| Project/Area Number |
60870013
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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 |
General medical chemistry
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| Research Institution | Osaka University |
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Principal Investigator |
TAKIGAWA Masaharu Department of Bichemistyr, Faculty of Dentistry, 歯学部, 講師 (20112063)
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| Co-Investigator(Kenkyū-buntansha) |
SHIRAI Eiji Deparment of Biochemistry, Faculty of Dentistry, 歯学部, 助手 (70171030)
TAKANO Teruko Department of Orthodontics, Faculty of Dentistry, 歯学部, 助手 (00127250)
SUZUKI Jujio Department of Biochemistyr, Faculty of Dentistry, 歯学部, 教授 (40028717)
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| Project Period (FY) |
1985 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1987: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1986: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1985: ¥3,000,000 (Direct Cost: ¥3,000,000)
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| Keywords | Parathyoid Hormone / Bioassay / Otnithine Devatboxylase / Cyclic AMP / Cultured Chondcocyted / 樹立細胞株 / 軟骨培養細胞株 / 軟骨細胞 |
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| Research Abstract |
1. Bioassay for parathyrois hormone (PTH) using rabbit costal growth cartilage (RGC) celle in culture. A simple procedure of bioassay of PTH using ornithine decarboxylase induction obsarved 4 hr after PTH addition was developed. In this method, RGC cells in primary cultyres were suspended in hypotonic standard buffer and directely incubated wirh^<14>Cornithine. The simi-log dose-dependent curve using PTH preparation of MRC research Standard A was linear between 0.01 to 1 IU/ml. The actibeties of various synthetic analogs and framents of PTH determined by this method almost corresponded to those obtained by in vivo assay and renal afenylate cyclase assat.
The stimulation of intracelluar cyclic AMP (cAMP) level obsearved 2-5 min after the addition of PTH was also found to be used as a new system fro the bioassay of PTH. Its semi-log dose-dependent curve was linear bvetween 10^<-9>M and 10^<-7>M. The activities of various synthetic analogs and fragments of PTH determined by this method als
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o corresponded to those obtained by other assay. The change in cAMP level after PTH addition was too fast to be used as a procedure by which many samples were assayed at the same time, byt this problem was solved using isobutylmethylxamthine which maintained the PTH-stimulated cAMP level at least for 30 min.
2. Establishment of a PTH-responsive clonal cell line from mouse growth cartilage. A clonal cell line that respond to PTH was isolated from a cell line established from secondary cultures of mouse growth cartilage. The change in the level of cAMP after addition of PTH to this clonal line was similar to that in primary cultures of RGC cells. The dose response vurce of change in the cAm@p level was also similat to that in RGC cells. However, ther extent of stimulation in the clonal cells was graster than that in RGC cells. The clonal cell line is now in passage 50 and is thought to be immortalized. Therefore, this cell line will be very useful for a simple, easy and time-sdabvign bioassay of PTH. Less
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