| 1. |
- Loskutoff, D J, et al.
(författare)
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Fibrinolytic system of cultured endothelial cells : regulation by plasminogen activator inhibitor.
- 1986
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 32:4, s. 273-80
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Tidskriftsartikel (refereegranskat)abstract
- Cultured bovine aortic endothelial cells have a relatively complex fibrinolytic system that is responsive to both the physiological state of the cell itself and to a variety of agents added to the culture medium. The fibrinolytic activity of these cells results from the production of both urokinase-type and tissue-type plasminogen activators and is regulated by an inhibitor capable of neutralizing their activities. The properties of these fibrinolytic components will be reviewed, and their respective roles in initiating and regulating the fibrinolytic activity of the cells will be summarized. A cDNA coding for the inhibitor has been isolated, and its sequence will be compared to that of other serine proteinase inhibitors.
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| 2. |
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| 3. |
- Ny, Tor, et al.
(författare)
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Cloning and sequence of a cDNA coding for the human beta-migrating endothelial-cell-type plasminogen activator inhibitor.
- 1986
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - 0027-8424 .- 1091-6490. ; 83:18, s. 6776-80
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Tidskriftsartikel (refereegranskat)abstract
- A lambda gt11 expression library containing cDNA inserts prepared from human placental mRNA was screened immunologically using an antibody probe developed against the beta-migrating plasminogen activator inhibitor (beta-PAI) purified from cultured bovine aortic endothelial cells. Thirty-four positive clones were isolated after screening 7 X 10(5) phages. Three clones (lambda 1.2, lambda 3, and lambda 9.2) were randomly picked and further characterized. These contained inserts 1.9, 3.0, and 1.9 kilobases (kb) long, respectively. Escherichia coli lysogenic for lambda 9.2, but not for lambda gt11, produced a fusion protein of 180 kDa that was recognized by affinity-purified antibodies against the bovine aortic endothelial cell beta-PAI and had beta-PAI activity when analyzed by reverse fibrin autography. The largest cDNA insert was sequenced and shown to be 2944 base pairs (bp) long. It has a large 3' untranslated region [1788 bp, excluding the poly(A) tail] and contains the entire coding region of the mature protein but lacks the initiation codon and part of the signal peptide coding region at the 5' terminus. The two clones carrying the 1.9-kb cDNA inserts were partially sequenced and shown to be identical to the 3.0-kb cDNA except that they were truncated, lacking much of the 3' untranslated region. Blot hybridization analysis of electrophoretically fractionated RNA from the human fibrosarcoma cell line HT-1080 was performed using the 3.0-kb cDNA as hybridization probe. Two distinct transcripts, 2.2 and 3.0 kb, were detected, suggesting that the 1.9-kb cDNA may have been copied from the shorter RNA transcript. The amino acid sequence deduced from the cDNA was aligned with the NH2-terminal sequence of the human beta-PAI. Based on this alignment, the mature human beta-PAI is 379 amino acids long and contains an NH2-terminal valine. The deduced amino acid sequence has extensive (30%) homology with alpha 1-antitrypsin and antithrombin III, indicating that the beta-PAI is a member of the serine proteinase inhibitor (serpin) superfamily.
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| 4. |
- Sawdey, M, et al.
(författare)
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Messenger RNA for plasminogen activator inhibitor.
- 1986
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Ingår i: Thrombosis Research. - 0049-3848 .- 1879-2472. ; 41:2, s. 151-60
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Tidskriftsartikel (refereegranskat)abstract
- We report here the identification and preliminary characterization of the messenger RNA coding for a Mr 50,000 plasminogen activator inhibitor (PAI) synthesized by cultured bovine aortic endothelial cells. Polyadenylated RNA was prepared from these cells and translated in a rabbit reticulocyte lysate in vitro translation system. When the 35S methionine labeled translation products were immunoprecipitated with monospecific antiserum to PAI and analyzed by SDS-PAGE and autoradiography, a single major polypeptide of Mr 40,000 was revealed. Competition experiments were performed to determine the relationship of the immunoprecipitated polypeptide to the PAI. The amount of 35S-labeled immunoprecipitate was greatly decreased by the presence of the purified PAI, consistent with the conclusion that the Mr 40,000 band represented the translation product of PAI mRNA. This mRNA migrated with a sedimentation coefficient of 22s when analyzed by sucrose gradient centrifugation. The in vitro translation assay was used to determine the relative amount of PAI mRNA in cells cultured in calf serum purchased from different vendors. The level of PAI mRNA varied by at least eight-fold depending on the serum employed, suggesting that expression of the PAI gene is subject to regulation by external factors.
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