| Project/Area Number |
10470185
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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 |
Dermatology
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| Research Institution | Hirosaki University |
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Principal Investigator |
TAMAI Katsuto Hirosaki University School of Medicine, Dermatology, Associate Professor, 医学部, 助教授 (20236730)
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| Co-Investigator(Kenkyū-buntansha) |
KON Atsushi Hirosaki University School of Medicine, Dermatology, Assistant Professor, 医学部附属病院, 講師 (60271798)
澤村 大輔 弘前大学, 医学部・附属病院, 講師 (60196334)
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| Project Period (FY) |
1998 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥14,000,000 (Direct Cost: ¥14,000,000)
Fiscal Year 2001: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2000: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1998: ¥5,600,000 (Direct Cost: ¥5,600,000)
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| Keywords | Keratinocytes / POU domain transcriptional factor / Skn-1n / Octanmer motif / in situ RT-PCR / POU ドメインファクター / RT-PCR / in situ / skn-1a / NLS / skn1a / 類天疱瘡抗原 / sknla |
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| Research Abstract |
1) Expression pattern of skn-1a in the normal and psoriatic skin.
Skn-1a expression pattern in the normal and psoriatic epidermis was evaluated by immunohistochemistry using skn-1a monoclonal antibody. In the normal human epidermis, skn-1a was stained mainly in the cytosol of the basal keratinocytes. In the suprabasal layers of the epidermis, skn-1a accumulated clearly in the nuclei, indicating differentiation-specific nuclear translocation of skn-1a. In the psoriatic skin, however, the epidermis of the elongated rate ridge showed sustained cytosolic staining of skn-1a, and only very upper layers revealed nuclear translocation of the skn-1a, suggesting aberrant signal transduction affecting nuclear translocation of the skn-1a in the psoriatic skin.
2) Molecular mechanism of nuclear translocation of skn-1a.
Computer search identified canonical nuclear localization signal (NLS) in the primary amino-acid sequence of skn-1a. Transient transfection studies with expression vector of the NLS-GFP
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fusion protein revealed accumulation of the fusion protein in the nuclei of cultured normal human epidermal keratinocytes (NHEK), indicating active function of the NLS in the skn-1a. Site-directed mutagenesis studies indicated that serine and threonine residues locating just beside the skn-1a NLS at C-terminal side function to regulate NLS activity. Substitution of both residues to alanine accelerates nuclear translocation, suggesting that phosphorylation/dephosphorylation regulation of the serine and/or threonine residues precisely regulates skn-1a NLS activity.
3) cDNA and genomic DNA cloning of human skn-1a.
For the characterization of exon-intron structure and regulatory region of human skn-1a gene, cDNA cloning and 5'RACE were performed. In the course of this study, novel splice variant of skn-1a, which contains an additional exon in the intervening sequence between exon 1 and exon 2 of skn-1a, was isolated, and designated as skn-1n. RT-PCR studies indicate actual expression of skn-1n in cultured normal human epidermal keratinocytes.
4) Expression analysis of skn-1a and skn-1n in vitro and in vivo.
Elevation of calcium concentration from 0.03 mM to 2.0 mM in culture media dose-and time-dependently down-regulated mRNA expression of both skn-1a and skn-1n in cultured normal human epidermal keratinocytes. At 2.0 mM calcium concentration, about 90% of the mRNA inhibition was observed by RT-PCR. In situ RT-PCR study exhibited skn-1n mRNA expression from basal to mid-spinous layers of the normal human epidermis, whereas whole epidermis showed skn-1a mRNA, suggesting differential splice regulation of skn-1a and skn-1n in vivo. Less
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