| Project/Area Number |
09670159
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Sapporo Medical University |
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Principal Investigator |
KUROKI Yoshio Sapporo Medical University, School of Medicine, Professor, 医学部, 教授 (70161784)
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| Co-Investigator(Kenkyū-buntansha) |
SANO Hitomi Sapporo Medical University, School of Medicine, Research Associate, 医学部, 助手 (80295344)
SOHMA Hitoshi Sapporo Medical University, School of Medicine, Assistant Professor, 医学部, 講師 (70226702)
小笠原 由法 札幌医科大学, 医学部, 講師 (20224090)
秋野 豊明 札幌医科大学, 医学部, 教授 (80045377)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | pulmonary surfactant / SP-A / SP-D / mannose-binding protein / collectin / lipopolysaccharides / CD14 / TNFalpha / サーファクタント蛋白質A / ホスファチジルコリン / ホスファチジルイノシトール / サーファクタント蛋白質 / C型レクチン / 肺胞II型細胞 / 糖鎖認識領域 |
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| Research Abstract |
1. Pulmonary surfactant proteins A and D (SP-A and SP-D) and mannose-binding protein A(MBP-A) are collectins in the C-type lectin superfamily. These collectins exhibit unique lipid binding properties. The structure-function relationship was investigated by using SP-ASP-D chimeras, It was found that the SP-A region of Glu^<195>- Phe^<228> is required for lipid and type II cell interactions and that the SP-D region of Cys^<261>-Phe^<355> is required for optimal lipid interactions.
Monoclonal antibodies (mAbs PElO and PC6) that recognize human SP-A inhibit the interactions of SP-A with lipids and alveolar type II cells. We mapped the epitopes for antihuman SP-A mAbs by a phage display peptide library. Phage selected by mAbs displayed the consensus peptide sequences that are nearly identical to ^<184>TPVNYTNWYRG^<194> of human SP-A.Chimeric proteins were generated in which the rat SP-A region Thr^<174>-Gly^<194> was replaced with the MBP-A region Thr^<164>-Asp^<184> (rat ama4, respectively)
… More
. Rat ama4 bound to an affinity matrix on mannose-sepharose but lost all of the SP-A functions except carbohydrate binding and Ca^<2+> independent GalCer binding. Strikingly, the rat ama4 chimera acquired the PI binding property that MBP-A exhibits. This study demonstrates that the amino acid residues 174-194 of SP-A and the corresponding region of MBP-A are critical for SP-A-type II cell interaction and Ca^<2+>-dependent lipid binding of collectins.
2. Pulmonary surfactant protein A (SP-A) plays an important part in antibody independent host defense mechanisms of the lung. SP-A bound to rough forms but not to smooth forms of LPS.When U937 cells and rat alveolar macrophages were preincubated with SP-A ; smooth LPS failed to induce TNFalpha secretion while rough LPS-induced TNFalpha secretion was modestly increased. Western blot analysis revealed that CD14 was one of the SP-A-binding proteins isolated from solubilized U937 cells. In addition, SP-A directly bound to recombinant soluble CD14 (rsCDl4). When rsCDl4 was preincubated with SP-A, the binding of rsCDl4 to smooth LPS was significantly reduced but the association of rsCDl4 with rough LPS was augmented. These results demonstrate the different actions of SP-A upon distinct serotypes of LPS, and indicate that the direct interaction of SP-A with CD14 constitutes a likely mechanism by which SP-A modulates LPS-elicited cellular responses. Less
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