| 1. |
- Bicsak, T A, et al.
(author)
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Tissue-type plasminogen activator in rat oocytes : expression during the periovulatory period, after fertilization, and during follicular atresia.
- 1989
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In: Endocrinology. - 0013-7227 .- 1945-7170. ; 124:1, s. 187-94
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Journal article (peer-reviewed)abstract
- The regulation of tissue-type plasminogen activator (tPA) in rat oocytes during the periovulatory period, in early embryos, and in oocytes during induced follicular atresia was studied using a quantitative chromogenic substrate assay. Oocytes and early embryos were collected from three ovulation models: 1) intact immature female rats treated with PMSG, followed by hCG 48 h later; 2) hypophysectomized immature rats treated with PMSG, followed by a GnRH agonist (GnRHa) 56 h later; and 3) adult cyclic rats on the mornings of proestrus and estrus and up to 5 days after fertilization. In addition, follicular atresia was induced by either withdrawal of diethylstilbestrol (DES) for 2 days or injection of GnRHa for 2 days in hypophysectomized DES-implanted immature rats. Treatment with PMSG alone did not increase oocyte tPA content (5-20 microIU/oocyte) in either immature rat model, but treatment with either hCG or GnRHa induced meiotic maturation and ovulation and increased tPA activity to 80 and 140 microIU/oocyte 24 h after hCG and GnRHa treatment, respectively. Northern blot analysis of total RNA extracted from oocytes of PMSG-treated rats indicated the presence of a specific tPA message at 22S. tPA levels were low in preovulatory oocytes obtained on proestrus morning and increased in ovulated oocytes on estrus morning. After fertilization, tPA levels remained high in the embryos on days 1-4 of pregnancy, but dropped dramatically on day 5. Furthermore, oocytes from atretic follicles of hypophysectomized DES-implanted rats after either DES withdrawal or GnRHa treatment contained elevated levels of tPA, coincident with germinal vesicle breakdown (GVBD). Immunohistochemical staining revealed tPA antigen only in those oocytes that had undergone apparent meiotic maturation, as confirmed by GVBD. Thus, oocytes contain tPA mRNA and synthesize the active protease under a variety of stimuli which result in GVBD. The observed periovulatory increase in oocyte tPA activity, its maintenance until day 5 of pregnancy, and expression of tPA in nonovulatory oocytes of atretic follicles suggest diverse functions for the oocyte and embryo tPA.
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| 2. |
- Borg, H, et al.
(author)
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Evidence for IFN-beta heterogeneity in a substrain of Namalwa cells.
- 1985
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In: Applied Biochemistry and Biotechnology. - 0273-2289 .- 1559-0291. ; 11:2, s. 111-22
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Journal article (peer-reviewed)abstract
- A substrain of Namalwa cells, denoted substrain B, was grown in fermentors up to the 100-L scale, and was induced with Sendai virus to produce interferon (IFN). The titer of the crude IFN varied extensively between different batches; part of the variation was caused by a differential expression of IFN-alpha and IFN-beta. More than 80% of the IFN activity was IFN-beta by several criteria. A two-step purification procedure was developed and the resulting preparation had a specific activity of approximately 10(6) U/mg protein. The IFN-beta type was found to be heterogeneous, and could be separated into several components, which probably represented post-translational modifications of one molecule.
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| 3. |
- Erickson, L A, et al.
(author)
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The fibrinolytic system of the vascular wall.
- 1985
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In: Clinics in haematology. - 0308-2261. ; 14:2, s. 513-30
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Journal article (peer-reviewed)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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| 4. |
- Galway, A B, et al.
(author)
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Epidermal growth factor stimulates tissue plasminogen activator activity and messenger ribonucleic acid levels in cultured rat granulosa cells : mediation by pathways independent of protein kinases-A and -C.
