specific activity of PCF-III was quite high, but PCF-III did not exert its activity at higher concentrations. PCF-III purified from 470 L culture medium was subjected to SDS-PAGE under reducing conditions. PCF-III band was in-gel digested with trypsin, and amino acid sequences of the liberated peptide fragments were determined. PCF-III was identified as human ubiquitin. Commercially available bovine ubiquitin and r human His-ubiquitin were found to exert the same activity as that of PCF-III. The treatment of these ubiquitin with trypsin abolished their activity completely. However, the mechanisms by which intracellular protein, ubiquitin, acts on MKs and stimulates MKs PPF, and its physiological significance remain to be elucidated.
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