Purification and cloning of a streptokinase from Streptococcus uberis.
(1/1360)
A bovine plasminogen activator was purified from the culture supernatant of the bovine pathogen Streptococcus uberis NCTC 3858. After the final reverse-phase high-performance liquid chromatography step a single protein with a molecular mass of 32 kDa was detected in the active fraction. A partial peptide map was established, and degenerate primers were designed and used for amplification of fragments of the gene encoding the activator. Inverse PCR was subsequently used for obtaining the full-length gene. The S. uberis plasminogen activator gene (skc) encodes a protein consisting of 286 amino acids including a signal peptide of 25 amino acids. In an amino acid sequence comparison the cloned activator showed an identity of approximately 26% to the streptokinases isolated from Streptococcus equisimilis and Streptococcus pyogenes. Interestingly, the activator from S. uberis was found to lack the C-terminal domain possessed by the streptokinase from S. equisimilis. This is apparently a general feature of the streptokinases of this species; biochemical and genetic analysis of 10 additional strains of S. uberis revealed that 9 of these were highly similar to strain NCTC 3858. Sequencing of the skc gene from three of these strains indicated that the amino acid sequence of the protein is highly conserved within the species. (+info)
Expression of the plague plasminogen activator in Yersinia pseudotuberculosis and Escherichia coli.
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Enteropathogenic yersiniae (Yersinia pseudotuberculosis and Yersinia enterocolitica) typically cause chronic disease as opposed to the closely related Yersinia pestis, the causative agent of bubonic plague. It is established that this difference reflects, in part, carriage by Y. pestis of a unique 9.6-kb pesticin or Pst plasmid (pPCP) encoding plasminogen activator (Pla) rather than distinctions between shared approximately 70-kb low-calcium-response, or Lcr, plasmids (pCD in Y. pestis and pYV in enteropathogenic yersiniae) encoding cytotoxic Yops and anti-inflammatory V antigen. Pla is known to exist as a combination of 32.6-kDa (alpha-Pla) and slightly smaller (beta-Pla) outer membrane proteins, of which at least one promotes bacterial dissemination in vivo and degradation of Yops in vitro. We show here that only alpha-Pla accumulates in Escherichia coli LE392/pPCP1 cultivated in enriched medium and that either autolysis or extraction of this isolate with 1.0 M NaCl results in release of soluble alpha and beta forms possessing biological activity. This process also converted cell-bound alpha-Pla to beta-Pla and smaller forms in Y. pestis KIM/pPCP1 and Y. pseudotuberculosis PB1/+/pPCP1 but did not promote solubilization. Pla-mediated posttranslational hydrolysis of pulse-labeled Yops in Y. pseudotuberculosis PB1/+/pPCP1 occurred more slowly than that in Y. pestis but was otherwise similar except for accumulation of stable degradation products of YadA, a pYV-mediated fibrillar adhesin not encoded in frame by pCD. Carriage of pPCP by Y. pseudotuberculosis did not significantly influence virulence in mice. (+info)
Macrophage plasminogen activator: induction by asbestos is blocked by anti-inflammatory steroids.
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Intraperitoneal injection of asbestos fibres into mice induces the formation of exudates containing macrophages that produce plasminogen activator. Like-wise, in vitro addition of asbestos to macrophage cultures stimulates plasminogen activator secretion; the synthesis and secretion of lysozyme and lysosomal enzymes are not changed under these conditions. The enhanced secretion of plasminogen activator by macrophages exposed to asbestos is suppressed by low concentrations of anti-inflammatory steroids. (+info)
Isolation of SMTP-3, 4, 5 and -6, novel analogs of staplabin, and their effects on plasminogen activation and fibrinolysis.
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Four novel triprenyl phenol metabolites, designated SMTP-3, -4, -5, and -6, have been isolated from cultures of Stachybotrys microspora IFO 30018 by solvent extraction and successive chromatographic fractionation using silica gel and silica ODS columns. A combination of spectroscopic analyses showed that SMTP-3, -4, -5, and -6 are staplabin analogs, containing a serine, a phenylalanine, a leucine or a tryptophan moiety in respective molecules in place of the N-carboxybutyl portion of the staplabin molecule. SMTP-4, -5, and -6 were active at 0.15 to 0.3 mM in enhancing urokinase-catalyzed plasminogen activation and plasminogen binding to fibrin, as well as plasminogen- and urokinase-mediated fibrinolysis. On the other hand, the concentration of staplabin required to exert such effects was 0.4 to 0.6 mM, and SMTP-3 was inactive at concentrations up to 0.45 mM. (+info)
Interaction between group A streptococci and the plasmin(ogen) system promotes virulence in a mouse skin infection model.
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Group A streptococci are capable of acquiring a surface-associated, unregulatable plasmin-like enzymatic activity when incubated in human plasma. The effect of this enzymatic activity on virulence of group A isolate CS101 was examined in a mouse skin infection model. Initial studies demonstrated enhanced virulence for bacteria preincubated in human plasma but not in plasminogen-depleted plasma. A direct correlation between surface-associated enzymatic activity and virulence was not observed; however, an association between virulence and the assembly of a surface-associated plasminogen activator that could activate mouse plasminogen was noted. This activity enhanced virulence in wild type but not in plg-/- plasminogen-deficient mice. These results support the hypothesis that acquisition of a surface-associated plasmin(ogen)-dependent enzymatic activity can contribute to the virulence of group A streptococcal invasive infections. (+info)
Carotid endarterectomy and intracranial thrombolysis: simultaneous and staged procedures in ischemic stroke.
