| Project/Area Number |
01571025
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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 |
Functional basic dentistry
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| Research Institution | Iwate Medical University |
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Principal Investigator |
SATO Nobuko Iwate Medical University School of Dentistry Department of Biochemistry Associate Professor, 歯学部・口腔生化学講座, 助教授 (00048399)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1990: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1989: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Mouse / Submandibular gland / Androgen / Chromosomal protein / Histone / Nonhistone protein / Phosphorylation / Protein kinase / プロテインカイネ-ス |
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| Research Abstract |
1 Intranuclear distribution of androgen receptor binding sites Nuclei from mouse submandibular gland after administration of testosterone propionate were digested with micrococcal nuclease and resulting active chromatin fraction was analyzed by glycerol gradient centrifugation. Endogenous androgen receptor complexes associated with chromatin binding sites were detected by an in vitro exchange assay using a [ ^3H]mibolerone (synthetic androgen) as a ligand. The level of the androgen receptor complex in dinucleosomes increased 1 h after the testosterone treatment and the androgen level in the mono- and trinucleosomes increased 3 h after the testosterone treatment ; indicating that the androgen receptor associated with chromatin and this association occurred in transcriptionally active chromatin regions which were preferentially sensitive to micrococcal nuclease.
2 Phosphorylation of chromosomal proteins in nuclear fraction and active chromatin fraction
Female mice were injected with testos
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terone propionate. Nuclear and active chromatin fractions were prepared from submandibular glands and incubated with [gamma-^<32>P]ATP. The nuclear fraction rapidly incorporated ^<32>P from [gamma-^<32>P]ATP Bin vitro into endogenous chromosomal proteins. Testosterone resulted in an increase in the rate of phosphorylation at 3 h and reduced to the control level at 6 h ; indicating that nuclear-associated proteins were phosphorylated by protein kinase intrinsic to nuclei. The level of phosphorylation in the active chromat in fraction increased 3 h after the testosterone treatment and this stimulation was similar to that of ^<32>P incorporation into nuclear phosphoproteins ; suggesting that the protein kinase which was sensitive to androgen was present in the active chromatin fraction.
3 Activities of protein kinase in nuclear and active chromatin fractions
The activities of protein kinase were measured using histone as a substrate. The activities of protein kinase in the unclear fraction stimulated at 3 h after testosterone treatment and a similar effect of androgen was observed in the active chromatin fraction ; indicating the androgenic response of phosphorylation of active chromatin-associated proteins.
4 SDS-Page analysis of phosphorylated nuclear proteins
Nonhistone proteins were isolated from phosphorylated nuclear proteins and separated by SDS-Page followed by autoradiography. Autoradiograms of ^<32>P labeled proteins showed phosphorylated nonhistone proteins bands with molecular mass ranging from 20 KD to 40 KD. Less
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