| 1. |
- 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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| 2. |
- Liu, Y X, et al.
(författare)
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Identification and regulation of tissue plasminogen activator activity in rat cumulus-oocyte complexes.
- 1986
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Ingår i: Endocrinology. - 0013-7227 .- 1945-7170. ; 119:4, s. 1578-87
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Tidskriftsartikel (refereegranskat)abstract
- Plasminogen activators convert plasminogen into plasmin, a serine protease that initiates extracellular proteolysis. Two types of plasminogen activator activities have recently been demonstrated in granulosa cells, and the proteolysis-inducing enzymes are believed to be involved in ovulation. However, little attention has been paid to the presence of these enzymes in oocytes. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique, we studied plasminogen activator activity in oocytes. Denuded oocytes collected from ovaries of hypophysectomized, estrogen-treated immature rats contained a tissue-type plasminogen activator (tPA), but not urokinase (uPA). In contrast, oocyte-free granulosa cells in these preantral follicles contained uPA, but not tPA. The tPA activity found in oocytes was plasminogen-dependent; incubation with increasing numbers (25-200) of denuded oocytes resulted in a dose-dependent increase in fibrinolysis only in the presence of plasminogen. Cellular localization of tPA was studied in the preantral follicles using an immuno-cytochemical method. Positive tPA staining was detected in the cytoplasm, but not in the germinal vesicle or zona pellucida of the oocytes. Furthermore, analysis using a reverse fibrin-overlay method did not reveal the presence of a plasminogen activator inhibitor. Culturing of denuded oocytes for 24 h increased the cellular content of tPA, but the enzyme activity was not further enhanced by treatment with FSH or forskolin. Also, no tPA activity was detected in the medium. We further studied plasminogen activator activities in the cumulus-oocyte complexes. Although only tPA activity was detected in freshly obtained cumulus-oocyte complexes, incubation for 24 h increased both tPA and uPA activity. Furthermore, tPA, but not uPA, activity was stimulated by treatment with FSH or forskolin. This was accompanied by the secretion of tPA into the medium. The identity of tPA and uPA in the cumulus-oocyte complexes was further confirmed by immunoprecipitation with specific antibodies. Isolation of denuded oocytes and cumulus cells after hormonal stimulation of the cumulus-oocyte complexes suggested that tPA activity was stimulated in both cell types and that the cumulus cells may mediate the action of FSH and forskolin on oocytes. In conclusion, the detection and regulation of tPA activity in cumulus-oocyte complexes suggest possible involvement of this enzyme in ovulation or the process of cumulus cell expansion and dispersion. Changes in oocyte tPA content may also serve as an indicator of oocyte development.
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| 3. |
- 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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| 4. |
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| 5. |
- Ny, Tor, et al.
(författare)
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Cultured granulosa cells produce two plasminogen activators and an antiactivator, each regulated differently by gonadotropins.
- 1985
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Ingår i: Endocrinology. - 0013-7227 .- 1945-7170. ; 116:4, s. 1666-8
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Tidskriftsartikel (refereegranskat)abstract
- Although treatment of cultured granulosa cells with gonadotropins increases their fibrinolytic activity, the biochemical nature of this effect is unclear. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fibrin autography techniques to characterize the fibrinolytic components secreted by granulosa cells. The fibrinolytic activity of these cells results from the production of both a tissue-type plasminogen activator (t-PA) and a urokinase-like activator (u-PA). The cells also produce an inhibitor of fibrinolysis (antiactivator). FSH and LH stimulate t-PA activity and suppress antiactivator activity, while u-PA activity is not affected by the gonadotropins. The differential regulation of these molecules by the gonadotropins may be essential for ovulation.
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| 6. |
- Erickson, L A, et al.
(författare)
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The fibrinolytic system of the vascular wall.
- 1985
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Ingår i: Clinics in haematology. - 0308-2261. ; 14:2, s. 513-30
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Tidskriftsartikel (refereegranskat)abstract
- The vascular endothelium produces both PAs and a PAI. The activities of these components in the circulation must be regulated precisely to ensure that normal vascular homeostasis is not compromised. The blood contains a number of molecules that may function in this way by either promoting or inhibiting the synthesis, release and/or activity of the PAs and PAI. It is clear that the regulation of this system is considerably more complex than previously thought. For example, the initiation of fibrin dissolution is influenced by a number of additional factors including fibrin itself, pro-activators, PAI, platelet components (including the PAI), and possibly by APC generated at the endothelial cell surface. Despite the many recent advances discussed above, little is known about the temporal control of the events leading to plasminogen activation during thrombus formation and dissolution. Obviously, such information must be obtained before more effective treatments of abnormal vascular fibrinolytic activity can be developed. In this chapter, we have described a number of reagents and assays that should aid in the quantification of the PAs and the PAI in plasma. Eventual utilization of these assays in a clinical setting may be valuable for the diagnosis and subsequent treatment of abnormalities of the vascular fibrinolytic system.
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| 7. |
- 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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