Project/Area Number |
10557253
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Research Category |
Grant-in-Aid for Scientific Research (B).
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Allocation Type | Single-year Grants |
Section | 展開研究 |
Research Field |
Laboratory medicine
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Research Institution | KANSAI MEDICAL UNIVERSITY |
Principal Investigator |
TAKAHASHI Hakuo KANSAI MEDICAL UNIVERSITY, FACULTY OF MEDICINE, PROFESSOR, 医学部, 教授 (80094431)
|
Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Kouji SEKISUI CHEMICALS CO.LTD., MEDICAL INSTITUTION, SENIOR INVESTIGATOR, メディカル研究所, 副主任研究員
MASUDA Midori KANSAI MEDICAL UNIVERSITY, FACULTY OF MEDICINE, ASSOCIATE PROFESSOR, 医学部, 講師 (50173753)
KOMIYAMA Yutaka KANSAI MEDICAL UNIVERSITY, FACULTY OF MEDICINE, ASSOCIATE PROFESSOR, 医学部, 講師 (40140264)
|
Project Period (FY) |
1998 – 2000
|
Project Status |
Completed (Fiscal Year 2000)
|
Budget Amount *help |
¥5,800,000 (Direct Cost: ¥5,800,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1998: ¥2,500,000 (Direct Cost: ¥2,500,000)
|
Keywords | ENDOGENOUS DIGITALIS / OUABAIN / CULTURED CELLS / ADRENAL MEDULLA / NEURONAL TISSUE / PROGESTERONE / MASS SPECTROMETRY |
Research Abstract |
Ouabainlike factor (OLF) has been suggested to be a steroid, and may be produced in adrenals. Thereby, progesterone could be a substrate for the OLF.However, there is no evidence that OLF is endogenously produced in mammalian cells. We have found Ouabainlike immunoreactivity in the adrenal medulla by immunohistochemistry, the pheochromocytoma tissue, adrenal cortex and medulla by enzyme-linked immunosorbent assay for ouabain. Since PC12 cells are of rat pheochromocytoma origin, we explored the possible production of OLF by using cultured PC12 cells by adding progesterone in the culture medium. Progesterone increased Ouabainlike immunoreactivity in the culture medium of PC12 cells dose-dependently. OLI in the culture medium was purified by 5 steps of high-performance liquid chromatography (HPLC). The final fraction of OLI was applied to HPLC/mass spectrometry (LC/MS) to analyze the molecular structure, and the OLI was found to be identical to authentic ouabain. This is the first direct evidence to show that mammalian cells produce ouabain, identical to that of plant origin, because the culture system is apparently out of contamination of ouabain of plant origin. It may be a novel finding that cells of neuronal origin can produce biologically active steroids. Furthermore, we are trying to develop a sensitive and selective assay method by using LC/MS with a selected ion mode. So far, it became possible to measure OLF and a digoxinlike factor from 5ml of plasma. To explore the possible existence of other digitalislike factors, we are developing a novel biological assay system for digitalislike bioactivity by measuring intracellular calcium ions in endothelial cells.
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