Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The chimeric molecule K1K2Pu, comprising the two kringle domains (K1 and K2) of tissue-type plasminogen activator (t-PA) and the COOH-terminal region with the serine protease domain (Pu) of urokinase-type plasminogen activator (u-PA), was previously shown to have a 5- to 10-fold reduced clearance rate with maintained specific thrombolytic activity, resulting in an increased thrombolytic potency in animal models of venous and arterial thrombosis. To document the thrombolytic potential of K1K2Pu, the thrombolytic potency and fibrin specificity were studied in a combined platelet-rich arterial eversion graft thrombosis and venous whole blood clot model in heparinized dogs (100 U/kg bolus and 50 U/kg per h infusion). Dose-response effects of bolus injections of K1K2Pu (0.032 to 0.25 mg/kg) were compared with those of recombinant t-PA (rt-PA) and of recombinant single chain u-PA (rscu-PA) (0.25 to 1.0 mg/kg each) in groups of five or six dogs, each given heparin with or without the thromboxane synthase inhibitor/prostaglandin endoperoxide receptor antagonist ridogrel. Heparin and ridogrel in the absence of a thrombolytic agent did not produce arterial reflow or venous clot lysis in five dogs. Addition of K1K2Pu, rt-PA or rscu-PA resulted in a dose-dependent induction of arterial reflow and of venous clot lysis in the absence of systemic fibrinolytic activation and fibrinogen breakdown. Consistent arterial reflow required 0.063 mg/kg of K1K2Pu and 0.5 mg/kg of rt-PA or of rscu-PA. The thrombolytic potency for venous clot lysis, expressed as percent lysis per mg compound administered per kg body weight, was (mean +/- SEM) 750 +/- 160 for K1K2Pu, 68 +/- 17 for rscu-PA (p less than 0.001 vs. K1K2Pu) and 110 +/- 29 for rt-PA (p less than 0.001 vs. K1K2Pu). The plasma clearance rates were significantly lower for K1K2Pu than for rscu-PA and rt-PA. In the absence of ridogrel, arterial reflow was significantly slower and was followed by cyclic reocclusion and reflow; however, venous clot lysis was unaffected. Template bleeding times were not significantly altered in the absence but were markedly prolonged in the presence of ridogrel. These results confirm and establish that, when given as a bolus injection, K1K2Pu has an approximately 10-fold higher thrombolytic potency for arterial and venous thrombolysis than does rt-PA or rscu-PA. Thrombolysis with K1K2Pu is obtained in the absence of systemic fibrinolytic activation and fibrinogen breakdown. These properties suggest that K1K2Pu offers potential for thrombolytic therapy by bolus administration in patients with thromboembolic disease.
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PMID:Comparative thrombolytic properties of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (u-PA) and K1K2Pu (a t-PA/u-PA chimera) in a combined arterial and venous thrombosis model in the dog. 134 79

We have recently shown that spermatozoa of various species contain both types of plasminogen activator, the tissue-type (t-PA) and the urokinase-type (u-PA). In the present study, the localization of t-PA and u-PA in plasma membrane and outer acrosomal membrane of human and boar spermatozoa has been investigated. The identification of the type of the plasminogen activator (t-PA or u-PA) was made immunologically. In human spermatozoa, the outer acrosomal membrane and plasma membrane contained both types of plasminogen activator (t-PA and u-PA); in addition, t-PA antigen was measured. In boar spermatozoa, the outer acrosomal membrane contained only t-PA, whereas plasma membrane contained both types of plasminogen activator (t-PA and u-PA). Plasminogen activator inhibition (PAI) has also been demonstrated in plasma and outer acrosomal membranes of both species and identified as PAI-1 in membranes of human spermatozoa.
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PMID:Plasminogen activator: the identification of an additional proteinase at the outer acrosomal membrane of human and boar spermatozoa. 135 44

The present study examined secretion of urokinase and tissue-plasminogen activator by epidermal cells in the presence of psoriatic or uninvolved skin fibroblast-conditioned medium. Using zymographic analyses, a 54kD lysis band and a small 110kD band derived from urokinase could be detected in the harvest fluid from keratinocytes treated with both psoriatic and uninvolved fibroblast-conditioned medium, as well as very weak lysis bands of 63kD and 120kD derived from tissue-plasminogen activator in the harvest fluid treated with psoriatic fibroblast-conditioned medium, but not with uninvolved fibroblast-conditioned medium.
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PMID:Secretion of urokinase and tissue-plasminogen activator by epidermal cells in the presence of psoriatic fibroblast-conditioned medium. 136 28