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PURPOSE: The feasibility and safety of combining carotid surgery and thrombolysis for occlusions of the internal carotid artery (ICA) and the middle cerebral artery (MCA), either as a simultaneous or as a staged procedure in acute ischemic strokes, was studied. METHODS: A nonrandomized clinical pilot study, which included patients who had severe hemispheric carotid-related ischemic strokes and acute occlusions of the MCA, was performed between January 1994 and January 1998. Exclusion criteria were cerebral coma and major infarction established by means of cerebral computed tomography scan. Clinical outcome was assessed with the modified Rankin scale. RESULTS: Carotid reconstruction and thrombolysis was performed in 14 of 845 patients (1.7%). The ICA was occluded in 11 patients; occlusions of the MCA (mainstem/major branches/distal branch) or the anterior cerebral artery (ACA) were found in 14 patients. In three of the 14 patients, thrombolysis was performed first, followed by carotid enarterectomy (CEA) after clinical improvement (6 to 21 days). In 11 of 14 patients, 0.15 to 1 mIU urokinase was administered intraoperatively, ie, emergency CEA for acute ischemic stroke (n = 5) or surgical reexploration after elective CEA complicated by perioperative intracerebral embolism (n = 6). Thirteen of 14 intracranial embolic occlusions and 10 of 11 ICA occlusions were recanalized successfully (confirmed with angiography or transcranial Doppler studies). Four patients recovered completely (Rankin 0), six patients sustained a minor stroke (Rankin 2/3), two patients had a major stroke (Rankin 4/5), and two patients died. In one patient, hemorrhagic transformation of an ischemic infarction was detectable postoperatively. CONCLUSION: Combining carotid surgery with thrombolysis (simultaneous or staged procedure) offers a new therapeutic approach in the emergency management of an acute carotid-related stroke. Its efficacy should be evaluated in interdisciplinary studies. (+info)
Glycosylation of asparagine-28 of recombinant staphylokinase with high-mannose-type oligosaccharides results in a protein with highly attenuated plasminogen activator activity.
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The properties of recombinant staphylokinase (SakSTAR) expressed in Pichia pastoris cells have been determined. The single consensus N-linked oligosaccharide linkage site in SakSTAR (at Asn28 of the mature protein) was occupied in approximately 50% of the expressed protein with high-mannose-type oligosaccharides. The majority of these glycans ranged in polymerization state from Man8GlcNAc2 to Man14GlcNAc2, with the predominant species being Man10GlcNAc2 and Man11GlcNAc2. Glycosylated SakSTAR (SakSTARg) did not differ from its aglycosyl form in its aggregation state in solution, its thermal denaturation properties, its ability to form a complex with human plasmin (hPm), the amidolytic properties of the respective SakSTAR-hPm complexes, or its ability to liberate the amino-terminal decapeptide required for formation of a functional SakSTAR-hPm plasminogen activator complex. However, this latter complex with SakSTARg showed a greatly reduced ability to activate human plasminogen (hPg) as compared with the same complex with the aglycosyl form of SakSTAR. We conclude that glycosylation at Asn28 does not affect the structural properties of SakSTAR or its ability to participate in the formation of an active enzymatic complex with hPm, but it is detrimental to the ability of the SakSTAR-hPm complex to serve as a hPg activator. This is likely due to restricted access of hPg to the active site of the SakSTARg-hPm complex. (+info)
The plasminogen-plasminogen activator (PA) system in neuroblastoma: role of PA inhibitor-1 in metastasis.
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Proteases of the plasminogen-plasminogen activator (PA) system play an important role in cancer metastasis. We have examined the expression of these proteases and their cell surface receptors and inhibitors in neuroblastoma, a tumor that originates in cells of the neural crest and is the second most common solid tumor in children. This analysis was performed in seven established human cell lines and 20 primary tumor specimens. Urokinase PA and, in particular, tissue-type PA were expressed in cell lines and in tumor tissues; however, their levels of expression did not correlate with clinical stage. There was little evidence suggesting that neuroblastoma cells concentrate PA activity at their cell surface because urokinase-type PA receptor mRNA was detected in two cell lines and in 5 of 20 tumor samples by reverse transcription-PCR only. PA inhibitor (PAI)-2 was absent in all cell lines and tumor tissue samples examined. However, PAI-1, which was not expressed by the cell lines, was expressed by stromal cells and, specifically, endothelial cells in tumor tissue. By extending the analysis of PAI-1 expression in 64 primary tumor specimens, we found that high PAI-1 expression paradoxically correlated with metastatic stage and tumor recurrence. In vitro experiments indicated that the expression of PAI-1 by human microvascular endothelial cells was stimulated in the presence of SK-N-BE(2) human neuroblastoma cells and neuroblastoma culture medium. Recombinant PAI-1 also promoted SK-N-BE(2) cell detachment from vitronectin and migration from vitronectin toward fibronectin. From these data, we conclude that the up-regulation of PAI-1 expression in endothelial cells may promote rather than inhibit metastasis in neuroblastoma. (+info)