| Budget Amount *help |
¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1994: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1993: ¥600,000 (Direct Cost: ¥600,000)
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| Research Abstract |
We have attempted to identify and purify a novel peptide that stimulates prostacyclin (PGl_2) production by vascular endothelial cells, with conditioned medium from cultured human diploid fibroblast cells. High pressure liquid chromatographic purification of the active peptide was succeeded using DEAE-5PW,Heparin-5PW,Protein-Pak 300, and lnsulin-like growth factor-1 ligand affinity column, respectively. The purified prostacyclin-stimulating factor (PSF) showed a single band with approximate molecular weight of 31 kDa on SDS PAGE electrophoresis. Additionally, the purity of the PSF was confirmed by C_4 reversed-phase chromatography, showing a single peak in the 31% (V/V) fraction under acetonitrile gradient. The purified PSF became 8000-fold in the activity that stimulates PGL_2 production by cultured bovine aortic endothelial cells as compared to the conditioned medium. The maximal stimulation by the purified PSF was shown at a concentration of 25 ng/ml added. Thereafter, a cDNA coding
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for the PSF was cloned and sequenced, indicating that the PSF is a novel bioactive peptide with no obvious sequence similarity to known proteins. Next, using Pvull/Smal digested fragment excised from the PSF cDNA as a probe, we examined the PSF expression by the Northern blot analysis on tissues of Wistar rats and on cultured vascular cells such as endothelial cells (ECs) and smooth muscle cells (SMCs). The expression of the PSF was found in many tissues of Wistar rats such as brain, lung, liver, kidney, skeketal muscle, and fat tissue. Particularly, lung and kidney showed marked expressions of the PSF.Furthermore, the PSF was extremely expressed in cultured vascular ECs and SMCs, as well as skin fibroblast cells. Moreover, our preliminary immunohistochemical study demonstrated that the PSF staining was found in the ECs and SMCs of tissue arteries. These results suggest that the PSF may be conserve over species and play a significant role in the regulation of PGl_2 production by vascular wall with autocrine and/or paracrine fashion. Less
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