The increasing incidence of thromboembolic diseases has sustained the search for new agents able to stimulate the natural fibrinolytic system. The first generation of antithrombotic agents include bacterial streptokinase and human urine urokinase. Because these molecules lack specificity for the fibrin clot, important efforts have been made to produce, using recombinant DNA technology, agents presenting higher fibrin clot selectivity such as t-PA (tissue-type plasminogen activator) and scu-PA (single chain urokinase-type plasminogen activator). In parallel, several laboratories are presently attempting to create mutants and hybrids plasminogen activators displaying improved thrombolytic properties with respect to the natural molecules. In this paper, we describe briefly the mechanisms of fibrinolysis and the role of the different natural thrombolytic agents. In addition, we review the possibilities of genetic engineering for the production of natural and novel plasminogen activators.
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PMID:Development of new thrombolytic agents using recombinant DNA technology. 136 29

The expression of recombinant single-chain urokinase-like plasminogen activator (rscuPA) in Escherichia coli was optimized by fusing the puk gene to different promoters and ribosome binding sequences. Comparison of the tac, trp and lambda PL promoters showed that expression was maximal under tac control. Variation in the ribosome binding sequence and its distance to the AUG start codon yielded a further slight improvement of expression. The largest increase in rscuPA expression was achieved by variations in the host strain and growth conditions. In E. coli DG75 grown at 37 degrees C maximal expression was achieved 30 min after induction and decreased gradually until 240 min after induction. Growth at 30 degrees C yielded maximal expression 60 min after induction and resulted in reduced activity at longer times. Western blot analysis of the products showed that degradation of rscuPA was much larger at 37 degrees C than at 30 degrees C. Using E. coli CAG630 carrying the htpR mutation, which avoids heat shock response, for expression of rscuPA eliminated the instability of the product at both temperatures. Expression in this strain was even more efficient than in E. coli JM101 carrying the lon mutation. It is concluded that induction of the general heat-shock response in E. coli must be avoided to obtain stabilization of rscuPA. This drastically improves the overall yield of rscuPA from recombinant E. coli strains.
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PMID:Optimizing the promoter and ribosome binding sequence for expression of human single chain urokinase-like plasminogen activator in Escherichia coli and stabilization of the product by avoiding heat shock response. 136 23

Urokinase-type plasminogen activator (uPA) is a proteolytic enzyme able to convert the zymogen plasminogen into the strong protease plasmin. The availability of very sensitive tests to measure the enzymatic activity of a plasminogen activator renders the corresponding gene an ideal candidate for the detection of promoter activity. In this paper we describe the utilization of the human uPA gene as detector of tissue-specificity of the murine whey acidic protein (WAP) expression signals in transgenic mice. The WAP promoter has been previously investigated for the production of foreign proteins in the milk of transgenic animals. In our genetic constructions, the human uPA cDNA was linked to the promoter region as well as to 3'-end distal sequences of the WAP gene. Five transgenic lines were obtained in which, however, expression levels of human uPA in the milk were still quite low. Surprisingly, four of these five positive transgenic mice show a consistent activity of the WAP promoter in brain extracts compared to other tissues.
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PMID:Use of the urokinase-type plasminogen activator gene as a general tool to monitor expression in transgenic animals: study of the tissue-specificity of the murine whey acidic protein (WAP) expression signals. 136 47

