| Project/Area Number |
10470150
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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 |
Respiratory organ internal medicine
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| Research Institution | Tohoku University |
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Principal Investigator |
NUKIWA Toshihiro ROMM, IDAC, Tohoku University, Professor, 加齢医学研究所, 教授 (40129036)
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| Co-Investigator(Kenkyū-buntansha) |
EBINA Masahito ROMM, IDAC, Tohoku University, Assistant Professor, 加齢医学研究所, 助手 (10280885)
HAGIWARA Kohichi ROMM, IDAC, Tohoku University, Senior Assistant Professor, 加齢医学研究所, 講師 (00240705)
ABE Tatsuya ROMM, IDAC, Tohoku University, Senior Assistant Professor, 加齢医学研究所, 講師 (70222651)
MIYAZAKI Junichi Molecular Applied Medicine, Osaka University, Professor, 大学院・医学系研究科, 教授 (10200156)
MIKI Makoto ROMM, IDAC, Tohoku University, Assistant Professor, 加齢医学研究所, 助手 (30312656)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥13,100,000 (Direct Cost: ¥13,100,000)
Fiscal Year 1999: ¥7,300,000 (Direct Cost: ¥7,300,000)
Fiscal Year 1998: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Keywords | protease inhibitor / SLPI / elafin / targeting / airway defense system / WAP motif / mouse EST database / multi-disulfide bridge structure / 気道防御 |
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| Research Abstract |
While SLPI (secretory leukoprotease inhibitor) was found as a protease inhibitor (PI) produced in respiratory tracts or reproductive organs, SLPI belongs to the proteins that have characteristic WAP (whey acidic protein) motif of multi-disulfide bridge. The aim of this study is to clarify the physiologic role of both SLPI and elafin, PIs and WAP motif proteins, by molecular cloning and targeting of mouse SLPI and elafin genes. The mouse SLPI cDNA was cloned by usage of the SLPI was expressed in non-epithelial cells in the germinal center of mouse spleen as well as neutrophils and macrophages in the pneumonic alveoli. It is of interest in this context that we have also confirmed human recombinant SLPI induces multi-organotrophic factor, HGF, in human lung fibroblasts, which opens a novel fields of SLPI function. We obtained the genomic mouse SLPI gene structure and identified the location of both human SLPI gene (chromosome 20q12-13.2) and mouse SLPI gene (chromosome 2H). With the infor
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mation of mouse SLPI genomic structure, we constructed a targeting plasmid with PGKNEO and DTA for mouse SLPI gene, transfected with 129/SvJ ES cells, and obtained chimeric SLPI +/- mice and then SLPI -/- mice for the study of their characteristic phenotype. As for the mouse elafin cDNA, failure of the cloning by the same strategy using homologous base sequence among species lead us to access the mouse EST database with WAP motif and found novel ELM1 and ELM2 genes. By way of 5'RACE method we obtained full length cDNA of ELM1 and ELM2, and subsequently revealed the genomic structure of ELM1 and ELM2. Northern blot analysis revealed high expression of ELM1 in kidney, and similar induction pattern of lung ELM1 gene as that of SLP1 gene upon bacterial pneumoria. Because antimicrobial peptides including defensins are also among WAP motif proteins with growing concern for their role in the innate immunity through evolution, our study pioneered the way to understand WAP motif PIs of SLPI and elafin in the context of their physiologic functions. Less
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