- 1989
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In: Endocrinology. - 0013-7227 .- 1945-7170. ; 125:1, s. 126-35
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Journal article (peer-reviewed)abstract
- Recent reports suggest that epidermal growth factor (EGF) or related peptides may act as local hormones to regulate granulosa cell differentiation. While FSH and GnRH are known to stimulate accumulation of tissue-type plasminogen activator (tPA) mRNA in granulosa cells, studies using nonovarian cells have shown stimulation of tPA by EGF. In this study, the effect of EGF and its structural analog transforming growth factor-alpha (TGF alpha) on ovarian tPA mRNA and activity was investigated. Granulosa cells obtained from immature estrogen-treated rats were cultured with FSH or increasing doses of EGF or TGF alpha before analysis of tPA activity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique. Like FSH and GnRH, EGF and TGF alpha stimulated the secretion of tPA activity in a dose- and time-dependent manner (onset, 12 h; maximum, 48 h). Northern blot hybridization of total RNA using a rat cRNA probe for tPA showed the accumulation of a 22S species mRNA in cells treated with EGF or TGF alpha, but not with nerve growth factor, suggesting increased expression of the tPA gene. Furthermore, slot blot hybridization of RNA from these cells confirmed a time-dependent increase in tPA mRNA preceding that in enzyme activity. Cotreatment of a saturating dose of EGF with phorbol myristate acetate (PMA) or GnRH resulted in additive increases in both tPA enzyme activity and mRNA levels. In addition, pretreatment with PMA desensitized the cells to subsequent treatment with PMA or GnRH, but did not diminish EGF-induced tPA mRNA, suggesting that EGF acts through a pathway independent of protein kinase-C. Also, extracellular cAMP levels did not increase with EGF treatment in the presence or absence of a phosphodiesterase inhibitor, suggesting the lack of involvement of the protein kinase-A pathway. Suppression of protein synthesis by cycloheximide inhibited the induction of tPA mRNA by EGF, whereas similar treatment resulted in the superinduction of tPA mRNA in FSH-treated cells, suggesting that EGF and FSH do not share the same pathway. These results suggest that EGF and TGF alpha induce tPA mRNA and activity in granulosa cells through a pathway independent of protein kinases-A (FSH) and -C (GnRH and phorbol ester), providing an interesting model for future elucidation of the molecular mechanism involved in tPA gene expression.
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| 5. |
- Gustafsson, A, et al.
(author)
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Similar effects of treatment with alpha interferon on the protein synthesis of human large granular lymphocytes, T cells, and monocytes.
- 1985
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In: Scandinavian Journal of Immunology. - 0300-9475 .- 1365-3083. ; 22:5, s. 519-28
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Journal article (peer-reviewed)abstract
- Preparations of human large granular lymphocytes (LGL), T cells, and monocytes (MC) were obtained through centrifugation on Percoll gradients and preparative E-rosetting. The different preparations contained more than 80% of the appropriate cell type, as judged by their ability to lyse 51Cr-labelled K562 cells, cell morphology, and the presence of cell surface structures recognized by the OKT3, OKT10, Leu 7 and OKM1 monoclonal antibodies. The protein synthesis is unstimulated and alpha interferon (IFN-alpha)-treated cells of the different types was studied by subjecting 35S-methionine-labelled cell extracts to two-dimensional gel electrophoresis. The general pattern of protein synthesis in LGL and T cells was virtually identical, whereas at least 7 major proteins were synthesized at a higher rate in monocytes. The effects of IFN-alpha on the protein synthesis of LGL and T cells were identical, IFN-alpha increasing the rate of synthesis of 9 proteins. These proteins were also expressed, but not always IFN-augmentable, in monocytes. No additional, cell-type associated, IFN-inducible proteins were found. This suggests that the augmenting effect of IFN-alpha on the cytotoxic capacity of LGL, T cells, and monocytes may be to affect common steps in their lytic machineries.
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| 6. |
- Hsueh, A J, et al.
(author)
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Gonadotropin-releasing hormone induces ovulation in hypophysectomized rats : studies on ovarian tissue-type plasminogen activator activity, messenger ribonucleic acid content, and cellular localization.
- 1988
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In: Endocrinology. - 0013-7227 .- 1945-7170. ; 122:4, s. 1486-95
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Journal article (peer-reviewed)abstract
- GnRH and its agonists are known to induce ovulation in hypophysectomized rats by acting directly at the ovary. Because tissue-type plasminogen activator (tPA) has been implicated in the gonadotropin induction of ovulation, we examined the effect of an ovulatory dose of GnRH on ovarian tPA activity, mRNA content, and cellular localization. Hypophysectomized immature rats were injected sc with 20 IU PMSG and a single dose of a GnRH agonist (GnRHa; des-Gly10,DLeu6(N alpha Me)Leu7,Pro9NHEt-GnRH) 58 h later. At different times after treatment, ovaries were prepared for morphological analysis. Using a fibrin overlay method, tPA activities were measured in ovarian homogenates and cumulus-oocyte complexes, whereas granulosa cells were cultured for 24 h to estimate tPA secretion. Total ovarian RNA was prepared for hybridization analysis of tPA message levels, and tPA localization was studied by immunohistochemistry of ovarian sections. GnRHa induced ovulation in PMSG-primed hypophysectomized rats 14-16 h after injection in a dose-dependent manner, and the GnRHa action was blocked by concomitant treatment with a GnRH antagonist. GnRHa stimulated the induction of tPA, but not urokinase-type PA, activity in ovarian homogenates and granulosa cell-conditioned medium in a time-dependent manner, reaching a maximum before ovulation. tPA activity in cumulus-oocyte complexes was also increased before ovulation, but this increase was sustained. Hybridization analysis of steady state tPA mRNA levels was performed using a rat cRNA probe. Northern blot analysis of total ovarian RNA demonstrated that GnRHa stimulated tPA mRNA levels 12 h after treatment, with a subsequent decrease 24 h after treatment. Immunohistochemistry indicated substantial increases in tPA staining in granulosa cells and oocytes of preovulatory follicles before ovulation. Thus, GnRHa acts through specific receptors to increase ovarian tPA enzyme activity, mRNA content, as well as immunostaining in granulosa cells and oocytes. Like gonadotropins, GnRH may induce ovulation by directly stimulating tPA levels in the ovary.
