| Project/Area Number |
60570526
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
内分泌・代謝学
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| Research Institution | The Research Institute of Environmental Medicine, Nagoya University |
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Principal Investigator |
MATSUI Nobuo The Research Institute of Environmental Medicine, Nagoya University, 環境医学研究所, 教授 (50023643)
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| Co-Investigator(Kenkyū-buntansha) |
MURATA Yoshiharu The Research Institute of Environmeental Medicine, Nagaya University, 環境医学研究所, 助手 (80174308)
SEO Hisao The Research Institute of Environmental Medicine, Nagoya University, 環境医学研究所, 助教授 (40135380)
KAMBE Fukushi The Research Institute of Environmental Medicine, Nagoya University (00211871)
SUEDA Kaori The Department of Home-economics, Nagoya Women's University
OGAWA Katsuhito Internal Medicine, National Toyohashi Hospital
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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 |
¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1987: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1986: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1985: ¥600,000 (Direct Cost: ¥600,000)
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| Keywords | Transcortin / Cell-free translation / Role of glycosylation / Solid phase RIA / Secretion of corticosterone / Micreheterogeneity of glucoprotein / 分子進化 / トランスコルチンの糖鎖 / 糖鎖付加阻害 / 糖鎖と分泌 / 糖鎖と分解 / ヒトトランスコルチン / cDNA |
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| Research Abstract |
1) Studies on the biosynthetic processes of human transcortin (hTr). Poly (A)-RNA extracted from human liver was translated employing rabbit reticulocyte lysate. hTr was identified in the translation products by binding to its specific antiserum and displacement by hTr purified from plasma. The translated Tr, pre-transcortin was 45.7kd and was able to bind to cortisol. Biosynthetic process of hTr was studied using a human hepatoma cell line, HepG2. Cells were pulse labeled and chased, and Tr in cells and media were analyzed by SDS-PAGE. Tr appeared in the cells as 56kd Tr at first, thereafter it decreased during chase while 69kd Tr increased in the media with time. In the presence of Tunicamycin, an inhibitor of core glycosylation, only 40kd Tr was accumulated in both cells and media. These results indicate that Tr is synthesized as 40kd polypeptide and 2 steps of glycosylation complete its molecule, and that glycosylation is snot indispensable for the secretion of Tr.
2) Cloning of hTr
… More
-cDNA. We tried to obtain hTr-cDNA from HepG2 cDNA library. cDNAs prepared from reverse transcription of HepG2 mRNA were inserted into Pst-I site of pBR 322. Host E.coli was transformed by the cDNA library. Among 5,000 recombinant colonies, however, no Tr-positive colony was found. In the next step, expression cDNA library of human liver constructed in gt11 bacteriophage was utilized. Host bacteria was infected by the phage and 4 Tr-positive clones were obtained. Nucleotide sequence was analyzed for one of the positive clones, however, the sequence homology to the reported one was not detected. Analysis of nucleotide sequence of 3 other clones is under way.
3) Others: A simple RIA method to determine hTr concentration using solid phase antibody method was established. Influence of transcortin on secretion of corticosteroids from adrenal cells was studied using monolayer cultured rat adrenal cells. Additon of hTr in the medium did not significantly increase corticosterone concentration. Biochemical, physiological and immunological properties of rat Tr were investigated. Evolutional changes of Tr were studied in primate and some vertebrates. Less
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