We have constructed a shuttle plasmid for Bacillus megaterium and Escherichia coli that contains the promoter and repressor gene of the B. megaterium-borne operon for xylose utilization. A polylinker downstream of the promoter allows versatile cloning of genes under its transcriptional control. We have placed gdhA (encoding glucose dehydrogenase) from B. megaterium, lacZ (encoding beta-galactosidase) from E. coli, mro (encoding mutarotase) from Acinetobacter calcoaceticus, and human puk (encoding single-chain urokinase-like plasminogen activator, rscuPA) under xylose control in this vector. All four genes were between 130-fold and 350-fold inducible by 0.5% xylose in the growth medium in B. megaterium. Enzymatically active glucose dehydrogenase and mutarotase accumulated to 20% and 30% of the total soluble protein, respectively. beta-Galactosidase and rscuPA were also expressed at a high level. A gel analysis of the products demonstrated their proteolytic stability in the cytoplasm, even up to 5 h after induction. The expression properties of this new host-vector system are discussed in comparison to the ones available for B. subtilis and E. coli.
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PMID:Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon. 136 76

The anti-urokinase-IgG-resistant plasminogen activator secreted by human embryonic lung diploid fibroblasts, IMR-90 cells (ATCC, CCL186) was purified to homogeneity from serum-free conditioned medium by a four-step procedure. The fibroblast plasminogen activator was identified as tissue plasminogen activator (t-PA) by the N-terminal sequence of the purified material and the complete amino acid sequence deduced from its complementary DNA (cDNA). The apparent molecular weight was the range of 64,000 to 68,000 by SDS-PAGE and was in the range of 69,000 to 72,000 by gel filtration. The fibroblast t-PA showed a stricter substrate specificity than urokinase in enzymatic hydrolysis of various chromogenic substrates. Compared to urokinase, the fibrobrast t-PA was more stable by heating at 95 degrees C for five min and was stable from pH 5 to 10. The fibrorast t-PA had a higher affinity for fibrin than urokinase.
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PMID:Purification and characterization of tissue plasminogen activator secreted by human embryonic lung diploid fibroblasts, IMR-90 cells. 136 81

mRNA levels for urokinase type plasminogen activator (uPA), tissue type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) were examined in human diploid (neonatal foreskin) fibroblasts grown in 200-ml microcarrier suspension culture. Four different substrates were used. These included gelatin-coated polystyrene plastic, DEAE-dextran, glass-coated polystyrene plastic and uncoated polystyrene plastic. Our previous studies have shown that culture fluids from diploid fibroblasts grown on DEAE-dextran contained higher levels of plasminogen-dependent fibrinolytic activity than culture fluids from the same cells grown on other substrates. The increased plasminogen activator activity was due largely to elevated amounts of tPA (In Vitro Cell. Develop. Biol. 22: 575-582, 1986). The present study shows that there is a corresponding elevation of tPA mRNA in diploid fibroblasts cultured on DEAE-dextran relative to the other substrates. There does not appear to be any difference in uPA mRNA or in mRNA for PAI-1 or PAI-2 produced by the same cells on the four substrates. These data suggest that the influence of the substrate on plasminogen activator production is mediated at the genetic level.
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PMID:Expression of plasminogen activator and plasminogen activator inhibitor mRNA in human fibroblasts grown on different substrates. 136 69

The Escherichia coli-derived tet regulatory elements from Tn10 have been used to construct vectors allowing the regulated, inducible, high-level expression of foreign genes in Bacillus subtilis. While the wild-type tet promoters are inactive in B. subtilis, a synthetic mutant tet sequence with improved promoter consensus sequences and upstream poly A blocks shows activity in B. subtilis. The expression of an indicator cat gene is inducible by sublethal amounts of tetracycline, indicating that the Tet repressor protein and the tet operator sequences are functional. However, the inducibility and maximal expression are not sufficient in this construct. To improve these properties a tet operator sequence was placed between the -35 and -10 boxes of the B. subtilis-derived very strong xyl promoter. In the presence of a tetR gene this construct is about 100-fold inducible and has high promoter strength, but some basal expression. This is avoided by placing a second tet operator downstream resulting in no detectable basal expression at the expense of reduced inducibility. Using the system with a single tet operator inducible expression of glucose dehydrogenase from B. megaterium was obtained at a very high level, and inducible expression of human single-chain urokinase-like plasminogen activator was achieved at the same level as in E. coli. Unlike in E. coli, the product was not degraded up to 4 h after induction in B. subtilis. These results demonstrate that the regulated expression vector described here should be very useful for production of foreign gene products from B. subtilis cultures.
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PMID:Regulated expression of heterologous genes in Bacillus subtilis using the Tn10 encoded tet regulatory elements. 136 98


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