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| 7. |
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| 8. |
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| 9. |
- Lawrence, D, et al.
(author)
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Purification of active human plasminogen activator inhibitor 1 from Escherichia coli. Comparison with natural and recombinant forms purified from eucaryotic cells.
- 1989
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In: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 186:3, s. 523-33
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Journal article (peer-reviewed)abstract
- Plasminogen activator inhibitor 1 (PAI-1) inhibits both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) and, therefore, is an important regulator of plasminogen activation. We have developed eucaryotic and procaryotic expression systems for PAI-1 and characterized the recombinant glycosylated and non-glycosylated products, together with a non-recombinant natural control, produced in the histosarcoma cell line HT 1080. For eucaryotic expression, the PAI-1 cDNA was stably transfected into chinese hamster ovary cells (CHO cells), while procaryotic expression in Escherichia coli was examined after inserting the DNA sequence encoding the mature PAI-1 protein into an inducible expression vector. Recombinant PAI-1 from CHO cells was purified approximately 50-fold in two steps and was indistinguishable from natural PAI-1. Between 3% and 4% of total cellular protein in the procaryotic expression system consisted of PAI-1, from which it was purified approximately 30-fold, with yields of between 15% and 20%. This PAI-1 formed 1:1 complexes with uPA and also with the single- and two-chain forms of tPA. Kinetic analysis demonstrated that the procaryote-produced PAI-1 had an inhibitory activity towards all three forms of PA that resembled that of natural PAI-1 with association rate constants of approximately 10(7) M-1 s-1. In contrast to PAI-1 from eucaryotic cells, the PAI-1 from E. coli had an inherent activity equal to that of guanidine/HCl-activated natural PAI-1. The activity could not be increased by treatment with denaturants suggesting that the latent form of PAI-1 was absent. However, at 37 degrees C the procaryote-produced PAI-1 lost activity at the same rate as natural PAI-1, with approximately 50% of the activity remaining after 3 h. This activity could be partially restored by treatment with 4 M guanidine/HCl. E. coli-derived PAI-1, added to human plasma and fractionated by Sephacryl S-200 chromatography, eluted in two peaks that were similar to those obtained with guanidine-activated PAI-1 from eucaryotic cells, suggesting that it bound to the PAI-1-binding protein (vitronectin).
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| 10. |
- Liu, Y X, et al.
(author)
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Gonadotropin regulation of tissue-type and urokinase-type plasminogen activators in rat granulosa and theca-interstitial cells during the periovulatory period.
- 1987
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In: Molecular and Cellular Endocrinology. - 0303-7207 .- 1872-8057. ; 54:2-3, s. 221-9
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Journal article (peer-reviewed)abstract
- Plasminogen activators (PAs) are believed to be involved in ovulation. Because both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are secreted by cultured rat granulosa cells, we have examined the activities of these proteins in ovarian homogenates as well as granulosa and theca-interstitial (TI) cells during gonadotropin-induced ovulation. Immature rats were injected with 20 IU pregnant mare serum gonadotropin (PMSG) to initiate follicle development, followed by treatment with 10 IU hCG 48 h later to induce ovulation. Ovarian proteins were separated by SDS-PAGE and PA activity determined by fibrin overlay. The activity of tPA, but not uPA, was stimulated following PMSG treatment in ovarian homogenates. Subsequent hCG injection further increased the tPA activity in a time-dependent manner, reaching a maximum (12 h after hCG treatment) immediately prior to ovulation and declined thereafter. Similar preovulatory increases in tPA activity were detected in isolated granulosa cells. Although both tPA and uPA activities were increased in TI cells after PMSG administration, no further increases were detected after hCG treatment. To estimate enzyme secretion, ovarian cells obtained at various preovulatory periods were incubated for 24 h in vitro. The ability of granulosa cells to secrete tPA, but not uPA, increased following in vivo PMSG and hCG treatment in a time-dependent manner, reaching a maximum immediately prior to ovulation. During the preovulatory period, an abrupt increase in tPA secretion by TI cells was also detected. Using immunohistochemical staining for tPA, it was found that ovarian sections from preovulatory rats at 12 h after hCG injection stained positively in granulosa, theca interna, and interstitial gland cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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